US 2004O101874A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0101874 A1 Ghosh et al. (43) Pub. Date: May 27, 2004

(54) TARGETS FOR THERAPEUTIC (22) Filed: Apr. 4, 2003 INTERVENTION IDENTIFIED IN THE MTOCHONDRIAL PROTEOME Related U.S. Application Data (75) Inventors: Soumitra S. Ghosh, San Diego, CA (60) Provisional application No. 60/412,418, filed on Sep. (US); Eoin D. Fahy, San Diego, CA 20, 2002. Provisional application No. 60/389,987, (US); Bing Zhang, Spring, TX (US); filed on Jun. 17, 2002. Provisional application No. Bradford W. Gibson, Berkeley, CA 60/372,843, filed on Apr. 12, 2002. (US); Steven W. Taylor, San Diego, CA (US); Gary M. Glenn, Encinitas, Publication Classification CA (US); Dale E. Warnock, San Diego, CA (US); Sara P. Gaucher, (51) Int. Cl." ...... C12O 1/68; C12N 1/00 Castro Valley, CA (US) (52) U.S. Cl...... 435/6; 435/317.1

Correspondence Address: ABSTRACT SEED INTELLECTUAL PROPERTY LAW (57) GROUP PLLC 701 FIFTHAVE Mitochondrial targets for drug Screening assays and for SUTE 6300 therapeutic intervention in the treatment of diseases associ SEATTLE, WA 98104-7092 (US) ated with altered mitochondrial function are provided. Com plete sequences SEQ ID NOS:1-3025 of (73) Assignees: MitoKor Inc., San Diego, CA (US); The polypeptides that comprise the heart mitochondrial Buck Institute for Age Research, proteome are provided, using fractionated derived Novato, CA (US) from highly purified mitochondrial preparations, to identify previously unrecognized mitochondrial molecular compo (21) Appl. No.: 10/408,765 nentS. Patent Application Publication May 27, 2004 Sheet 1 of 5 US 2004/0101874 A1

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TARGETS FOR THERAPEUTIC INTERVENTION mtDNA encodes 22 tRNAS, two ribosomal RNAS (12s and IDENTIFIED IN THE MITOCHONDRIAL 16S rRNA) and only 13 polypeptides, which are enzymes of PROTEOME the (ETC), the elaborate multi Subunit complex mitochondrial assembly where, for CROSS-REFERENCES TO RELATED example, respiratory oxidative phosphorylation takes place. APPLICATIONS (See, e.g., Wallace et al., in Mitochondria & Free Radicals in Neurodegenerative Diseases, M. F. Beal, N. Howell and 0001) This application claims the benefit of U.S. Provi I. BodisWollner, eds., 1997 Wiley-Liss, Inc., New York, pp. sional Patent Applications No. 60/412,418, filed Sep. 20, 283-307, and references cited therein; see also, e.g., Schef 2002; 60/389,987, filed Jun. 17, 2002; and 60/372,843, filed fler, I. E., Mitochondria, 1999Wiley-Liss, Inc., New York.) Apr. 12, 2002. Mitochondrial DNA thus includes Sequences encoding seven subunits of NADH dehydrogenase, also known as STATEMENT REGARDING SEQUENCE ETC Complex I (ND1, ND2, ND3, ND4, ND4L, ND5 and LISTING SUBMITTED ON CD-ROM ND6); one subunit of ETC Complex III (ubiquinol: cyto 0002 The Sequence Listing associated with this applica chrome c oxidoreductase, Cytb); three cytochrome c oxidase tion is provided on CD-ROM in lieu of a paper copy, and is (ETC Complex IV) subunits (COX1, COX2 and COX3); hereby incorporated by reference into the Specification. and two proton-translocating ATP synthase (Complex V) Three CD-ROMs are provided, containing identical copies subunits (ATPase6 and ATPase8). All other mitochondrial of the sequence listing: CD-ROM No. 1 is labeled COPY 1, constituent polypeptides are presumed to be encoded by contains the file 465.app.txt which is 14.4 MB and created of the extramitochondrial genome, and the number on Apr. 4, 2003; CD-ROM No.2 is labeled COPY 2, contains and identities of a large number of these polypeptides remain the file 465.app.txt which is 14.4 MB and created on Apr. 4, unknown. Accordingly, for most of the estimated 25,000 2003; CD-ROM No. 3 is labeled CRF, contains the file 40,000 proteins encoded by the human nuclear genome 465.app.txt which is 14.4 MB and created on Apr. 4, 2003. (Venter et al., 2001 Science 291: 1304; Lander et al., 2001 Nature 409:860) little is known regarding subcellular local BACKGROUND OF THE INVENTION ization, for example, which proteins may be molecular components of mitochondria. 0003) 1. Field of the Invention 0008 Mitochondria contain an outer mitochondrial mem 0004. The present invention relates generally to compo brane that Serves as an interface between the organelle and Sitions and methods for identifying mitochondrial proteins the cytosol, a highly folded inner mitochondrial membrane that are useful as targets for therapeutic intervention in that appears to form attachments to the Outer membrane at treating diseases associated with altered mitochondrial func multiple Sites, and an intermembrane Space between the two tion. More Specifically, the invention is directed to pro mitochondrial membranes. The Subcompartment within the teomic profiling of proteins and polypeptides of mitochon inner mitochondrial membrane is commonly referred to as dria and to uses of mitochondrial polypeptides in Screening the mitochondrial matrix (for review, See, e.g., Ernster et al., assays for, and as targets of, therapeutic agents. 1981 J. Cell Biol. 91:227s.) The cristae, originally postu 0005 2. Description of the Related Art lated to occur as infoldings of the inner mitochondrial 0006 Mitochondria are the complex subcellular membrane, have recently been characterized using three organelles that manufacture bioenergetically essential dimensional electron tomography as also including tube-like adenosine triphosphate (ATP) by oxidative phosphorylation, conduits that may form networks, and that can be connected and that promote direct and indirect biochemical regulation to the inner membrane by open, circular (30 nm diameter) of a wide array of cellular respiratory, oxidative and meta junctions (Perkins et al., 1997, JI. of Struct. Biol. 119:260). bolic processes, including aerobic respiration and intracel While the outer membrane is freely permeable to ionic and lular calcium regulation. For example, mitochondria provide non-ionic Solutes having molecular weights less than about the Subcellular site for physiologically important processes ten kilodaltons, the inner mitochondrial membrane exhibits Such as the Krebs cycle, the , fatty acid B-Oxida Selective and regulated permeability for many Small mol tion, and heme Synthesis. Mitochondria also participate in ecules, including certain cations, and is impermeable to mechanisms of apoptosis, or programmed cell death (e.g., large (greater than about 10 kD) molecules. Newmeyer et al., Cell 79:353-364, 1994; Liu et al., Cell 0009 Four of the five multisubunit complexes 86:147-157, 1996), which is apparently required for, inter (Complexes I, III, IV and V) that mediate ETC activity are alia, normal development of the nervous System and proper localized to the inner mitochondrial membrane. The remain functioning of the immune System. ing ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place 0007 Functional mitochondria contain gene products within the ETC, protons are moved from the mitochondrial encoded by mitochondrial genes situated in mitochondrial matrix, across the inner membrane, to the intermembrane DNA (mtDNA) and by extramitochondrial (e.g., nuclear) Space. This disequilibrium of charged Species creates an genes not situated in the circular mitochondrial genome. electrochemical membrane potential of approximately 220 While it has been estimated that a functional human mito mV referred to as the “protonmotive force” (PMF). The chondrion contains on the order of 1,000-1,500 distinct PMF, which is often represented by the notation Ap, corre proteins (Lopez et al., 2000 Electrophoresis 21:3427; Schef sponds to the Sum of the electric potential (Alpm) and the pH fler, I. E., Mitochondria, 1999 Wiley-Liss, Inc., New York; differential (ApH) across the inner membrane according to Rabilloud et al., 1998 Electrophoresis 19:1006; Schefflere the equation tal., 2001 1:161; Schatz, G., 1995 Biochem. Biophys. Acta Mol. Basis Dis. 1271:123), the 16.5 kb Ap=AGom-ZApH US 2004/0101874 A1 May 27, 2004

0010 wherein Z stands for -2.303 RT/F. The value of Z 0013 For instance, free radical production in biological is -59 at 25 C. when Ap and Alpm are expressed in mV and Systems is known to result in the generation of reactive ApH is expressed in pH units (See, e.g., Ernster et al., J. Cell Species that can chemically modify molecular components Biol. 91:227s, 1981 and references cited therein). of cells and tissues. Such modifications can alter or disrupt Structural and/or functional properties of these molecules, 0.011 Aum provides the energy for phosphorylation of leading to compromised cellular activity and tissue damage. adenosine diphosphate (ADP) to yield ATP by ETC Com Mitochondria are a primary Source of free radicals in bio pleX V, a process that is coupled Stoichiometrically with logical Systems (see, e.g., Murphy et al., 1998 in Mitochon transport of a proton into the matrix. Alm is also the driving dria and Free Radicals in Neurodegenerative Diseases, force for the influx of cytosolic Ca" into the mitochondrion. Beal, Howell and Bodis-Woliner, Eds., Wiley-Liss, New Under normal metabolic conditions, the inner membrane is York, pp. 159-186 and references cited therein), and altered impermeable to proton movement from the intermembrane mitochondrial function, Such as failure at any Step of the Space into the matrix, leaving ETC Complex V as the Sole mitochondrial electron transport chain (ETC), may also lead means whereby protons can return to the matrix. When, to the generation of highly reactive free radicals. Thus, free however, the integrity of the inner mitochondrial membrane radicals generated in biological Systems, including free is compromised, as occurs during mitochondrial permeabil radicals resulting from altered mitochondrial function or from extramitochondrial Sources, include reactive oxygen ity transition (MPT) that accompanies certain diseases asso Species (ROS), for example, Superoxide, peroxynitrite and ciated with altered mitochondrial function, protons are able hydroxyl radicals, and potentially other reactive Species that to bypass the conduit of Complex V without generating ATP, may be toxic to cells. Diseases associated with altered thereby uncoupling respiration. During MPT, Aum col mitochondrial function therefore include disorders in which lapses and mitochondrial membranes lose the ability to free radicals contribute to pathogenesis at the molecular Selectively regulate permeability to Solutes both Small (e.g., level (see, e.g., Halliwell B. and J. M. C. Gutteridge, Free ionic Cat", Na", K" and H") and large (e.g., proteins). Radicals in Biology and Medicine, 1989 Clarendon Press, 0012. A number of diseases, disorders or conditions, Oxford, UK). including degenerative diseases, are thought to be caused by, 0014) A particularly prevalent example of a disease asso or are associated with, alterations in mitochondrial function ciated with altered mitochondrial function is type 2 diabetes as provided herein. These disorders include Alzheimer's mellitus, or “late onset' diabetes, a common, degenerative Disease (AD), diabetes mellitus, Parkinson's Disease (PD), disease affecting 5 to 10 percent of the population in Huntington's disease, Freidreich's ataxia, atherosclerosis, developed countries. The propensity for developing type 2 hypertension, ischemia-reperfusion injury, Osteoarthritis, diabetes mellitus (“type 2 DM”) is reportedly maternally inflammatory diseases, amyotrophic lateral Sclerosis (ALS), inherited, Suggesting a mitochondrial genetic involvement. Wilson disease, autosomal recessive hereditary Spastic (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178–1180 paraplegia, Leigh Syndrome, benign and fatal infantile myo (1991); Reny, S. L., International J. Epidem. 23:886-890 pathies, multiple Sclerosis, dystonia, Leber's hereditary (1994)). Diabetes is a heterogeneous disorder with a strong optic neuropathy, Schizophrenia, cancer, psoriasis, Down's genetic component, monozygotic twins are highly concor Syndrome, hyperproliferative disorders, mitochondrial dia dant and there is a high incidence of the disease among first betes and deafness (MIDD) and myodegenerative disorders degree relatives of affected individuals. Such as “mitochondrial encephalopathy, lactic acidosis, and 0015. At the cellular level, the degenerative phenotype stroke” (MELAS), and “myoclonic epilepsy ragged red fiber that may be characteristic of late onset diabetes mellitus syndrome” (MERRF), as well as other mitochondrial respi includes indicators of altered mitochondrial respiratory ratory chain diseases (reviewed in Chinnery et al., 1999.J. function, for example impaired insulin Secretion, decreased Med. Genet. 36:425; see also references cited therein). ATP synthesis and increased levels of reactive oxygen Diseases associated with altered mitochondrial function thus species. Studies have shown that type 2 DM may be pre include these and other diseases in which one or more levels ceded by or associated with certain related disorders. For of an indicator of altered mitochondrial function differ in a example, it is estimated that forty million individuals in the Statistically significant manner from the corresponding indi U.S. suffer from impaired glucose tolerance (IGT). Follow cator levels found in clinically normal Subjects known to be ing a glucose load, ciruculating glucose concentrations in free of a presence or risk of Such disease. Other diseases IGT patients rise to higher levels, and return to baseline involving altered or respiration within cells may levels more slowly, than in unaffected individuals. A small also be regarded as diseases associated with altered mito percentage of IGT individuals (5-10%) progress to non chondrial function, for example, those in which free radicals insulin dependent diabetes (NIDDM) each year. This form Such as reactive oxygen species (ROS) contribute to patho of diabetes mellitus, type 2 DM, is associated with decreased genesis. Certain diseases associated with altered mitochon release of insulin by pancreatic beta cells and a decreased drial function appear to involve States of insufficient apop end-organ response to insulin. Other Symptoms of diabetes tosis (e.g., cancer and autoimmune diseases) or excessive mellitus and conditions that precede or are associated with levels of apoptosis (e.g., Stroke and neurodegeneration). For diabetes mellitus include obesity, Vascular pathologies, a general review of apoptosis, and the role of mitochondria peripheral and Sensory neuropathies and blindness. therein, see, e.g., Green and Reed, Science 281: 1309-1312, 1998; Green, Cell 94:695-698, 1998 and Kromer, Nature 0016 Despite intense effort, nuclear genes that segregate Medicine 3:614-620, 1997. The extensive list of additional with diabetes mellitus are rare and include, for example, diseases associated with altered mitochondrial function con mutations in the insulin gene, the insulin receptor gene and tinues to expand as aberrant mitochondrial or mitonuclear the glucokinase gene. By comparison, although a number of activities are implicated in particular disease processes. altered mitochondrial genes that Segregate with diabetes US 2004/0101874 A1 May 27, 2004

mellitus have been reported (see generally e.g., PCT/US95/ embodiments the disease associated with altered mitochon 04063), relationships amongst mitochondrial and extrami drial function is Alzheimer's disease, diabetes mellitus, tochondrial factors that contribute to cellular respiratory Parkinson's disease, Huntington's disease, osteoarthritis, and/or metabolic activities as they pertain to diabetes remain dystonia, Leber's hereditary optic neuropathy (LHON), poorly understood. mitochondrial encephalopathy, lactic acidosis, and Stroke 0017 Current pharmacological therapies for type 2 DM (MELAS), myoclonic epilepsy ragged red fiber Syndrome include injected insulin, and oral agents that are designed to (MERRF) or cancer. In certain embodiments the modifica lower blood glucose levels. Currently available oral agents tion is an amino acid Substitution, an amino acid insertion, include (i) the Sulfonylureas, which act by enhancing the an amino acid deletion, a posttranslational modification or Sensitivity of the pancreatic beta cell to glucose, thereby an altered expression level, and in certain further embodi increasing insulin Secretion in response to a given glucose ments the posttranslational modification is , load; (ii) the biguanides, which improve glucose disposal phosphorylation, nitration, nitrosylation, amidation, fatty rates and inhibit hepatic glucose output; (iii) the thiazo acylation or oxidative modification, including, for example, lidinediones, which improve peripheral insulin Sensitivity oxidative post-translational modification of tryptophan resi through interaction with nuclear peroxisome proliferator dues. activated receptors (PPAR, see, e.g., Spiegelman, 1998 0021. In certain other embodiments the present invention Diabetes 47:507-514; Schoonjans et al., 1997 Curr. Opin. provides a method of identifying an agent for treating a Lipidol. 8:159-166; Staels et al., 1997 Biochimie 79.95-99), disease associated with altered mitochondrial function, com (iv) repaglinide, which enhances insulin Secretion through prising (a) contacting a candidate agent with a biological interaction with ATP-dependent potassium channels; and (v) Sample from a Subject having a disease associated with acarbose, which decreases intestinal absorption of carbohy altered mitochondrial function, wherein the Sample com drates. It is clear that none of the current pharmacological prises at least one polypeptide that exhibits altered biologi therapies corrects the underlying biochemical defect in type cal activity which accompanies the disease and wherein the 2 DM. Neither do any of these. currently available treat polypeptide is (i) a polypeptide having an amino acid ments improve all of the physiological abnormalities in type sequence as set forth in any one of SEQID NOS 1-3025, or 2 DM Such as impaired insulin Secretion, insulin resistance (ii) a modified polypeptide that comprises at least one and/or excessive hepatic glucose output. In addition, treat modification to a polypeptide having an amino acid ment failures are common with these agents, Such that sequence as set forth in any one of SEQID NOS 1-3025; and multi-drug therapy is frequently necessary. (b) determining an increase or decrease in the altered bio logical activity of the polypeptide in the presence of the 0.018 Clearly there is a need for improved diagnostic candidate agent relative to the level of the altered biological methods for early detection of a risk for developing a disease activity in the absence of the candidate agent, and therefrom asSociated with altered mitochondrial function, and for bet identifying an agent for treating a disease associated with ter therapeutics that are specifically targeted to correct altered mitochondrial function. biochemical and/or metabolic defects responsible for Such disease, regardless of whether Such a defect underlying 0022. In certain embodiments the altered biological altered mitochondrial function may have mitochondrial or activity is an indicator of altered mitochondrial function that extramitochondrial origins. The present invention provides is ATP biosynthesis (e.g., an ATP biosynthesis factor), compositions and methods related to identification of mito oxidative phosphorylation, mitochondrial calcium uptake, chondrial targets for therapeutic intervention in treating mitochondrial calcium release, maintenance of inner mito these diseases, and offers other related advantages. chondrial membrane potential, mitochondrial permeability transition, ETC-mediated electron transport or mitochon BRIEF SUMMARY OF THE INVENTION drial intermembrane Space protein release. In certain other embodiments the Sample is a cell, a mitochondria enriched 0019. The present invention provides the identities of Sample, an isolated mitochondrion or a Submitochondrial 3025 polypeptide sequences SEQ ID NOS:1-3025) that are particle. In certain embodiments the disease associated with constituents of the human mitochondrial proteome. It is altered mitochondrial function is Alzheimer's disease, dia therefore an aspect of the present invention to provide a betes mellitus, Parkinson's disease, Huntington's disease, method for identifying a mitochondrial target for therapeutic Osteoarthritis, dystonia, Leber's hereditary optic neuropathy intervention in treatment of a disease associated with altered (LHON), mitochondrial encephalopathy, lactic acidosis, and mitochondrial function, comprising (a) determining a pres Stroke (MELAS), myoclonic epilepsy ragged red fiber Syn ence, in a biological Sample from a Subject known to have drome (MERRF) or cancer. or Suspected of having a disease associated with altered 0023. According to certain other embodiments there is mitochondrial function, of at least one modified polypeptide, provided by the present invention a method of treating a the modified polypeptide comprising at least one modifica disease associated with altered mitochondrial function com tion to a polypeptide having an amino acid Sequence as Set prising administering to a Subject in need thereof an agent forth in any one of SEQ ID NOS 1-3025; and (b) correlating that compensates for at least one biological activity of a the modification with at least one disease associated with polypeptide that exhibits altered biological activity which altered mitochondrial function, and therefrom identifying a accompanies the disease, wherein the polypeptide is (i) a mitochondrial target for therapeutic intervention. polypeptide having an amino acid Sequence as Set forth in 0020. In certain embodiments the modified polypeptide any one of SEQID NOS 1-3025, or (ii) a modified polypep exhibits altered biological activity. In certain embodiments tide that comprises at least one modification to a polypeptide the biological Sample is Selected from the group consisting having an amino acid Sequence as Set forth in any one of of blood, Skin, Skeletal muscle, liver and cartilage. In certain SEO ID NOS 1-3025. In another embodiment the invention US 2004/0101874 A1 May 27, 2004 provides a method for identifying a risk for having or a brane potential, mitochondrial permeability transition, ETC presence of a disease associated with altered mitochondrial mediated electron transport or intermembrane Space protein function, comprising (a) determining a presence, in a bio release. In another further embodiment the Sample is a cell, logical Sample from a Subject Suspected of having a disease a mitochondria enriched Sample, an isolated mitochondrion asSociated with altered mitochondrial function, of at least or a Submitochondrial particle. In certain other further one modified polypeptide, the modified polypeptide com embodiments, the disease associated with altered mitochon prising at least one modification to a polypeptide having an drial function is Alzheimer's disease, diabetes mellitus, amino acid sequence as set forth in any one of SEQ ID NOS Parkinson's disease, Huntington's disease, osteoarthritis, 1-3025, wherein the modification correlates with at least one dystonia, Leber's hereditary optic neuropathy (LHON), disease associated with altered mitochondrial function, and mitochondrial encephalopathy, lactic acidosis, and Stroke therefrom identifying a risk for or presence of disease. (MELAS), myoclonic epilepsy ragged red fiber Syndrome 0024 Certain other embodiments of the invention pro (MERRF), or cancer. vide a method of identifying an agent for treating a disease 0026. These and other aspects of the present invention asSociated with altered mitochondrial function, comprising will become evident upon reference to the following detailed (a) contacting a candidate agent with an isolated polypeptide description and attached drawings. In addition, various that exhibits altered biological activity which accompanies a references are set forth below which describe in more detail disease associated with altered mitochondrial function, certain procedures or compositions and are therefore incor wherein the polypeptide is Selected from the group consist porated by reference in their entireties. ing of (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025 and (ii) a modified BRIEF DESCRIPTION OF THE DRAWINGS polypeptide that comprises at least one modification to a polypeptide having an amino acid Sequence as Set forth in 0027 FIG. 1 shows representative western immunoblot any one of SEQ ID NOS 1-3025; and (b) determining an analysis (FIG. 1A) of indicated mitochondrial ETC proteins increase or decrease in the altered biological activity of the in Sucrose density gradient fractionated isolated human heart polypeptide in the presence of the candidate agent relative to mitochondria, following resolution of proteins by one-di the level of the altered biological activity in the absence of mensional polyacrylamide gel electrophoresis (FIG. 1B). the candidate agent, and therefrom identifying an agent for 0028 FIG. 2 shows a representative MALDI mass spec treating a disease associated with altered mitochondrial trum for a single band excised from a one-dimensional function. In certain further embodiments the disease asso polyacrylamide gel following electrophoretic resolution of ciated with altered mitochondrial function is Alzheimer's proteins from Sucrose density gradient fractionated isolated disease, diabetes mellitus, Parkinson's disease, Hunting human heart mitochondria. Peptides are from indicated ton's disease, osteoarthritis, dystonia, Leber's hereditary mitochondrial proteins as follows: B=ATP synthase beta optic neuropathy (LHON), mitochondrial encephalopathy, subunit, y=ATP synthase gamma Subunit, eCoA=enlyl-CoA lactic acidosis, and stroke (MELAS), myoclonic epilepsy hydratase, and Vd=Voltage dependent anion channel 1 ragged red fiber syndrome (MERRF), or cancer. In other (VDAC-1). (K=keratin.) further embodiments the isolated polypeptide is present in a preparation that is a Submitochondrial particle, a proteoli 0029) FIG. 3 shows products of tryptophan oxidation in poSome or a mitochondrial protein fraction. proteins. 0.025 In another embodiment the invention provides a 0030 FIG. 4 shows MALDI-TOF mass spectrometry of method of identifying an agent for treating a disease asso two peptides from complex I subunit NDUFS4 displaying ciated with altered mitochondrial function, comprising (a) (A) tryptophan and (B) methionine oxidation. The samples administering a candidate agent to a Subject having a disease were as follows (i) human heart mitochondria complex I associated with altered mitochondrial function; and (b) (HHM individual #1) prepared by Sucrose density gradient determining, in a first biological Sample obtained from the fractionation (SDG) and 1D electrophoresis; (ii) HHM indi Subject prior to the Step of administering the candidate agent vidual #1 prepared by immunocapture and ID electrophore and in a Second biological Sample obtained from the Subject sis (iii) HHM individual #2 prepared by immunocapture and Subsequent to the Step of administering the candidate agent, 1D electrophoresis; (iv) HHM individuals #3.4.5 (pooled) wherein each of Said first and Second Samples comprises at prepared by SDG and 1D electrophoresis, (v) bovine heart least one polypeptide that exhibits altered biological activity mitochondria (BHM animal #1) prepared by SDG and 1D which accompanies said disease and wherein the polypep electrophoresis; (vi) (BHM animal #2) prepared by SDG and tide is selected from the group consisting of (i) a polypeptide 2D electrophoresis. having an amino acid Sequence as Set forth in any one of 0031 FIG. 5 shows a comparison of the distribution of SEQ ID NOS 1-3025 and (ii) a modified polypeptide that (a) tryptophan and (b) methionine oxidation for complex I comprises at least one modification to a polypeptide having Subunit peptides. an amino acid Sequence as Set forth in any one of SEQ ID NOS 1-3025, an increase or decrease in the altered biologi cal activity of the polypeptide in the Second Sample relative DETAILED DESCRIPTION OF THE to the level of the altered biological activity in the first INVENTION Sample, and therefrom identifying an agent for treating a 0032. The present invention provides a method for iden disease associated with altered mitochondrial function. In a tifying mitochondrial polypeptide targets for therapeutic further embodiment, the altered biological activity is an intervention in the treatment of diseases associated with indicator of altered mitochondrial function that is ATP altered mitochondrial function, and a method for identifying biosynthesis, oxidative phosphorylation, calcium uptake, agents for treating Such diseases, as well as other related calcium release, maintenance of inner mitochondrial mem advantages. US 2004/0101874 A1 May 27, 2004

0033. The invention derives from characterization of the embodiments a biological Sample comprises a cybrid cell human heart mitochondrial proteome as described herein, to line having nuclear and mitochondrial DNAS of differing arrive at the Surprising discovery and recognition for the first biological origins, which in certain embodiments may be a time that polypeptides having the amino acid Sequences Set human cell, an immortal cell, a neuronal cell, a neuroblas forth in SEO ID NOS:1-3025 are mitochondrial molecular toma or other transformed cell, for example, a SH-SY5Y components. This unexpected determination, that isolated human neuroblastoma cell. In certain other particularly human mitochondria comprise polypeptides having the preferred embodiments a biological Sample comprises a amino acid sequences set forth in SEQ ID NOS:1-3025, is Sample readily obtained from a Subject or biological Source, usefully combined with methods for determining the pres Such as blood, skin, Skeletal muscle, liver or cartilage. ence of a disease associated with altered mitochondrial 0036 By way of background, mitochondria are com function, and with methods for determining modification to, prised of “mitochondrial molecular components”, which and altered biological activity of, a polypeptide, to provide may be any protein, polypeptide, peptide, amino acid, or targets for drug-Screening assays and for therapeutic agents. derivative thereof; any lipid, fatty acid or the like, or According to certain embodiments, the invention relates to derivative thereof; any carbohydrate, Saccharide or the like determination of at least one modified polypeptide that or derivative thereof, any , nucleotide, nucleo comprises a modification to a polypeptide having an amino Side, purine, pyrimidine or related molecule, or derivative acid sequence as set forth in any one of SEQ ID NOS:1- thereof, or the like, or any other biological molecule that is 3025, and according to certain other embodiments the inven a constituent of a mitochondrion, which may include mol tion relates to determination of a profile comprising a ecules that are integral or stable components of mitochon plurality (e.g., two or more) of polypeptides having distinct drial Structure, and may also include molecules that may amino acid Sequences wherein at least one Such polypeptide transiently associate with mitochondria under certain con has one of the amino sequences set forth in SEQ ID ditions, for example, regulated intracellular events that NOS:1-3025, and has not been previously identified as a involve mitochondria. In the most preferred embodiments, mitochondrial component. the present invention is directed to compositions and meth 0034. Thus, it is an aspect of the present invention to ods that relate to those mitochondrial molecular components provide a method for identifying a mitochondrial target for that are mitochondrial polypeptides or proteins, although the therapeutic intervention in treatment of a disease associated invention need not be So limited. with altered mitochondrial function, comprising (a) deter mining a presence, in a biological Sample from a Subject 0037. In certain preferred embodiments of the present known to have or Suspected of having a disease associated invention, a mitochondrial protein fraction is derived from with altered mitochondrial function, of at least one modified the biological sample as provided herein. A protein fraction polypeptide, the modified polypeptide comprising at least may be any preparation that contains at least one protein that one modification to a polypeptide having an amino acid is present in the Sample and which may be obtained by sequence as set forth in any one of SEQID NOS 1-3025; and processing a biological Sample according to any biological (b) correlating the modification with at least one disease and/or biochemical methods useful for isolating or otherwise asSociated with altered mitochondrial function, and there Separating a protein from its biological Source. Those famil from identifying a mitochondrial target for therapeutic inter iar with the art will be able to Select an appropriate method vention. depending on the biological Starting material and other factors. Such methods may include, but need not be limited 0.035 Biological samples may comprise any tissue or cell to, cell fractionation, density Sedimentation, differential preparation containing mitochondria. Biological Samples extraction, Salt precipitation, ultrafiltration, gel filtration, may be provided by obtaining a blood Sample, biopsy ion-exchange chromatography, partition chromatography, Specimen, tissue explant, organ culture or any other tissue or hydrophobic chromatography, reversed-phase chromatogra cell preparation from a Subject or a biological Source. The Subject or biological Source may be a human or non-human phy, one- and two-dimensional electrophoresis, affinity tech animal, a primary cell culture or culture adapted cell line niqueS or any other Suitable Separation method. including but not limited to genetically engineered cell lines 0038. It will be noted that in certain particularly preferred that may contain chromosomally integrated or episomal embodiments of the present invention, at least one Sample as recombinant nucleic acid Sequences, immortal, immortal described herein comprises a “mitochondria enriched” ized or immortalizable cell lines (e.g., capable of at least ten Sample, which refers to a Sample that comprises one or more cell doublings in vitro), Somatic cell hybrid or cytoplasmic mitochondria and that is Substantially depleted (i.e., partially hybrid “cybrid” cell lines (including mitochondrial cybrid or fully depleted, where the degree of depletion of a given cells having nuclear and mitochondrial DNAS of differing component can be quantified to show that its presence has biological origins, see, e.g., U.S. Pat. No. 5,888,498 and been reduced in a statistically significant manner) of one or International Publication No. WO95/26793), differentiated more non-mitochondrial marker proteins to the extent Such or differentiatable cell lines, transformed cell lines and the markers can be removed from a preparation and are detect like. In certain preferred embodiments of the invention, the able, as described herein and known to the art. Thus, for Subject or biological Source may be Suspected of having or example, cell fractionation techniques for the enrichment being at risk for having a disease associated with altered and detection of mitochondria, and/or biochemical markers mitochondrial function, including, for example, altered characteristic of these and other defined organelles, may be mitochondrial molecular composition or constitution, or used to determine that a particular Subcellular fraction oxidative modification of one or more mitochondrial pro containing one or more detectable organelle-Specific or teins, and in certain preferred embodiments of the invention organelle-associated markers or polypeptides, as provided the Subject or biological Source may be known to be free of herein, is Substantially enriched in mitochondria (See, e.g., a risk or presence of Such a disease. In certain other preferred Ernster et al., 1981 J. Cell Biol. 91:227s; see also, e.g., US 2004/0101874 A1 May 27, 2004

Rickwood et al., 1987, Mitochondria, a practical approach natural System, is isolated. Thus, for example, Such proteins (Darley-Usman, R., Wilson, Ed.), IRL Press; Storrie and could be part of a multisubunit complex or a membrane Madden, 1990 Methods in Enzymology 182, 203-225). vesicle, and/or Such peptides could be part of a composition, 0.039 For example, and in certain preferred embodiments and Still be isolated in that Such complex, vesicle or com including methods for determining the presence in a bio position is not part of its natural environment. logical Sample of a mitochondrial target polypeptide for 0042 “Biological activity” of a protein may be any therapeutic intervention-or for Screening a candidate agent detectable parameter that directly relates to a condition, for its ability to alter the biological activity of Such a target, process, pathway, dynamic structure, State or other activity a mitochondrial molecular component Such as any protein or involving the protein and that permits detection of altered polypeptide having an amino acid Sequence as Set forth in protein function in a biological Sample from a Subject or any one of SEQ ID NOS:1-3025 may be obtained from a biological Source, or in a preparation of the protein isolated preparation of isolated mitochondria and/or from a prepa therefrom. The methods of the present invention thus pertain ration of isolated submitochondrial particles (SMP). Tech in part to Such correlation where the protein having biologi niques for isolating mitochondria and for preparing SMP are cal activity may be, for example, an enzyme, a structural well known to the perSon having ordinary skill in the art and protein, a receptor, a ligand, a membrane channel, a regul may include certain minor modifications as appropriate for latory protein, a Subunit, a complex component, a chaperone the particular conditions Selected (e.g., Smith, A. L., Meths. protein, a binding protein or a protein having a biological Enzymol. 10:81-86; Darley-Usman et al., (eds.), Mitochon activity according to other criteria including those provided dria. A Practical Approach, IRL Press, Oxford, UK; Storrie herein. Such activity may include the amount of a protein et al., 1990 Meths. Enzymol. 182:203-255). Cell or tissue that is present, or the amount of a given protein's function lysates, homogenates, extracts, Suspensions, fractions or the that is detectable. like, or other preparations containing partially or fully purified mitochondrial molecular components Such as mito 0043 “Altered biological activity” of a protein may refer chondrial proteins (e.g., MCA) may also be useful in these to any condition or State, including those that accompany a and related embodiments. According to certain other related disease associated with altered mitochondrial function, for embodiments, one or more isolated mitochondrial molecular example, a disease or disorder characterized by altered (e.g., components Such as isolated targets for therapeutic inter increased or decreased in a Statistically significant manner vention in the treatment of a disease associated with altered relative to an appropriate control) mitochondrial molecular mitochondrial function may be present in membrane composition or constitution or by modification of a mito vesicles Such as uni- or multilamellar membrane vesicles, or chondrial protein as provided herein (and in particular, e.g., reconstituted into naturally derived or Synthetic liposomes or a modification to a polypeptide that in its unmodified form proteoliposomes or similar membrane-bounded compart comprises an amino acid Sequence as Set forth in any one of ments, or the like, according to generally accepted method SEQ ID NOS:1-3025), where any structure or activity that ologies (e.g., Jezek et al., 1990 J. Biol. Chem. 265:10522 is directly or indirectly related to a particular protein's 10526). function (or multiple functions) has been changed in a Statistically Significant manner relative to a control or Stan 0040 Affinity techniques are particularly useful-in the dard. context of the present invention, and may include any method that exploits a specific binding interaction with a 0044 Altered biological activity may have its origin in mitochondrial protein or peptide to effect a separation. Other deletion, Substitution or insertion of one or more amino acids useful affinity techniques include immunological techniques in a mitochondrial protein; in posttranslational modification for isolating specific proteins or peptides, which techniques of a mitochondrial protein; in an altered expression level rely on Specific binding interaction between antibody com (e.g., a statistically significant increase or decrease in the bining sites for antigen and antigenic determinants present in amount present) of a mitochondrial protein; in oxidatively the proteins or peptides. Immunological techniques include, modified Structures or oxidative events as well as in Oxida but need not be limited to, immunoaffinity chromatography, tion-independent Structures or events, in direct interactions immunoprecipitation, Solid phase immunoadsorption or between mitochondrial and extramitochondrial genes and/or other immunoaffinity methods. See, for example, Scopes, R. their gene products, or in Structural or functional changes K., Protein Purification: Principles and Practice, 1987, that occur as the result of interactions between intermediates Springer-Verlag, NY; Weir, D. M., Handbook of Experimen that may be formed as the result of Such interactions, tal Immunology, 1986, Blackwell Scientific, Boston; Deut including metabolites, catabolites, Substrates, precursors, scher, M. P., Guide to Protein Purification, 1990, Methods cofactors and the like. According to certain embodiments as in Enzymology Vol. 182, Academic Press, New York; and provided herein, altered biological activity of a protein may Hermanson, G. T. et al., Immobilized Afinity Ligand Tech also result from direct or indirect interaction of a biologi niques, 1992, Academic Press, Inc., California; which are cally active protein with an introduced agent Such as an hereby incorporated by reference in their entireties, for agent for treating a disease associated with altered mito details regarding techniques for isolating and characterizing chondrial function as described herein, for example, a Small proteins and peptides, including affinity techniques. molecule. 0041. The term "isolated” means that the material is 0045. Additionally, altered biological activity of a mito removed from its original environment (e.g., the natural chondrial protein (including proteins having any amino acid environment if it is naturally occurring). For instance, a sequence set forth in SEQ ID NOS:1-3025 or modified naturally occurring protein or peptide present in a living forms of Such proteins as provided herein) may result in animal is not isolated, but the Same protein or peptide, altered respiratory, metabolic or other biochemical or bio Separated from Some or all of the co-existing materials in the physical activity in Some or all cells of a biological Source US 2004/0101874 A1 May 27, 2004

having a disease associated with altered mitochondrial func 0048. The polypeptides of the present invention are pref tion. AS non-limiting examples, markedly impaired ETC erably provided in an isolated form, and in certain preferred activity may be related to altered biological activity of at embodiments are purified to homogeneity. The terms "frag least one protein, as may be generation of increased free ment,”“derivative” and “analog when referring to mito radicals Such as reactive oxygen species (ROS) or defective chondrial proteins Such as polypeptides identified herein as oxidative phosphorylation. AS further examples, altered mitochondrial components and having amino acid mitochondrial membrane potential, induction of apoptotic sequences as set forth in at least one of SEQ ID NOS:1- pathways and formation of a typical chemical and biochemi 3025, or when referring to modified polypeptides that com cal crosslinked Species within a cell, whether by enzymatic prise at least one modification to a polypeptide having an or non-enzymatic mechanisms, may all be regarded as amino acid Sequence as Set forth in any one of SEQ ID indicative of altered protein biological activity. Non-limiting NOS:1-3025 as provided herein, refers to any polypeptide or examples of altered protein biological activity are described protein that retains essentially the same biological function in greater detail below. or activity as Such polypeptide. Thus, an analog includes a 0046) Thus, by way of non-limiting examples, coordi proprotein which can be activated by cleavage of the pro nated replication of nuclear and mitochondrial DNA protein portion to produce an active polypeptide. (reviewed in Clayton, D. A., 1992, Int. Rev. Cytol. 141, 0049. The polypeptide (e.g., a human mitochondrial pro 217-232; and Shadel and Clayton, 1997, Annu. Rev. Bio tein or polypeptide having an amino acid Sequence Set forth chem. 66,409-435), or mitochondrial DNA transcription and in SEQ ID NOS:1-3025) of the present invention may be a RNA processing (Shadel and Clayton, 1996, Methods Enzy naturally occurring, a recombinant polypeptide or a Syn mol. 264,149-158; Micol et al., 1996, Methods Enzymol. thetic polypeptide, and is preferably an isolated, naturally 264,158-173) both incompletely understood processes occurring polypeptide. Modified polypeptides according to involving a large number of mitochondrial and extramito the present invention comprise at least one modification chondrial proteins, may be altered mitochondrial functions (e.g., a structural change that occurs with Statistical signifi in certain diseases associated with altered mitochondrial cance in a disease associated with altered mitochondrial function as provided herein. According to these examples, function) to a protein or polypeptide having an amino acid the disclosure herein-that polypeptides Such as those listed sequence as set forth in any one of SEQ ID NOS:1-3025. in Table 2 alongside the functional classifications Such as The protein or polypeptide may therefore be an unmodified “carrier”, “DNA synthesis”, “nucleotide metabolism”, “tran polypeptide or may be a polypeptide that has been post Scription' and "transport', are mitochondrial components translationally modified, for example by glycosylation (e.g., provides targets for therapeutic intervention in Such dis N-linked glycosylation via asparagines residues, or O-linked eases. In like manner, the disclosure herein that other glycoslyation via Serine or threonine residues or post-bio polypeptides having amino acid Sequences as Set forth in Synthetic glycation, etc.), phosphorylation, oxidation or oxi SEQ ID NOS:1-3025 are mitochondrial components also dative modification, nitration, nitrosylation, amidation, fatty identifies these proteins as targets for therapeutic interven acylation including glycosylphosphatidylinositol anchor tion in a disease associated with altered mitochondrial modification or the like, phospholipase cleavage Such as function. Moreover, functional classifications of these pro phosphatidylinositol-specific phospholipase c mediated teins as recited in Tables 1 and 2 and in the GenBank hydrolysis or the like, protease cleavage, dephosphorylation annotations cited therein (which are incorporated by-refer or any other type of protein posttranslational modification ence) provides further guidance to those familiar with the art Such as a modification involving formation or cleavage of a regarding how readily and without undue experimentation to covalent chemical bond, although the invention need not be Select a biological activity for interrogation, to determine So limited and also contemplates non-covalent associations whether Such activity is altered in a Sample according to art of proteins with other biomolecules (e.g., lipoproteins, met accepted methodologies. alloproteins, etc.). Methods for determining the presence of 0047 According to certain embodiments of the inven Such modifications are well known in the art (e.g., Scopes, tion, a mitochondrial polypeptide is isolated from a biologi R. K., Protein Purification: Principles and Practice, 1987, cal Sample following exposure of the Sample to a “biological Springer-Verlag, NY; Angeletti, Ed., Techniques in Protein Stimulus', which may include any naturally occurring or Chemistry III, Academic Press, Inc., New York, 1993; artificial (including recombinant) compound that is capable Baynes et al., 1991 Diabetes 40:405; Baynes et al., 1999 of inducing altered biological activity of a mitochondrial Diabetes 48:1; Yamakura et al., 1998 J. Biol. Chem. molecular component which is, in preferred embodiments, a 273:14085; MacMillan et al., 1998 Biochem. 37:1613; see mitochondrial polypeptide. Thus, a biological Stimulus may also PCT/US01/14066). be employed, according to certain of the Subject invention 0050 Afragment, derivative or analog of a mitochondrial methods, to effect a perturbation of the biological Status of molecular component polypeptide or protein may be (i) one a cell in a manner that alters biological activity of a in which one or more of the amino acid residues are mitochondrial polypeptide, Such that the altered activity can Substituted with a conserved or non-conserved amino acid be detected using any methodology described or referred to residue (preferably a conserved amino acid residue) and herein or known to the art, for example, according to the Such Substituted amino acid residue may or may not be one mass spectrometric fingerprinting methods described herein encoded by the genetic code, or (ii) one in which one or and in the cited references. Non-limiting examples of bio more of the amino acid residues includes a Substituent logical Stimuli include antibodies, hormones, cytokines, group, which may include a posttranslational modification chemokines, biologically active polypeptides and peptides or an adduct (e.g., an oxidative adduct), or (iii) one in which and other Soluble mediators, apoptogens, Signal transduction one or more of the amino acid residues are deleted, or (iv) agents, Small molecules, cations and ionophores, physical one in which additional amino acids are fused to the and chemical StreSSors, and the like. polypeptide, including a signal Sequence, a leader Sequence US 2004/0101874 A1 May 27, 2004 or a proprotein Sequence or the like, and also including protein assemblies, factors or complexes involved in DNA additional peptide or non-peptide moieties that may be (including mtDNA) replication or transcription or in trans added to proteins Such as ubiquitin, glutathione, thioredoxin lation of mRNA, cellular receptorS, G-proteins or G-protein and the like. Such fragments, derivatives and analogs are coupled receptors, kinases, phosphatases, ion channels, deemed to be within the scope of those skilled in the art from lipases, phosholipases, nuclear receptors and factors, intra the teachings herein. cellular structures, components of Signal transduction and apoptotic pathways, and the like. 0051. The polypeptides of the present invention include mitochondrial polypeptides and proteins having amino acid 0055 Methods for identifying a mitochondrial target Sequences that are identical or Similar to Sequences known (e.g., a pharmaceutical target Such as a target for therapeutic in the art. As known in the art “similarity” between two intervention in a disease associated with altered mitochon polypeptides is determined by comparing the amino acid drial function as provided herein, for instance, diabetes Sequence and conserved amino acid Substitutes thereto of the mellitus, a neurodegenerative disease, a disease associated polypeptide to the Sequence of a Second polypeptide. Frag with inappropriate cell proliferation or cell Survival, or a ments or portions of the polypeptides of the present inven cardiovascular condition) include providing a compound tion may be employed for producing the corresponding that modulates expression level, Structure and/or activity of full-length polypeptide by peptide Synthesis, therefore, the a particular mitochondrial protein (e.g., a component of the fragments may be employed as intermediates for producing human mitochondrial proteome Such as any one or more of the full-length polypeptides. the proteins having amino acid Sequences Set forth in SEQ ID NOS:1-3025) and identifying the cellular component(s) 0.052 AS described herein, isolation of a mitochondrial that binds to the compound to form a molecular complex, polypeptide component Such as a mitochondrial molecular preferably through a specific interaction. component with which an agent identified according to the methods of the invention interacts refers to physical Sepa 0056 “Altered mitochondrial function” may refer to any ration of Such a complex from its biological Source, and may condition or State, including those that accompany a disease be accomplished by any of a number of well known tech asSociated with altered mitochondrial function, where any niques including but not limited to those described herein, Structure or activity that is directly or indirectly related to a and in the cited references. Without wishing to be bound by mitochondrial function has been changed in a Statistically theory, a compound that “binds a mitochondrial component' Significant manner relative to a control or Standard. Altered can be any discrete molecule, agent compound, composition mitochondrial function may have its origin in extramito of matter or the like that may, but need not, directly bind to chondrial Structures or events as well as in mitochondrial a mitochondrial molecular component, and may in the Structures or events, in direct interactions between mito alternative bind indirectly to a mitochondrial molecular chondrial and extramitochondrial genes and/or their gene component by interacting with one or more additional products, or in Structural or functional changes that occur as components that bind to a mitochondrial molecular compo the result of interactions between intermediates that may be nent. These or other mechanisms by which a compound may formed as the result of Such interactions, including metabo bind to and/or associate with a mitochondrial molecular lites, catabolites, Substrates, precursors, cofactors and the component are within the Scope of the claimed methods. like. Binding to a mitochondrial component may under certain conditions result in altered biological activity of the mito 0057 Additionally, altered mitochondrial function may chondrial component. include altered respiratory, metabolic or other biochemical or biophysical activity in one or more cells of a biological 0.053 According to certain preferred embodiments of the Sample or a biological Source. AS non-limiting examples, present invention, proteins and polypeptides comprising one markedly impaired ETC activity may be related to altered or more of the amino acid sequences set forth in SEQ ID mitochondrial function, as may be generation of increased NOS:1-3025, which include polypeptides not previously reactive oxygen species (ROS) or defective oxidative phos known to be mitochondrial components, may be targets for phorylation. AS further examples, altered mitochondrial drug Screening and/or for therapeutic intervention. A “tar membrane potential, induction of apoptotic pathways and get refers to a biochemical entity involved in a biological formation of a typical chemical and biochemical crosslinked process, typically a protein that plays a useful role in the Species within a cell, whether by enzymatic or non-enzy physiology or biology of a Subject or biological Source. A matic mechanisms, may all be regarded as indicative of therapeutic composition or compound may bind to, alter the altered mitochondrial function. These and other non-limiting conformation of, impair or enhance the activity of or oth examples of altered mitochondrial function are contem erwise influence a target to alter (e.g., increase or decrease plated by the present invention. in a Statistically significant manner relative to an appropriate untreated control) its function. AS used herein, targets can 0058 For instance, altered mitochondrial function may include, but need not be limited to, proteins having a be related, interalia, to altered intracellular calcium regula mitochondrial function classification as Summarized in tion that may accompany loss of mitochondrial membrane Table 2 and as described in greater detail below. electrochemical potential by intracellular calcium flux, by mechanisms that include free radical oxidation, defects in 0.054 For example, targets may include proteins that are transmitochondrial membrane Shuttles and transporterS Such components of, or that associate with, mitochondrial ETC as the adenine nucleotide transporter or the malate-aspartate complexes, Krebs cycle or TCA cycle components including shuttle, by defects in ATP biosynthesis, by impaired asso any molecules functionally linked (e.g., as Substrates, cofac ciation of hexokinases and/or other enzymes with porin at tors, intermediates, biochemical donor or acceptor Species, the inner mitochondrial membrane, or by other events. or the like) to Such components, transport protein or carrier Altered intracellular calcium regulation and/or collapse of US 2004/0101874 A1 May 27, 2004

mitochondrial inner membrane potential may result from Such an enzyme may be a mitochondrial enzyme or an ATP direct or indirect effects of mitochondrial genes, gene prod biosynthesis factor that is an enzyme, for example an ETC ucts or related downstream mediator molecules and/or extra enzyme or a Krebs cycle enzyme. mitochondrial genes, gene products or related downstream 0064. Reference herein to “enzyme quantity”, “enzyme mediators, or from other known or unknown causes. catalytic activity” or “enzyme expression level” is meant to 0059 Thus, an “indicator of altered mitochondrial func include a reference to any of a mitochondrial enzyme tion' may be any detectable parameter that directly relates to quantity, activity or expression level or an ATP biosynthesis a condition, process, pathway, dynamic Structure, State or factor quantity, activity or expression level; either of which other activity involving mitochondria and that permits detec may further include, for example, an ETC enzyme quantity, tion of altered mitochondrial function in a biological Sample activity or expression level or a Krebs cycle enzyme quan from a Subject or biological Source. According to non tity, activity or expression level. In the most preferred limiting theory, altered mitochondrial function therefore embodiments of the invention, an enzyme is a natural or may also include altered mitochondrial permeability to recombinant protein or polypeptide that has enzyme cata calcium or to mitochondrial molecular components involved lytic activity as provided herein. Such an enzyme may be, by in apoptosis (e.g., cytochrome c), or other alterations in way of non-limiting examples, an enzyme, a holoenzyme, an mitochondrial respiration, or any other altered biological enzyme complex, an enzyme Subunit, an enzyme fragment, activity as provided herein that is a mitochondrially associ derivative or analog or the like, including a truncated, ated activity. processed or cleaved enzyme. 0060. In certain preferred embodiments of the invention, 0065 A“mitochondrial enzyme” that may be an indicator an enzyme is the indicator of altered mitochondrial function of altered mitochondrial function as provided herein refers as provided herein. The enzyme may be a mitochondrial to a mitochondrial molecular component that has enzyme enzyme, which may further be an ETC enzyme or a Krebs catalytic activity and/or functions as an enzyme cofactor cycle enzyme. The enzyme may also be an ATPbiosynthesis capable of influencing enzyme catalytic activity. AS used factor, which may include an ETC enzyme and/or a Krebs herein, mitochondria are comprised of “mitochondrial cycle enzyme, or other enzymes or cellular components molecular components”, which may be a protein, polypep related to ATP production as provided herein. A “non tide, peptide, amino acid, or derivative thereof; a lipid, fatty enzyme” refers to an indicator of altered mitochondrial acid or the like, or derivative thereof; a carbohydrate, function that is not an enzyme (i.e., that is not a mitochon Saccharide or the like or derivative thereof, a nucleic acid, drial enzyme or an ATP biosynthesis factor as provided nucleotide, nucleoside, purine, pyrimidine or related mol herein). In certain other preferred embodiments, an enzyme ecule, or derivative thereof, or the like, or any covalently or is a co-indicator of altered mitochondrial function. The non-covalently complexed combination of these compo following enzymes may not be indicators of altered mito nents, or any other biological molecule that is a Stable or chondrial function according to the present invention, but transient constituent of a mitochondrion. may be co-indicators of altered mitochondrial function as provided herein: citrate synthase (EC 4.1.3.7), hexokinase II 0066. A mitochondrial enzyme that may be an indicator (EC 2.7.1.1, See, e.g., Kruszynska et al. 1.998), cytochrome of altered mitochondrial function or a co-indicator of altered c oxidase (EC 1.9.3.1), phosphofructokinase (EC 2.7.1.11), mitochondrial function as provided herein, or an ATP bio glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12), Synthesis factor that may be an indicator of altered mito glycogen phosphorylase (EC 2.4.1.1) creatine kinase (EC chondrial function as provided herein, may comprise an 2.7.3.2), NADH dehydrogenase (EC 1.6.5.3), glycerol ETC enzyme, which refers to any mitochondrial molecular 3-phosphate dehydrogenase (EC 1.1.1.8), triose phosphate component that is a mitochondrial enzyme component of the dehydrogenase (EC 1.2.1.12) and malate dehydrogenase mitochondrial electron transport chain (ETC) complex asso (EC 1.1.1.37). ciated with the inner mitochondrial membrane and mito chondrial matrix. An ETC enzyme may include any of the 0061. In other highly preferred embodiments, the indica multiple ETC subunit polypeptides encoded by mitochon tor of altered mitochondrial function is any ATPbiosynthesis drial and nuclear genes. The ETC is typically described as factor as described below. In other preferred embodiments, comprising complex I (NADH:ubiquinone reductase), com the indicator is ATP production. In other preferred embodi plex II (Succinate dehydrogenase), complex III (ubiquinone: ments, the indicator of altered mitochondrial function may be mitochondrial mass or mitochondrial number. According cytochrome c oxidoreductase), complex IV (cytochrome c to the present invention, mitochondrial DNA content may oxidase) and complex V (mitochondrial ATP synthetase), not be an indicator of altered mitochondrial function but where each complex includes multiple polypeptides and may be a co-predictor of altered mitochondrial function or a cofactors (for review see, e.g., Walker et al., 1995 Meths. co-indicator of altered mitochondrial function, as provided Enzymol. 260:14; Ernster et al., 1981 J. Cell Biol. 91:227s herein. In other preferred embodiments the indicator of 255s, and references cited therein). altered mitochondrial function may be free radical produc 0067. A mitochondrial enzyme that may be an indicator tion, a cellular response to elevated intracellular calcium or of altered mitochondrial function as provided herein, or an a cellular response to an apoptogen. ATP biosynthesis factor that may be an indicator of altered mitochondrial function as provided herein, may also com 0062) INDICATORS OF ALTERED MITOCHON prise a Krebs cycle enzyme, which includes mitochondrial DRIAL FUNCTION THAT ARE ENZYMES molecular components that mediate the Series of biochemi 0.063 AS provided herein, in certain preferred embodi cal/bioenergetic reactions also known as the ments, an altered biological activity comprises an indicator or the tricarboxylic acid cycle (see, e.g., Lehninger, Bio of altered mitochondrial function that may be an enzyme; chemistry, 1975 Worth Publishers, NY; Voet and Voet, US 2004/0101874 A1 May 27, 2004

Biochemistry, 1990 John Wiley & Sons, NY; Mathews and ATP production may be accomplished using any known van Holde, Biochemistry, 1990 Benjamin Cummings, method for quantitative ATP detection, for example by way Menlo Park, Calif.). Krebs cycle enzymes include subunits of illustration and not limitation, by differential extraction and cofactors of citrate Synthase, aconitase, isocitrate dehy from a Sample optionally including chromatographic isola drogenase, the C-ketoglutarate dehydrogenase complex, tion; by spectrophotometry; by quantification of labeled ATP Succinyl CoA synthetase, Succinate dehydrogenase, fuma recovered from a Sample contacted with a Suitable form of rase and malate dehydrogenase. Krebs cycle enzymes fur a detectably labeled ATP precursor molecule such as, for ther include enzymes and cofactors that are functionally example, P; by quantification of an enzyme activity asso linked to the reactions of the Krebs cycle, Such as, for ciated with ATP synthesis or degradation; or by other example, nicotinamide adenine dinucleotide, coenzyme A, techniques that are known in the art. Accordingly, in certain thiamine pyrophosphate, lipoamide, guanosine diphosphate, embodiments of the present invention, the amount of ATP in flavin adenine dinucloetide, acetyl-coA carboxylase (ACC) a biological Sample or the production of ATP (including the and nucleoside diphosphokinase. rate of ATP production) in a biological Sample may be an 0068 The methods of the present invention also pertain indicator of altered mitochondrial function. In one embodi in part to the correlation of mitochondrial associated disease ment, for instance, ATP may be quantified by measuring with an indicator of altered mitochondrial function that may luminescence of luciferase catalyzed Oxidation of D-lu be an ATP biosynthesis factor, an altered amount of ATP or ciferin, an ATP dependent process. an altered amount of ATP production. 0071. “Enzyme catalytic activity” refers to any function performed by a particular enzyme or category of enzymes 0069. An “ATP biosynthesis factor” refers to any natu rally occurring cellular component that contributes to the that is directed to one or more particular cellular function(s). efficiency of ATP production in mitochondria. Such a cel For example, “ATP biosynthesis factor catalytic activity” lular component may be a protein, polypeptide, peptide, refers to any function performed by an ATP biosynthesis amino acid, or derivative thereof; a lipid, fatty acid or the factor as provided herein that contributes to the production like, or derivative thereof; a carbohydrate, Saccharide or the of ATP. Typically, enzyme catalytic activity is manifested as like or derivative thereof, a nucleic acid, nucleotide, nucleo facilitation of a chemical reaction by a particular enzyme, Side, purine, pyrimidine or related molecule, or derivative for instance an enzyme that is an ATP biosynthesis factor, thereof, or the like. An ATP biosynthesis factor includes at wherein at least one enzyme Substrate or reactant is least the components of the ETC and of the Krebs cycle (see, covalently modified to form a product. For example, enzyme e.g., Lehninger, Biochemistry, 1975 Worth Publishers, NY; catalytic activity may result in a Substrate or reactant being Voet and Voet, Biochemistry, 1990 John Wiley & Sons, NY; modified by formation or cleavage of a covalent chemical Mathews and van Holde, Biochemistry, 1990 Benjamin bond, but the invention need not be so limited. Various Cummings, Menlo Park, Calif.) and any protein, enzyme or methods of measuring enzyme catalytic activity are known other cellular component that participates in ATP synthesis, to those having ordinary skill in the art and depend on the regardless of whether such ATP biosynthesis factor is the particular activity to be determined. product of a nuclear gene or of an extranuclear gene (e.g., a 0072 For many enzymes, including mitochondrial mitochondrial gene). Participation in ATP synthesis may enzymes or enzymes that are ATP biosynthesis factors as include, but need not be limited to, catalysis of any reaction provided herein, quantitative criteria for enzyme catalytic related to ATP synthesis, transmembrane import and/or activity are well established. These criteria include, for export of ATP or of an enzyme cofactor, transcription of a example, activity that may be defined by international units gene encoding a mitochondrial enzyme and/or translation of (IU), by enzyme turnover number, by catalytic rate constant Such a gene transcript. (K), by Michaelis-Menten constant (K), by Specific activity or by any other enzymological method known in the 0070 Compositions and methods for determining art for measuring a level of at least one enzyme catalytic whether a cellular component is an ATP biosynthesis factor activity. Specific activity of a mitochondrial enzyme, Such as are well known in the art, and include methods for deter mining ATP production (including determination of the rate an ATP biosynthesis factor, may be expressed as units of of ATP production in a sample) and methods for quantifying Substrate detectably converted to product per unit time and, ATP itself. The contribution of an ATPbiosynthesis factor to optionally, further per unit sample mass (e.g., per unit ATP production can be determined, for example, using an protein or per unit mitochondrial mass). isolated ATP biosynthesis factor that is added to cells or to 0073. In certain preferred embodiments of the invention, a cell-free system. The ATPbiosynthesis factor may directly enzyme catalytic activity may be expressed as units of or indirectly mediate a step or Steps in a biosynthetic Substrate detectably converted by an enzyme to a product pathway that influences ATP production. For example, an per unit time per unit total protein in a Sample. In certain ATP biosynthesis factor may be an enzyme that catalyzes a particularly preferred embodiments, enzyme catalytic activ particular chemical reaction leading to ATP production. AS ity may be expressed as units of Substrate detectably con another example, an ATP biosynthesis factor may be a verted by an enzyme to product per unit time per unit cofactor that enhances the efficiency of Such an enzyme. AS mitochondrial mass in a Sample. In certain highly preferred another example, an ATP biosynthesis factor may be an embodiments, enzyme catalytic activity may be expressed as exogenous genetic element introduced into a cell or a units of Substrate detectably converted by an enzyme to cell-free system that directly or indirectly affects an ATP product per unit time per unit mitochondrial protein mass in biosynthetic pathway. Those having ordinary skill in the art a Sample. Products of enzyme catalytic activity may be are readily able to compare ATP production by an ATP detected by Suitable methods that will depend on the quan biosynthetic pathway in the presence and absence of a tity and physicochemical properties of the particular prod candidate ATP biosynthesis factor. Routine determination of uct. Thus, detection may be, for example by way of illus US 2004/0101874 A1 May 27, 2004

tration and not limitation, by radiometric, calorimetric, involved in ATP synthesis and/or may alter the conformation Spectrophotometric, fluorimetric, immunometric or mass of the cellular component in a manner that promotes ATP Spectrometric procedures, or by other Suitable means that Synthesis. Further to this example, Such conformational will be readily apparent to a person having ordinary skill in alteration may be part of a signal transduction pathway, an the art. allosteric activation pathway, a transcriptional activation 0.074. In certain embodiments of the invention, detection pathway or the like, where an interaction between cellular of a product of enzyme catalytic activity may be accom components leads to ATP production. plished directly, and in certain other embodiments detection 0076 Thus, according to the present invention, an ATP of a product may be accomplished by introduction of a biosynthesis factor may include, as non-limiting examples, detectable reporter moiety or label into a Substrate or reac an ATP synthase, acetyl-coA carboxylase (ACC) a mito tant Such as a marker enzyme, dye, radionuclide, lumines chondrial matrix protein and a mitochondrial membrane cent group, fluorescent group or biotin, or the like. The protein. Suitable mitochondrial membrane proteins include amount of Such a label that is present as unreacted Substrate Such mitochondrial components as the adenine nucleotide and/or as reaction product, following a reaction to assay transporter (ANT; e.g., Fiore et al., 1998 Biochimie 8.0:137; enzyme catalytic activity, is then determined using a method Klingenberg 1985 Ann. N.Y. Acad. Sci. 456:279), the voltage appropriate for the Specific detectable reporter moiety or dependent anion channel (VDAC, also referred to as porin; label. For radioactive groups, radionuclide decay monitor e.g., Manella, 1997.J. Bioenergetics Biomembr. 29:525), the ing, Scintillation counting, Scintillation proximity assays malate-aspartate Shuttle, the mitochondrial calcium uni (SPA) or autoradiographic methods are generally appropri porter (e.g., Litsky et al., 1997 Biochem. 36:7071), uncou ate. For immunometric measurements, Suitably labeled anti pling proteins (UCP-1, -2, -3; see e.g., Jezek et al., 1998 Int. bodies may be prepared including, for example, those J. Biochem. Cell Biol. 30:1163), a hexokinase, a peripheral labeled with radionuclides, with fluorophores, with affinity benzodiazepine receptor, a mitochondrial intermembrane tags, with biotin or biotin mimetic Sequences or those creatine kinase, cyclophilin D, a Bcl-2 gene family encoded prepared as antibody-enzyme conjugates (see, e.g., Weir, D. polypeptide, the tricarboxylate carrier (e.g., lacobazi et al., M., Handbook of Experimental Immunology, 1986, Black 1996 Biochim. Biophys. Acta 1284.9; Bisaccia et al., 1990 well Scientific, Boston; Scouten, W. H., Methods in Enzy Biochim. Biophys. Acta 1019:250) and the dicarboxylate mology 135:30-65,1987; Harlow and Lane, Antibodies. A carrier (e.g., Fiermonte et al., 1998 J. Biol. Chem. Laboratory Manual, Cold Spring Harbor Laboratory, 1988; 273:24754; Indiveri et al., 1993 Biochim. Biophys. Acta Haugland, 1996 Handbook of Fluorescent Probes and 1143:310; for a general review of mitochondrial membrane Research Chemicals-Sixth Ed., Molecular Probes, Eugene, transporters, See, e.g., Zoratti et al., 1994. J. Bioenergetics Oreg.; Scopes, R. K., Protein Purification. Principles and Biomembr; 26:543 and references cited therein). Practice, 1987, Springer-Verlag, NY; Hermanson, G. T. et al., ImmobilizedAffinity Ligand Techniques, 1992, Academic 0077 “Enzyme quantity” as used herein refers to an Press, Inc., NY; Luo et al., 1998 J. Biotechnol. 65:225 and amount of an enzyme including mitochondrial enzymes or references cited therein). Spectroscopic methods may be enzymes that are ATP biosynthesis factors as provided used to detect dyes (including, for example, calorimetric herein, or of another ATPbiosynthesis factor, that is present, products of enzyme reactions), luminescent groups and i.e., the physical presence of an enzyme or ATPbiosynthesis fluorescent groups. Biotin may be detected using avidin or factor Selected as an indicator of altered mitochondrial Streptavidin, coupled to a different reporter group (com function, irrespective of enzyme catalytic activity. Depend monly a radioactive or fluorescent group or an enzyme). ing on the physicochemical properties of a particular Enzyme reporter groups may generally be detected by the enzyme or ATPbiosynthesis factor, the preferred method for addition of Substrate (generally for a specific period of time), determining the enzyme quantity will vary. In the most followed by Spectroscopic, Spectrophotometric or other highly preferred embodiments of the invention, determina analysis of the reaction products. Standards and Standard tion of enzyme quantity will involve quantitative determi additions may be used to determine the level of enzyme nation of the level of a protein or polypeptide using routine catalytic activity in a Sample, using well known techniques. methods in protein chemistry with which those having skill 0075 AS noted above, enzyme catalytic activity of an in the art will be readily familiar, for example by way of ATP biosynthesis factor may further include other functional illustration and not limitation, those described in greater activities that lead to ATP production, beyond those involv detail below. ing covalent alteration of a Substrate or reactant. For 0078. Accordingly, determination of enzyme quantity example by way of illustration and not limitation, an ATP may be by any suitable method known in the art for biosynthesis factor that is an enzyme may refer to a trans quantifying a particular cellular component that is an membrane transporter molecule that, through its enzyme enzyme or an ATP biosynthesis factor as provided herein, catalytic activity, facilitates the movement of metabolites and that in preferred embodiments is a protein or polypep between cellular compartments. Such metabolites may be tide. Depending on the nature and physicochemical proper ATP or other cellular components involved in ATP synthesis, ties of the enzyme or ATP biosynthesis factor, determination Such as gene products and their downstream intermediates, of enzyme quantity may be by densitometric, mass Spectro including metabolites, catabolites, Substrates, precursors, metric, Spectrophotometric, fluorimetric, immunometric, cofactors and the like. AS another non-limiting example, an chromatographic, electrochemical or any other means of ATP biosynthesis factor that is an enzyme may, through its quantitatively detecting a particular cellular component. enzyme catalytic activity, transiently bind to a cellular Methods for determining enzyme quantity also include component involved in ATP synthesis in a manner that methods described above that are useful for detecting prod promotes ATP synthesis. Such a binding event may, for ucts of enzyme catalytic activity, including those measuring instance, deliver the cellular component to another enzyme enzyme quantity directly and those measuring a detectable US 2004/0101874 A1 May 27, 2004 label or reporter moiety. In certain preferred embodiments of in Neurodegenerative Diseases, Beal, Howell and Bodis the invention, enzyme quantity is determined by immuno Wollner, Eds., Wiley-Liss, New York, pp.29-55; Radi et al., metric measurement of an isolated enzyme or ATP biosyn 1998 in Mitochondria & Free Radicals in Neurodegenera thesis factor. In certain preferred embodiments of the inven tive Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley tion, these and other immunological and immunochemical Liss, New York, pp. 57-89; Gunter and Pfeiffer, 1991, Am. techniques for quantitative determination of biomolecules J. Physiol. 27: C755; Gunter et al., 1994, Am. J. Physiol. such as an enzyme or ATP biosynthesis factor may be 267: 313). For example, fluctuating levels of mitochondrial employed using a variety of assay formats known to those of free Ca" may be responsible for regulating oxidative ordinary skill in the art, including but not limited to enzyme metabolism in response to increased ATP utilization, Via linked immunosorbent assay (ELISA), radioimmunoassay allosteric regulation of enzymes (reviewed by Crompton et (RIA), immunofluorimetry, immunoprecipitation, equilib al., 1993 Basic Res. Cardiol. 88: 513-523;) and the glyc rium dialysis, immunodiffusion and other techniques. (See, erophosphate shuttle (Gunter et al., 1994 J. Bioenerg. e.g., Harlow and Lane, Antibodies. A Laboratory Manual, Biomembr. 26: 471). Cold Spring Harbor Laboratory, 1988; Weir, D. M., Hand book of Experimental Immunology, 1986, Blackwell Scien 0082 Normal mitochondrial function includes regulation tific, Boston.) For example, the assay may be performed in of cytosolic free calcium levels by Sequestration of exceSS a Western blot format, wherein a preparation comprising Ca" within the mitochondrial matrix. Depending on cell proteins from a biological Sample is Submitted to gel elec type, cytosolic Ca" concentration is typically 50-100 nM. trophoresis, transferred to a Suitable membrane and allowed In normally functioning cells, when Ca" levels reach 200 to react with an antibody specific for an enzyme or an ATP 300 nM, mitochondria begin to accumulate Ca" as a biosynthesis factor that is a protein or polypeptide. The function of the equilibrium between influx via a Cat" presence of the antibody on the membrane may then be uniporter in the inner mitochondrial membrane and Ca" detected using a Suitable detection reagent, as is well known efflux via both Na' dependent and Na' independent calcium in the art and described above. carriers. In certain instances, Such perturbation of intracel lular calcium homeostasis is a feature of diseases associated 0079 INDICATORS OF ALTERED MITOCHON with altered mitochondrial function, regardless of whether DRIAL FUNCTION THAT ARE CELLULAR the calcium regulatory dysfunction is causative of, or a RESPONSES TO ELEVATED INTRACELLULAR CAL consequence of, altered mitochondrial function. CIUM 0083 Elevated mitochondrial calcium levels thus may 0080 According to certain embodiments of the present accumulate in response to an initial elevation in cytosolic invention, a method is provided that comprises in pertinent free calcium, as described above. Such elevated mitochon part determining a biological activity of a mitochondrial drial calcium concentrations in combination with reduced polypeptide by monitoring intracellular calcium homeosta ATP or other conditions associated with mitochondrial sis and/or cellular responses to perturbations of this homeo pathology, can lead to collapse of mitochondrial inner mem Stasis, including physiological and pathophysiological cal brane potential (see Gunter et al., 1998 Biochim. Biophys. cium regulation. In particular, according to these Acta 1366:5; Rottenberg and Marbach, 1990, Biochim. Bio embodiments, the method of the present invention is phys. Acta 1016:87). Generally, in order to practice the directed to comparing a cellular response to elevated intra Subject invention methods, the extramitochondrial (cytoso cellular calcium in a biological Sample in the presence and lic) level of Ca" in a biological Sample is greater than that absence of a candidate agent, or to comparing Such a present within mitochondria. For example, in the case of response in a Sample from a Subject known or Suspected of type 2 diabetes mellitus (type 2 DM), mitochondrial or having a disease associated with altered mitochondrial func cytosolic calcium levels may vary from the above ranges tion with that of a control subject. The range of cellular and may range from, e.g., about 1 nM to about 500 mM, responses to elevated intracellular calcium is broad, as is the more typically from about 10 nM to about 100 uM and range of methods and reagents for the detection of Such usually from about 20 nM to about 1 uM, where “about” responses. Many specific cellular responses are known to indicates t10%. A variety of calcium indicators are known those having ordinary skill in the art; these responses will in the art, including but not limited to, for example, fura-2 depend on the particular cell types present in a Selected (McCormacket al., 1989 Biochim. Biophys. Acta 973:420); biological Sample. It is within the contemplation of the magfura-2; BTC (U.S. Pat. No. 5,501,980); fluo-3, fluo-4 present invention to provide a method comprising compar and fluo-5N (U.S. Pat. No. 5,049,673); rhod-2; benzothiaza ing a cellular response to elevated intracellular calcium, 1; and benzothiaza-2 (all of which are available from where Such response is an indicator of altered mitochondrial Molecular Probes, Eugene, Oreg.). These or any other function as provided herein. AS non-limiting examples, means for monitoring intracellular calcium are contemplated cellular responses to elevated intracellular calcium include according to the Subject invention method for identifying a Secretion of Specific Secretory products, exocytosis of par risk for type 2 DM. ticular preformed components, increased glycogen metabo 0084. For monitoring an indicator of altered mitochon lism and cell proliferation (see, e.g., Clapham, 1995 Cell drial function that is a cellular response to elevated intrac 80:259; Cooper, The Cell A Molecular Approach, 1997 ellular calcium, compounds that induce increased cytoplas ASM Press, Washington, D.C.; Alberts, B., Bray, D., et al., mic and mitochondrial concentrations of Ca", including Molecular Biology of the Cell, 1995 Garland Publishing, calcium ionophores, are well known to those of ordinary skill in the art, as are methods for measuring intracellular 0081. As a brief background, normal alterations of intra calcium and intramitochondrial calcium (see, e.g., Gunter mitochondrial Cat" are associated with normal metabolic and Gunter, 1994J. Bioenerg. Biomembr. 26: 471; Gunter et regulation (Dykens, 1998 in Mitochondria & Free Radicals al., 1998 Biochim. Biophys. Acta 1366:5; McCormacket al., US 2004/0101874 A1 May 27, 2004

1989 Biochim. Biophys. Acta 973:420; Orrenius and Nico monitoring mitochondrial membrane potential are also dis tera, 1994.J. Neural. Transm. Suppl. 43:1; Leist and Nico closed in U.S. application Ser. No. 09/161,172. tera, 1998 Rev. Physiol. Biochem. Pharmacol. 132:79; and 0087 Mitochondrial membrane potential can also be Haugland, 1996 Handbook of Fluorescent Probes and measured by nonfluorescent means, for example by using Research Chemicals-Sixth Ed., Molecular Probes, Eugene, TTP (tetraphenylphosphonium ion) and a TTP-sensitive Oreg.). Accordingly, a person skilled in the art may readily electrode (Kamo et al., 1979 J. Membrane Biol. 49:105; Select a Suitable ionophore (or another compound that results Porter and Brand, 1995 Am. J. Physiol. 269:R1213). Those in increased cytoplasmic and/or mitochondrial concentra skilled in the art will be able to select appropriate detectable tions of Caf") and an appropriate means for detecting compounds or other appropriate means for measuring Alm. intracellular and/or intramitochondrial calcium for use in the By way of example and not limitation, TMRM is somewhat present invention, according to the instant disclosure and to preferable to TMRE because, following efflux from mito well known methods. chondria, TMRE yields slightly more residual signal in the 0085) Ca" influx into mitochondria appears to be largely endoplasmic reticulicum and than TMRM. dependent, and may be completely dependent, upon the 0088 As another non-limiting example, membrane negative transmembrane electrochemical potential (Au) potential may be additionally or alternatively calculated established at the inner mitochondrial membrane by electron from indirect measurements of mitochondrial permeability transfer, and Such influx fails to occur in the absence of Alp to detectable charged Solutes, using matrix Volume and/or even when an eight-fold Ca" concentration gradient is pyridine nucleotide redox determination combined with imposed (Kapus et al., 1991 FEBS Lett. 282:61). Accord Spectrophotometric or fluorimetric quantification. Measure ingly, mitochondria may release Ca' when the membrane ment of membrane potential dependent Substrate exchange potential is dissipated, as occurs with uncouplers like 2,4- diffusion across the inner mitochondrial membrane may also dinitrophenol and carbonyl cyanide p-trifluoro-methoX provide an indirect measurement of membrane potential. yphenylhydrazone (FCCP). Thus, according to certain (See, e.g., Quinn, 1976, The Molecular Biology of Cell embodiments of the present invention, collapse of Alp may Membranes, University Park Press, Baltimore, Md., pp. be potentiated by influxes of cytosolic free calcium into the mitochondria, as may occur under certain physiological 200-217 and references cited therein.) conditions including those encountered by cells of a Subject 0089 Excquisite sensitivity to extraordinary mitochon having type 2 DM. Detection of such collapse may be drial accumulations of Ca" that result from elevation of accomplished by a variety of means as provided herein. intracellular calcium, as described above, may also charac terize type 2 DM. Such mitochondrial sensitivity may pro 0.086 Typically, mitochondrial membrane potential may vide an indicator of altered mitochondrial function accord be determined according to methods with which those ing to the present invention. Additionally, a variety of skilled in the art will be readily familiar, including but not physiologically pertinent agents, including hydroperoxide limited to detection and/or measurement of detectable com and free radicals, may synergize with Ca" to induce col pounds Such as fluorescent indicators, optical probes and/or lapse of Alp (Novgorodov et al., 1991 Biochem. BiophyS. Sensitive pH and ion-Selective electrodes (See, e.g., Ernster Acta 1058: 242; Takeyama et al., 1993 Biochem. J. 294:719; et al., 1981 J. Cell Biol. 91:227s and references cited; see Guidox et al., 1993 Arch. Biochem. Biophys. 306:139). also Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals-Sixth Ed., Molecular Probes, Eugene, 0090) INDICATORS OF ALTERED MITOCHON Oreg., pp.266-274 and 589-594). For example, by way of DRIAL FUNCTION THAT ARE CELLULAR illustration and not limitation, the fluorescent probes 2-4- RESPONSES TO APOPTOGENIC STIMULI dimethylaminostyryl-N-methyl pyridinium (DASPMI) and 0091 Turning to another aspect, the present invention tetramethylrhodamine esters (such as, e.g., tetramethyl relates to the correlation of diseases associated with altered rhodamine methyl ester, TMRM; tetramethylrhodamine mitochondrial function with an indicator of altered mito ethyl ester, TMRE) or related compounds (see, e.g., chondrial function, involving programmed cell death or Haugland, 1996, Supra) may be quantified following accu apoptosis. In particular, according to this aspect, the present mulation in mitochondria, a process that is dependent on, invention is directed to a method comprising comparing a and proportional to, mitochondrial membrane potential (see, cellular response to an apoptosis-inducing ("apoptogenic') e.g., Murphy et al., 1998 in Mitochondria & Free Radicals Stimulus in a biological Sample from (i) a Subject believed to in Neurodegenerative Diseases, Beal, Howell and Bodis be at risk for disease, and (ii) a control Subject. The range of Wollner, Eds., Wiley-Liss, New York, pp. 159-186 and ref cellular responses to various known apoptogenic Stimuli is erences cited therein; and Molecular Probes On-line Hand broad, as is the range of methods and reagents for the book of Fluorescent Probes and Research Chemicals, at detection of Such responses. It is within the contemplation of http://www.probes.com/handbook/toc.html). Other fluores the present invention to provide a method for identifying a cent detectable compounds that may be used in the invention risk for disease by comparing a cellular response to an include but are not limited to rhodamine 123, rhodamine B apoptogenic Stimulus, where Such response is an indicator of hexyl ester, DiOC(3), JC-1 5.5",6,6'-Tetrachloro-1,1,3,3'- Tetraethylbezimidazolcarbocyanine Iodide (see Cossarizza, altered mitochondrial function as provided herein. et al., 1993 Biochem. Biophys. Res. Comm. 197:40; Reers et 0092. By way of background, mitochondrial dysfunction al., 1995 Meth. Enzymol. 260:406), rhod-2 (see U.S. Pat. No. is thought to be critical in the cascade of events leading to 5,049,673; all of the preceding compounds are available apoptosis in various cell types (Kroemer et al., FASEB J. from Molecular Probes, Eugene, Oreg.) and rhodamine 800 9:1277-87, 1995). Altered mitochondrial physiology may be (Lambda Physik, GmbH, Göttingen, Germany; see among the earliest events in programmed cell death Sakanoue et al., 1997 J. Biochem. 121:29). Methods for (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami US 2004/0101874 A1 May 27, 2004

et al., J. Exp. Med 181:1661-72, 1995) and elevated reactive 0095. In one embodiment of the subject invention method oxygen species (ROS) levels that result from such altered wherein the indicator of altered mitochondrial function is a mitochondrial function may initiate the apoptotic cascade cellular response to an apoptogen, cells in a biological (Ausserer et al., Mol. Cell. Biol. 14:5032-42.1994). In sev Sample that are Suspected of undergoing apoptosis may be eral cell types, reduction in the mitochondrial membrane examined for morphological, permeability or other changes potential (Alpm) precedes the nuclear DNA degradation that that are indicative of an apoptotic State. For example by way accompanies apoptosis. In cell-free Systems, mitochondrial, of illustration and not limitation, apoptosis in many cell but not nuclear, enriched fractions are capable of inducing types may cause altered morphological appearance Such as nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). plasma membrane blebbing, cell shape change, loSS of Perturbation of mitochondrial respiratory activity leading to Substrate adhesion properties or other morphological altered cellular metabolic States, Such as elevated intracel changes that can be readily detected by a perSon having lular ROS, may occur in certain diseases associated with ordinary skill in the art, for example by using light microS altered mitochondrial function (e.g., type 2 DM) and may copy. AS another example, cells undergoing apoptosis may further induce pathogenetic events via apoptotic mecha exhibit fragmentation and disintegration of , nisms. which may be apparent by microScopy and/or through the 0093. Oxidatively stressed mitochondria may release a use of DNA-Specific or chromatin-specific dyes that are pre-formed Soluble factor that can induce chromosomal known in the art, including fluorescent dyes. Such cells may condensation, an event preceding apoptosis (Marchetti et al., also exhibit altered plasma membrane permeability proper Cancer Res. 56:2033-38, 1996). In addition, members of the ties as may be readily detected through the use of Vital dyes Bcl-2 family of anti-apoptosis gene products are located (e.g., propidium iodide, trypan blue) or by the detection of within the Outer mitochondrial membrane (Monaghan et al., lactate dehydrogenase leakage into the extracellular milieu. J. Histochem. Cytochem. 40:1819-25, 1992) and these pro These and other means for detecting apoptotic cells by teins appear to protect membranes from Oxidative StreSS morphologic criteria, altered plasma membrane permeabil (Korsmeyer et al, Biochim. Biophys. Act. 1271:63, 1995). ity and related changes will be apparent to those familiar Localization of Bcl-2 to this membrane appears to be with the art. indispensable for modulation of apoptosis (Nguyen et al., J. 0096. In another embodiment of the subject invention Biol. Chem. 269:16521-24, 1994). Thus, changes in mito method wherein the indicator of altered mitochondrial func chondrial physiology may be important mediators of apop tion is a cellular response to an apoptogen, cells in a tosis. biological Sample may be assayed for translocation of cell 0094. Altered mitochondrial function, may therefore membrane phosphatidylserine (PS) from the inner to the lower the threshold for induction of apoptosis by an apop outer leaflet of the plasma membrane, which may be togen. A variety of apoptogens are known to those familiar detected, for example, by measuring outer leaflet binding by with the art (see, e.g., Green et al., 1998 Science 281:1309 the PS-specific protein annexin. (Martin et al., J. Exp. Med. and references cited therein) and may include by way of 182:1545, 1995; Fadok et al., J. Immunol. 148:2207,1992.) illustration and not limitation: tumor necrosis factor-alpha In still another embodiment of this aspect of the invention, (TNF-C); Fas ligand; glutamate; N-methyl-D-aspartate a cellular response to an apoptogen is determined by an (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et assay for induction of Specific protease activity in any al., 1997.J. Cell. Biol. 138:449-469); paraquat (Costantini et member of a family of apoptosis-activated proteases known al., 1995 Toxicology 99:1-2); ethylene glycols; protein as the caspases (see, e.g., Green et al., 1998 Science kinase inhibitors, Such as, e.g. staurosporine, calphostin C, 281: 1309). Those having ordinary skill in the art will be caffeic acid phenethyl ester, chelerythrine chloride, readily familiar with methods for determining caspase activ genistein; 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; ity, for example by determination of caspase-mediated N-2-((p-bromocinnamyl)amino)ethyl-5-5-isoquinoline cleavage of Specifically recognized protein SubStrates. These Sulfonamide, KN-93, quercitin; d-erythro-Sphingosine Substrates may include, for example, poly-(ADP-ribose) derivatives, UV irradiation, ionophores Such as, e.g.: iono polymerase (PARP) or other naturally occurring or synthetic mycin and Valinomycin; MAP kinase inducerS Such as, e.g.: peptides and proteins cleaved by caspases that are known in aniSomycin, anandamine; cell cycle blockerS Such as, e.g.: the art (see, e.g., Ellerby et al., 1997.J. Neurosci. 17:6165). aphidicolin, colcemid, 5-fluorouracil, homoharringtonine; The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ ID acetylcholinesterase inhibitorS Such as, e.g. berberine; anti NO: ;), wherein “Z” indicates a benzoyl carbonyl estrogens Such as, e.g.: tamoxifen; pro-Oxidants, Such as, moiety and AFC indicates 7-amino-4-trifluoromethylcou e.g.: tert-butyl peroxide, hydrogen peroxide, free radicals marin (Klucket al., 1997 Science 275:1132; Nicholson et al., Such as, e.g., , inorganic metal ions, Such as, e.g., 1995 Nature 376:37), is one such substrate. Other non cadmium; DNA Synthesis inhibitorS Such as, e.g.: actinomy limiting examples of Substrates include nuclear proteins cin D; DNA intercalatorS Such as, e.g., doxorubicin, bleo such as U1-70 kDa and DNA-PKcs (Rosen and Casciola mycin Sulfate, hydroxyurea, methotrexate, mitomycin C, Rosen, 1997.J. Cell. Biochem. 64:50; Cohen, 1997 Biochem. camptothecin, daunorubicin; protein Synthesis inhibitors J. 326:1). Such as, e.g., cycloheximide, puromycin, rapamycin; agents 0097 As described above, the mitochondrial inner mem that affect microtubulin formation or Stability Such as, e.g.: brane may exhibit highly Selective and regulated permeabil Vinblastine, Vincristine, colchicine, 4-hydroxyphenylretina ity for many Small Solutes, but is impermeable to large (>~10 mide, paclitaxel, Bad protein, Bid protein and Bax protein kDa) molecules. (See, e.g., Quinn, 1976 The Molecular (see, e.g., Jurgenmeier et al., 1998 Proc. Nat. Acad. Sci. USA Biology of Cell Membranes, University Park Press, Balti 95:4997-5002 and references cited therein); calcium and more, Md.). In cells undergoing apoptosis, however, col inorganic phosphate (Kroemer et al., 1998 Ann. Rev. lapse of mitochondrial membrane potential may be accom Physiol. 60:619). panied by increased permeability permitting macromolecule US 2004/0101874 A1 May 27, 2004

diffusion across the mitochondrial membrane. Thus, in of art accepted criteria for determining the presence of other another embodiment of the Subject invention method diseases associated with altered mitochondrial function as wherein the indicator of altered mitochondrial function is a provided herein. cellular response to an apoptogen, detection of a mitochon 0101 Hence, the person having ordinary skill in the art drial protein, for example cytochrome c that has escaped can “correlate' one or more parameters described herein from mitochondria in apoptotic cells, may provide evidence (e.g., mitochondrial functions) with Such a disease associ of a response to an apoptogen that can be readily deter ated with altered mitochondrial function, in view of the mined. (Liu et al., Cell 86:147, 1996) Such detection of present disclosure and based on familiarity with the art. cytochrome c may be performed Spectrophotometrically, Briefly, Statistically significant deviation from a normal, immunochemically or by other well established methods for disease-free range for any of a number of clinical Signs and determining the presence of a Specific protein. Symptoms and/or criteria for mitochondrial function, per mits determination of the Statistically significant coinci 0.098 For instance, release of cytochrome c from cells dence of Such parameter(s) with disease. Such deviation can challenged with apoptotic stimuli (e.g., ionomycin, a well further be confirmed, for instance, by comparing the same known calcium ionophore) can be followed by a variety of parameters and criteria that are detected in disease to those immunological methods. Matrix-assisted laser desorption in a Suitable control Sample, in this case a control derived ionization time-of-flight (MALDI-TOF) mass spectrometry from a Subject known to be free of a risk for having, or coupled with affinity capture is particularly Suitable for Such presence of, Such disease. analysis Since apo-cytochrome c and holo-cytochrome c can 0102) Accordingly, given the disclosure of the instant be distinguished on the basis of their unique molecular application, and in particular the identification of the weights. For example, the Surface-Enhanced Laser DeSorp polypeptide sequences set forth in SEQ ID NOS:1-3025 as tion/lonization (SELDITM) system (Ciphergen, Palo Alto, belonging to a defined human mitochondrial proteome, the Calif.) may be utilized to detect cytochrome c release from present invention provides a control Set of polypeptides Such mitochondria in apoptogen treated cells. In this approach, a that a Sample may be analyzed for the presence of at least cytochrome c Specific antibody immobilized on a Solid one modified polypeptide as described herein, in order to So Support is used to capture released cytochrome c present in “correlate” Such modification with a disease associated with a Soluble cell extract. The captured protein is then encased altered mitochondrial function. Establishing Such a correla in a matrix of an energy absorption molecule (EAM) and is tion then provides a target for Screening assays to identify an deSorbed from the Solid Support Surface using pulsed laser agent Suitable for therapeutic intervention, i.e., an agent that excitation. The molecular mass of the protein is determined beneficially counteracts the disease-associated alteration in by its time of flight to the detector of the SELDITM mass mitochondrial function. Without wishing to be bound by Spectrometer. theory, a target for therapeutic intervention preferably con tributes to the pathogenesis of disease by exhibiting unde 0099. A person having ordinary skill in the art will Sirably altered biological activity, Such that a therapeutic readily appreciate that there may be other Suitable tech agent reverses Such alteration to a control range. The inven niques for quantifying apoptosis, and Such techniques for tion need not, however, be So limited, as even in Situations purposes of determining an indicator of altered mitochon where the target identified according to the Subject invention drial function that is a cellular response to an apoptogenic method is a Surrogate marker of disease, Such a target stimulus are within the scope of the methods provided by the nevertheless may be restored to a normal control range by a present invention. therapeutic agent regardless of whether the interaction is direct, in a manner that ameliorates disease. In certain 0100. As noted above, an increasing number of diseases, embodiments the invention further provides for determina disorders and conditions have been identified as diseases tion of altered biological activity in Such a modified asSociated with altered mitochondrial function as provided polypeptide, as also described herein. herein, Such that given the present disclosure and the State of the art with respect to methods for assessing mitochondrial 0103). According to the present invention, there are pro function and with respect to clinical Signs and Symptoms of vided compositions and methods for the identification of Such diseases, the perSon having ordinary skill in the art can differential protein expression at the organellar proteome readily determine criteria for establishing a Statistically level (e.g., the mitochondrial proteome), in a Sub-proteomic, Significant deviation from a normal range for one or more complex mixture of proteins or at the level of a Single parameters that are appropriate to the definition of the targeted protein. The invention thus relates in pertinent part disease, in order to establish that a disease associated with to the unexpected advantages associated with the unique altered mitochondrial function is present. AS an illustrative physicochemical properties of particular organelle-derived example, where it is desirable to determine whether or not (e.g., mitochondria) polypeptides, peptides (e.g., peptide a Subject or biological Source falls within clinical parameters fragments) and proteins, in conjunction with biochemical indicative of type 2 diabetes mellitus, Signs and Symptoms (including immunochemical) methods, modern spectrom of type 2 diabetes that are accepted by those skilled in the art etry and protein bioinformatics Software tools to identify may be used to So designate a Subject or biological Source, peptides and proteins that are detected as differentially for example clinical signs referred to in Gavin et al. (Dia expressed products, and to identify previously unrecognized betes Care 22(suppl. 1):S5-S19, 1999, American Diabetes peptides and proteins as molecular components of a particu ASSociation Expert Committee on the Diagnosis and Clas lar organelle (e.g., mitochondrial molecular components as sification of Diabetes Mellitus) and references cited therein, provided herein). or other means known in the art for diagnosing type 2 0104. The invention also relates in pertinent part to the diabetes. Similarly, those familiar with the art will be aware Surprising advantages offered by the use of an organelle US 2004/0101874 A1 May 27, 2004 enriched sample fraction (e.g., a mitochondria enriched reasons discussed above but which, in any event, will permit Sample as provided herein). Determining the pattern of isolation of Such a polypeptide as provided herein. differential protein expression (e.g., absence or presence of one or more particular proteins in a Sample, Structural 0107 The isolated polypeptide is then contacted with a modification of a particular protein, or other altered expres proteolytic agent to generate a plurality of derivative peptide Sion Such as a Statistically significant increase or decrease in fragments, from which a mass spectrum can be generated to the amount of one or more particular proteins in a Sample permit determination of the presence, amount or Structure when normalized to a control) at the peptide and/or protein (e.g., level) of the polypeptide in the sample, which may level in a complex protein mixture obtained from a biologi then be compared to similarly obtained levels of a mito cal Sample as provided herein (i.e., at the proteomic level) chondrial polypeptide obtained from other Samples. provides, in certain embodiments, targets for drug Screening 0108. In an effort to better understand the molecular assays and for therapeutic intervention in Specific disease details of mitochondrial dysfunction as a contributing factor States. Accordingly, in certain embodiments the invention in disease, a high-resolution map of the human mitochon provides methods for evaluating the effects of candidate drial proteome is disclosed herein using human heart tissue therapeutic agents (e.g., drugs or biological Stimuli as pro as the Source of isolated mitochondria, which are further vided herein) on biological activity of a mitochondrial enriched on metrizamide density gradients, Solubilized and protein, for example, where the protein exhibits altered fractionated using Sucrose density gradients. Although a biological activity due to one or more of a modification Such protein map was previously generated using an only par as a mutation (insertion, deletion and/or Substitution of one tially enriched mitochondrial fraction from human placenta or more amino acids), a posttranslational modification or an (Rabilloud et al., 1998 Electrophor. 19:1006), no reliable altered level of protein expression. Thus, in certain embodi database cataloguing mitochondrial proteins is currently ments, Such candidate agents may cause one or more specific available (cf., e.g., Koc et al., 2000 J. Biol. Chem. alterations (e.g., increases or decreases in a statistically 275:32585; Lopez et al., 2000 Electrophor. 21:3427). Typi Significant manner) in the biological activity of a mitochon cally, mitochondria may be obtained from brain, heart, drial protein, preferably in Some beneficial fashion. skeletal muscle or liver, where they are most abundant, 0105. As also noted elsewhere herein, certain embodi although other Sources (e.g., blood platelets) may also be ments of the invention relate in pertinent part to isolating at used. According to the present invention there is provided a least one mitochondrial polypeptide according to any of a framework for investigating mitochondrial proteins, includ variety of biochemical Separation methodologies for isolat ing identifying previously unrecognized mitochondrial pro ing a polypeptide as known in the art and as provided herein teins (e.g., novel proteins or known proteins not previously (see, e.g., Scopes, 1987 Protein Purification. Principles and known to exist as mitochondrial molecular components) as Practice, Springer-Verlag, NY; Deutscher, 1990 Meths. well as those that are modified as provided herein as a Enzymol. Vol. 182; Nilsson et al., 2000 Mass Spectrom. Rev. correlate of disease, by mapping the human heart mitochon 19:390; Godovac-Zimmermann et al., 2001 Mass Spectrom. drial proteome. AS described in greater detail in the Re: 20:1; Gatlin et al., 2000 Anal. Chem. 72:757; Link et al., Examples, mitochondrial proteins in distinct Sucrose density 1999 Nat. Biotechnol. 17:676). Hence, as provided herein gradient fractions were Separated by one-dimensional poly and as known to the art, Such methodologies for isolating a acrylamide gel electrophoresis, and isolated proteins recov mitochondrial polypeptide may exploit physicochemical and ered from gels were analyzed as described below using hydrodynamic properties of the polypeptide, including, for matrix assisted laser desorption ionization (MALDI) and example, the approximate apparent molecular mass of the MALDI-post source decay (MALDI-PSD) techniques. (For polypeptide, the amino acid Sequence of the polypeptide, other MS methods for proteins, See, e.g., Godovac-Zimmer and in certain contemplated embodiments, the apparent mann et al., 2001 Mass Spectromet. Rev. 20:1-57; Nilsson et approximate isolelectric focusing point of the polypeptide. al., 2000 Mass Spectromet. Rev. 19:390-397.) Over 1400 0106 AS is well known to those having ordinary skill in proteins were identified in the NCBI (http://www.ncbi.nim the art, variability in biological Sample Source and condition, .nih.gov/Entrez/) and GenPept (http://www.ncbi.nlm.nih extraction reagents and methods, Separation media and .gov/Entrez/protein.html) databases. Alternative databases instrumentation, analytical apparatus and the like, may for identifying protein Sequences are known to the art and account for differences in values observed for Such proper include, for example, Swissprot (http://www.expasy.ch/ ties of polypeptides as molecular mass and isoelectric focus Sprot/sprot-top.html), and Owl (http://www.biochem.u- ing point. Hence, it will be understood that an “apparent cl.ac.uk/bsm/dbbrowser/OWL/OWL.html.) The data set so molecular mass or isoelectric focusing point refers to that obtained provides for the identification of proteins present in which is detected in a particular rendition of a particular mitochondria from human heart, a bioenergetically active isolation procedure, although the value detected for Such a tissue. parameter may vary among Separate isolations, Similarly 0109 AS described in greater detail below, the present those familiar with the art will appreciate that from among invention is also directed in pertinent part to the use of mass the variables listed above, including imprecision in instru spectrometry (MS), and in particular to the use of matrix mentation, apparent values may vary in a manner that assisted laser desorption ionization time-of-flight (MALDI renders a particular value that is detected only an “approxi TOF) mass spectrometry, for the analysis of mitochondrial mation' of the actual parameter being measured. Thus, proteins and peptides obtained from a Subject or biological according to certain embodiments of the present invention a Source as provided herein. mitochondrial polypeptide may be isolated on the basis of approximate apparent molecular mass, apparent approxi 0110. In particularly preferred embodiments of the mate isoelectric focusing point and/or amino acid Sequence, present invention, all or a portion of a protein fraction which parameters may be Susceptible to Some variability for derived from a biological Sample as provided herein may be US 2004/0101874 A1 May 27, 2004 contacted with one or more proteolytic agents under condi proteins may include Alzheimer's disease (AD), diabetes tions and for a time Sufficient to generate a plurality of mellitus, Parkinson's disease, amyotrophic lateral Sclerosis peptide fragments derived from the protein fraction. Peptide (ALS), atherosclerosis and other degenerative and inflam fragments are typically continuous portions of a polypeptide matory diseases. Those familiar with the art will be aware of chain derived from a protein of the protein fraction, which clinical criteria for diagnosing certain of these diseases, portions may be up to about 100 amino acids in length, which diagnostic criteria are augmented in View of the preferably up to about 50 amino acids in length, more Subject invention methods and compositions. preferably up to about 30 amino acids in length, and Still 0114 AS described in greater detail in the Examples, more preferably up to about 15-20 amino acids in length. In certain embodiments of the invention contemplate the unex particularly preferred embodiments peptide fragments are pected discovery that a mitochondrial protein or peptide 10-15 amino acids in length, and in other preferred embodi containing tryptophan may be oxidatively modified to yield ments peptide fragments may be 2-12 amino acids long. proteins or peptides containing this modified amino acid, 0111 A variety of proteolytic agents and suitable condi although the invention is not intended to be So limited and tions for using them are known in the art, any of which may as described herein contemplates mitochondrial proteins and be useful according to certain embodiments of the present peptides comprising a wide variety of other amino acids that invention wherein peptide fragments are generated. Particu may be oxidatively modified, according to oxidation reac larly preferred are proteolytic agents that are proteolytic tions Such as those described, for example, in Halliwell and enzymes or proteases, for example trypsin, Glu-C protease Gutteridge (Free Radicals in Biology and Medicine, 1989 (Staphylococcal V8 protease), Lys-C protease, Arg-C pro Clarendon Press, Oxford, UK). As described below, a num tease, or other proteases known in the art to cleave peptides ber of mitochondrial proteins have been identified in which at Specific amino acid linkages, typically at a relatively at least one tryptophan residue was doubly oxidized, thereby limited number of cleavage Sites within a protein or undergoing conversion to N-formylkynurenine. Accord polypeptide. Other useful proteolytic agents that are pro ingly, in certain embodiments the invention contemplates teolytic enzymes include Serine proteases, for example, determination of a modified polypeptide (e.g., SEQ ID chymotrypsin, elastase and trypsin; thiolproteases, Such as NOS:1-3025) comprising an oxidative modification that papain or yeast proteinase B, acid proteases, including, e.g., may, in certain further embodiments comprise an oxidized pepsin or cathepsin D; metalloproteinases (e.g., collagena trytophan residue, which may in certain Still further com ses, microbial neutral proteinases); carboxypeptidases; prise N-formylkynurenine. Identification and determination N-terminal peptidases or any other proteolytic enzymes that of oxidative modification of tryptophan in proteins and those having ordinary skill in the art will recognize may be peptides are well known to those familiar with the art (e.g., employed to generate peptide fragments as provided herein Halliwell and Gutteridge, pages 93-97; 315-320; 413-429). (see, e.g., Bell, J. E. and Bell, E.T., Proteins and Enzymes, 0115 For instance, the oxidation of tryptophan to 1988 Prentice-Hall, Englewood Cliffs, N.J.; Worthington N-formylkynurenine in proteins has been known for over 35 Enzyme Manual, V. Worthington, ed., 1993 Worthington years Since Previero et al. described it in hen's egg-white Biochemical Corp., Freehold, N.J.). lysozyme in anhydrous formic acid (1967 J. Mol. Biol. 0112 Alternatively, in certain embodiments it may be 24:261). Kuroda et al. (1975 J. Biochem. (Tokyo) 78:641) desirable to use proteolytic agents that are chemical agents, Subsequently found inactivation of lysozyme by OZone in for example HCl, CNBr, formic acid, N-bromosuccinimide, aqueous Solution occurred only when one critical tryptophan BNPS-skatole, o-iodosobenzoic acid/p-cresol, Cyssor, 2-ni residue was oxidized, thus providing the first evidence that tro-5-thiocyanobenzoic acid, hydroxylamine, pyridine/ace oxidation of a Specific tryptophan residue can impair enzyme function. These early reports relied on identification tic acid or other chemical cleavage procedures (See, e.g., of the tryptophan oxidation products by characteristic elec Bell and Bell, 1988, and references cited therein). tronic absorption spectra. Finley et al. (1998 Protein Sci. 0113 AS noted above, oxidative damage to proteins, Such 7:2391) exposed C-crystallin from bovine lens tissue to as protein modification that results from reactive free radical Fenton chemistry in Vitro and Separated the component activity in biological Systems, is an underlying feature in the tryptic peptides by HPLC. Tandem MS/MS spectrometry pathogenesis of a number of diseases. Accordingly, a disease was used to identify oxidized amino acid Sites by +16, +32 asSociated with altered mitochondrial function, for example and +4 u increases in the molecular mass of peptide frag a disease associated with altered mitochondrial constitution ment ions containing tryptophan residues. Structures corre or composition (e.g., a disorder or condition characterized sponding to those mass shifts are shown in FIG. 3. More by Statistically significant alterations in the quantity, Struc recently Thiede et al. (2000 Rapid Commun. Mass Spectrom. ture and/or activity of one or more mitochondrial molecular 14:496) described oxidatively modified tryptophan residues components as provided herein) may also include a “disease in peptides from human Jurkat T lymphoblastoid cells. asSociated with oxidative modification of a protein', Such as These workers described oxidatively modified tryptophan in any disease in which at least one protein or peptide is a peptide which, as shown by the Examples provided herein, oxidatively (e.g., covalently) and, in most cases, inappro shares Structure with a similar peptide derived from the priately modified. In highly preferred embodiments, at least mitochondrial voltage dependent anion channel-1 (VDAC1, one protein or peptide in a Subject or biological Source e.g., SEQ ID NO:2559) polypeptide (see Table 3, KLE having a disease associated with oxidative modification of a TAVNLAWTAGNSNTR). Certain embodiments of the protein includes a mitochondrial protein that has undergone present invention therefore contemplate expressly excluding disease-associated oxidative damage. Thus, Such a disease determination of the peptide KLETAVNLAWTAGNSNTR may have a basis in a respiratory or metabolic or other which comprises oxidatively modified tryptophan, certain defect, whether mitochondrial or extramitochondrial in ori other embodiments contemplate expressly excluding an oxi gin. Diseases associated with oxidative modification of datively modified VDAC1 polypeptide, and certain other US 2004/0101874 A1 May 27, 2004 embodiments of the present invention therefore contemplate 0119). In one embodiment of this aspect of the invention, expressly excluding a disease associated with altered mito therapeutic agents or combinations of agents that are tailored chondrial function that is T-cell lymphoma or leukemia. to effectively treat an individual patient's particular disease 0116. In order to determine whether a mitochondrial may be identified by routine Screening of candidate agents component may contribute to a particular disease associated on cybrid cells constructed with the patient's mitochondria. with Oxidative modification of a protein, it may be useful to In another embodiment, a method for identifying Subtypes construct a model System for diagnostic tests and for Screen of the particular disease is provided, for example, based on ing candidate therapeutic agents in which the nuclear genetic differential effects of individual candidate agents on cybrid background may be held constant while the mitochondrial cells constructed using mitochondria from different Subjects genome is modified. It is known in the art to deplete diagnosed with the same disease. mitochondrial DNA from cultured cells to produce m cells, 0120 MALDI thereby preventing expression and replication of mitochon drial genes and inactivating mitochondrial function. It is 0121 AS noted above, in certain preferred embodiments further known in the art to repopulate such m” cells with of the present invention there is provided a method for mitochondria derived from foreign cells in order to assess identifying at least one mitochondrial protein comprising the contribution of the donor mitochondrial genotype to the generating a mass Spectrum of a mitochondrial polypeptide respiratory phenotype of the recipient cells. Such cytoplas derived peptide fragment, wherein the mass spectrum is mic hybrid cells, containing genomic and mitochondrial preferably generated using MALDI-TOF. By way of back DNAS of differing biological origins, are known as cybrids. ground, in 1987, matrix-assisted laser desorption/ionization See, for example, International Publication Number WO mass spectrometry (MALDI) was introduced by Hillenkamp 95/26973 and U.S. Pat. No. 5,888,498 which are hereby and Karas, and Since has become a very powerful bioana incorporated by reference in their entireties, and references lytical tool (Anal. Chem. 60:2288-2301,1988; see also Bur cited therein. lingame et al., Anal. Chem. 68:599-651, 1996 and references cited therein). The success of MALDI in the area of protein 0117. According to the present invention, a level of at Science can be attributed to Several factors. The greatest of least one mitochondrial protein or peptide is determined in these is that MALDI can be rapidly (-5 minutes) applied as a biological Sample from a Subject or biological Source. For an analytical technique to analyze Small quantities of Virtu Subjects that are asymptomatic, that exhibit a pre-disease ally any protein (practical sensitivities of ~1 pmole protein phenotype or that meet clinical criteria for having or being loaded into the mass spectrometer). The technique is also at risk for having a particular disease, Such determination extremely accurate. Beavis and Chait demonstrated that the may have prognostic and/or diagnostic usefulness. For molecular weights of peptides and proteins can be deter example, where other clinical indicators of a given disease mined to within -0.01% by using methods in which internal are known, levels of at least one mitochondrial protein or mass calibrants (X-axis calibration) are introduced into the peptide in Subjects known to be free of a risk or presence of analysis (Anal. Chem, 62:1836-40, 1990). MALDI can also Such disease based on the absence of these indicators may be be made quantitative using a similar method in which determined to establish a control range for such level(s). The internal reference Standards are introduced into the analysis levels may also be determined in biological Samples for ion signal normalization (y-axis calibration). Quantita obtained from Subjects Suspected of having or being at risk tive determination of proteins and peptides is possible using for having the disease, and compared to the control range this approach with accuracies on the order of ~10% (Nelson determined in disease free Subjects. Those having familiarity et al., Anal. Chem. 66:1408-15, 1994). Finally, MALDI is with the art will appreciate that there may be any number of extremely tolerant of large molar excesses of buffer Salts variations on the particular Subjects, biological Sources and and, more importantly, the presence of other proteins. bases for comparing levels of at least one mitochondrial protein or peptide that are useful. beyond those that are 0.122 With the high tolerance towards buffer salts and expressly presented herein, and these additional uses are other biomolecular components comes the ability to directly within the Scope and Spirit of the invention. analyze complex biological mixtures. Many examples exist where MALDI is used to directly analyze the results of 0118 For instance, determination of levels of at least one proteolytic or chemical digestion of polypeptides (see Bur mitochondrial protein or peptide may take the form of a lingame et al., Supra). Other examples extend to elucidating prognostic or a diagnostic assay performed on a skeletal post-translational modifications (namely carbohydrate type muscle biopsy, on whole blood collected from a subject by and content), a process requiring the Simultaneous analysis routine venous blood draw, on buffy coat cells prepared from of components present in a heterogeneous glycoprotein blood or on biological Samples that are other cells, organs or mixture (Sutton et al., Techniques in Protein Chemistry III, tissue from a Subject. Alternatively, in certain situations it Angeletti, Ed., Academic Press, Inc., New York, pp. 109 may be desirable to construct cybrid cell lines using mito 116,1993). Arguably, the most impressive use of direct chondria from either control Subjects or Subjects Suspected mixture analysis is the Screening of natural biological fluids. of being at risk for a particular disease associated with In that application, proteins are identified, as prepared oxidative modification of proteins. Such cybrids may be directly from the host fluid, by detection at precise and used to determine levels of at least one mitochondrial characteristic mass-to-charge (m/z) values (Tempst et al., peptide or protein for diagnostic or predictive purposes, or Mass Spectrometry in the Biological Sciences, Burlingame as biological Sources for Screening assays to identify agents that may be Suitable for treating the disease based on their and Carr, Ed., Humana Press, Totowa, N.J., p. 105, 1996). ability to alter (e.g., to increase or decrease in a statistically 0123 The use of an affinity ligand-derivatized support to Significant manner) the levels of at least one mitochondrial selectively retrieve a target analyte specifically for MALDI protein or peptide in treated cells. analysis was first demonstrated by Hutchens and Yip (Rapid US 2004/0101874 A1 May 27, 2004

Commun. Mass Spectrom. 7:576-80, 1993). Those investi massive cluster impact (MCI-like SIMS using large cluster gators used single-Stranded DNA-derivatized agarose beads primary ions), laser desorption/ionization (LDI-laser light to Selectively retrieve a protein, lactoferrin, from pre-term is used to desorb/ionize species from a Surface), and matrix infant urine by incubating the beads with urine. The agarose assisted laser desorption/ionization (MALDI-like LDI beads were then treated as the MALDI analyte-a process except the Species are desorbed/ionized from a matrix involving mixing with a solution-phase MALDI matrix capable of assisting in the desorption and ionization events). followed by deposition of the mixture on a mass spectrom Any of the aforementioned desorption/ionization techniques eter probe. MALDI then proceeded in the usual manner. may be employed in the practice of the present invention. In Results indicated that the derivatized beads selectively a preferred embodiment, LDI is employed, and in a more retrieved and concentrated the lactoferrin; enough So to preferred embodiment, MALDI is utilized. For matrix enable ion signal in the MALDI mass spectrum adequate to assisted laser desorption ionization/time of flight (MALDI unambiguously identify the analyte at the appropriate m/z. TOF) analysis or other MS (mass spectrometry) techniques value (81,000 Da). A number of variations on this approach known to those skilled in the art, see, for example, U.S. Pat. have since been reported. These include the use of immu Nos. 5,622,824, 5,605,798 and 5,547,835. Alternatively, noaffinity precipitation for the MALDI analysis of transfer other Soft-ionization mechanisms that are not based on rins in serum (Nakanishi et al., Biol. Mass Spectrom. 23:230 particle bombardment but that are also capable of ionizing 33, 1994), screening of ascites for the production of peptides and/or proteins could be employed. Such methods monoclonal antibodies (Papac et al., Anal. Chem. 66:2609 include electrospray ionization (ESI, liquid flow containing 13, 1994), and the identification of linear epitope regions analyte sprayed from a nozzle or needle at high voltage) or within an antigen (Zhao et al., Anal. Chem. 66:3723-26, atmospheric pressure ionization (API). 1994). Even more recently, the affinity capture approaches have been made rigorously quantitative by incorporating 0127 Screening Assays and Agents mass-shifted variants of the analyte into the analysis (Nelson 0128. In certain embodiments, the present invention pro et al. Anal. Chem, 67: 1153-58, 1995). The variants are vides a method of identifying an agent for treating a disease retained throughout the analysis (in the same manner as the asSociated with altered mitochondrial function, comprising true analyte) and observed as unique (resolved) signals in (a) contacting a candidate agent with a biological sample the MALDI mass spectrum. Quantification of the analyte is from a Subject having a disease associated with altered performed by equating the relative ion signals of the analyte mitochondrial function, wherein the Sample comprises at and variant to an analyte concentration. least one polypeptide that exhibits altered biological activity 0124 Suitable mass spectrometers include, but are not which accompanies the disease and wherein the polypeptide limited to, a magnetic Sector mass spectrometer, a Fourier is (i) a polypeptide having an amino acid sequence as set transform ion cyclotron resonance (FTICR) mass spectrom forth in any one of SEQ ID NOS 1-3025, or (ii) a modified eter, a quadrupole (rods or ion trap) mass spectrometer and polypeptide that comprises at least one modification to a a time-of-flight (TOF) mass spectrometer, and/or various polypeptide having an amino acid Sequence as Set forth in hybrid instruments comprising combinations of any two or any one of SEQ ID NOS 1-3025; and (b) determining an more of Such types of mass analyzer (e.g., quadrupole/ increase or decrease in the altered biological activity of the orthogonal TOF, Qq/TOF, TOF/TOF, etc.). In a preferred polypeptide in the presence of the candidate agent relative to embodiment, the mass spectrometer is a time TOF mass the level-of the altered biological activity in the absence of Spectrometer. the candidate agent, and therefrom identifying an agent for treating a disease associated with altered mitochondrial 0.125 Since large molecules, such as peptides and pro function. teins, are generally too large to be desorbed/ionized intact, a matrix is used to assist laser desorption/ionization of the 0.129 Candidate agents for use in these and related meth Same. This technique is referred to as matrix assisted laser ods of Screening for a modulator of mitochondrial protein or desorption/ionization or (MALDI), and the matrix agent is peptide according to the present invention may be provided referred to as a “MALDI matrix.” In short, the analyte is as “libraries' or collections of compounds, compositions or contacted with a Suitable MALDI matrix and allowed to molecules. Such molecules typically include compounds crystallize. Suitable MALDI matrix materials are known to known in the art as “Small molecules” and having molecular those skilled in this field, and include, for example, deriva weights less than 10 daltons, preferably less than 10" tives of cinnamic acid Such as C-cyano-4-hydroxycinnamic daltons and still more preferably less than 10 daltons. For acid (ACCA) and Sinapinic acid (SA). example, members of a library of test compounds can be administered to a plurality of Samples, and then assayed for 0126. A first criterion to performing mass spectrometry their ability to increase or decrease the level of at least one on the analyte captured by the interactive Surface is the indicator of altered mitochondrial function. generation of vapor-phase ions. In the practice of this invention, Such species are generated by desorption/ioniza 0.130 Candidate agents further may be provided as mem tion techniques. Suitable techniques include desorption/ bers of a combinatorial library, which preferably includes ionization methods derived from impact of particles with the Synthetic agents prepared according to a plurality of prede sample. These methods include fast atom bombardment termined chemical reactions performed in a plurality of (FAB-impact of neutrals with a sample Suspended in a reaction vessels. For example, Various Starting compounds volatile matrix), secondary ion mass spectrometry (SIMS may be prepared employing one or more of Solid-phase impact of keV primary ions generating Secondary ions from Synthesis, recorded random mix methodologies and a surface), liquid SIMS (LSIMS-like FAB except the recorded reaction Split techniques that permit a given con primary species is an ion), plasma desorption mass spec Stituent to traceably undergo a plurality of permutations trometry (like SIMS except using MeV primary ions), and/or combinations of reaction conditions. The resulting US 2004/0101874 A1 May 27, 2004 20 products comprise a library that can be Screened followed by that correlation between levels of at least one mitochondrial iterative Selection and Synthesis procedures, Such as a Syn protein or peptide and any particular clinical Score used to thetic combinatorial library of peptides (see e.g., PCT/US91/ evaluate a particular disease may be monitored. For 08694, PCT/US91/04666, which are hereby incorporated by example, Stratification of an AD patient population accord reference in their entireties) or other compositions that may ing to levels of at least one mitochondrial protein or peptide include Small molecules as provided herein (see e.g., PCT/ may provide a useful marker with which to correlate the US94/08542, EP 0774464, U.S. Pat. No. 5,798,035, U.S. efficacy of any candidate therapeutic agent being used in AD Pat. No. 5,789,172, U.S. Pat. No. 5,751,629, which are Subjects. hereby incorporated by reference in their entireties). Those 0133. In certain other embodiments, the invention pro having ordinary skill in the art will appreciate that a diverse vides a method of treating a patient having a disease asSortment of Such libraries may be prepared according to asSociated with altered mitochondrial function by adminis established procedures, and tested for their influence on an tering to the patient an agent that that compensates for at indicator of altered mitochondrial function, according to the least one biological activity of a polypeptide that exhibits present disclosure. altered biological activity which accompanies the disease, 0131 The present invention provides compositions and wherein the polypeptide is (i) a polypeptide having an amino methods that are useful in pharmacogenomics, for the clas acid sequence as set forth in any one of SEQ ID NOS sification and/or Stratification of a Subject or patient popu 1-3025, or (ii) a modified polypeptide that comprises at least lation. In one embodiment, for example, Such Stratification one modification to a polypeptide having an amino acid may be achieved by identification in a Subject or patient sequence as set forth in any one of SEQ ID NOS 1-3025. As population of one or more distinct profiles of at least one known to the art, an agent that “compensates' for an altered mitochondrial protein or peptide that is modified (e.g., an biological activity of a polypeptide includes an agent that altered expression level, altered amino acid Sequence, counterbalances any Structural or functional defect or alter altered posttranslational modification or an oxidative modi ation in Such polypeptide, Such as an altered biological fication) or in which the biological activity is altered and that activity arising as the result of a modification as provided correlates with a particular disease associated with altered herein, where Such counterbalancing may be partial or full mitochondrial function. Such profiles may define parameters restoration of normal activity, or restoration to Supranormal indicative of a Subject's predisposition to develop the par levels, So long as an effect is demonstrable in a Statistically ticular disease, and may further be useful in the identifica Significant manner. In certain preferred embodiments the tion of novel subtypes of that disease. In another embodi agent Substantially restores at least one mitochondrial pro ment, correlation of one or more traits in a Subject with at tein or peptide to a level found in control or normal Subjects least one mitochondrial protein or peptide (e.g., expression (which in Some cases may be an undetectable level). In a levels of a mitochondrial protein that can be determined to most preferred embodiment, an agent that Substantially differ from a control in a statistically significant manner) restores (e.g., increases or decreases) at least one mitochon may be used to gauge the Subject's responsiveness to, or the drial protein or peptide to a normal level effects the return of efficacy of, a particular therapeutic treatment. Similarly, the level of that indicator to a level found in control subjects. where levels of at least one indicator mitochondrial protein In another preferred embodiment, the agent that Substan or peptide and risk for a particular disease associated with tially restores Such an indicator conferS a clinically benefi altered mitochondrial function are correlated, the present cial effect on the Subject. In another embodiment, the agent invention provides advantageous methods for identifying that Substantially restores the indicator promotes a Statisti agents Suitable for treating Such disease(s), where Such cally significant change in the level of at least one mito agents affect levels of at least one mitochondrial protein or chondrial protein or peptide. AS noted herein, those having peptide (or levels of a modification) in a biological Source. ordinary skill in the art can readily determine whether a Such Suitable agents will be those that alter (e.g., increase or change in the level of a particular mitochondrial protein or decrease) the level of at least one mitochondrial protein or peptide brings that level closer to a normal value and/or peptide in a Statistically significant manner. In certain pre clinically benefits the Subject, based on the present disclo ferred embodiments, a Suitable agent alters a mitochondrial Sure. Thus, an agent that Substantially restores at least one protein or peptide level in a manner that confers a clinical mitochondrial protein or peptide to a normal level may benefit, and in certain other, non-exclusive preferred include an agent capable of fully or partially restoring Such embodiments, a Suitable agent alters a mitochondrial protein level. These and related advantages will be appreciated by or peptide level by causing it to return to a level detected in those familiar with the art. control or normal (e.g., disease-free) Subjects. 0134) Any of the agents for treating a disease associated 0.132. As described herein, determination of levels of at with altered mitochondrial function (e.g., oxidative modifi least one mitochondrial protein or peptide may also be used cation of a protein), identified as described herein, are to stratify a patient population (i.e., a population classified as preferably part of a pharmaceutical composition when used having one or more diseases associated with altered mito chondrial function, for example, by oxidative modification in the methods of the present invention. The pharmaceutical of a protein). Accordingly, in another preferred embodiment composition will include at least one of a pharmaceutically of the invention, determination of levels of a mitochondrial acceptable carrier, diluent or excipient, in addition to one or protein or peptide in at least one protein or peptide in a more agents for treating a disease associated with oxidative biological Sample from an oxidatively Stressed Subject may modification of a protein, and, optionally, other components. provide a useful correlative indicator for that Subject. A 0.135 “Pharmaceutically acceptable carriers” for thera Subject So classified on the basis of mitochondrial protein peutic use are well known in the pharmaceutical art, and are expression levels may be monitored using any known clini described, for example, in Remingtons Pharmaceutical Sci cal parameters for a specific disease referred to above, Such ences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For US 2004/0101874 A1 May 27, 2004 example, Sterile Saline and phosphate-buffered Saline at acid or Sodium bisulfite, chelating agents Such as ethylene physiological pH may be used. Preservatives, Stabilizers, diaminetetraacetic acid; bufferS Such as acetates, citrates or dyes and even flavoring agents may be provided in the phosphates and agents for the adjustment of tonicity Such as pharmaceutical composition. For example, Sodium ben Sodium chloride or dextrose. The parenteral preparation can Zoate, Sorbic acid and esters of p-hydroxybenzoic acid may be enclosed in ampoules, disposable Syringes or multiple be added as preservatives. Id. at 1449. In addition, antioxi dose Vials made of glass or plastic Physiological Saline is a dants and Suspending agents may be used. Id. preferred adjuvant. An injectable pharmaceutical composi 0.136 “Pharmaceutically acceptable salt” refers to salts of tion is preferably Sterile. the compounds of the present invention derived from the 0141 Aliquid composition intended for either parenteral combination of Such compounds and an organic or inorganic or oral administration should contain an amount of agent(s) acid (acid addition Salts) or an organic or inorganic base for treating a disease associated with oxidative modification (base addition salts). The compounds of the present inven of a protein Such that a Suitable dosage will be obtained. tion may be used in either the free base or salt forms, with Typically, this amount is at least 0.01 wt % of an agent for both forms being considered as being within the Scope of the treating a disease associated with oxidative modification of present invention. a protein in the composition. When intended for oral admin istration, this amount may be varied to be between 0.1 and 0.137 The pharmaceutical compositions that contain one about 70% of the weight of the composition. Preferred oral or more agents for treating a disease associated with Oxida compositions contain between about 4% and about 50% of tive modification of a protein may be in any form which the agent for treating a disease associated with oxidative allows for the composition to be administered to a patient. modification of a protein. Preferred compositions and prepa For example, the composition may be in the form of a Solid, rations are prepared So that a parenteral dosage unit contains liquid or gas (aerosol). Typical routes of administration between 0.01 to 1% by weight of active compound. include, without limitation, oral, topical, parenteral (e.g., Sublingually or buccally), Sublingual, rectal, vaginal, intrath 0142. The pharmaceutical composition may be intended ecal and intranasal. The term parenteral as used herein for topical administration, in which case the carrier may includes Subcutaneous injections, intravenous, intramuscu Suitably comprise a Solution, emulsion, ointment or gelbase. lar, intrasternal, intracavernous, intrameatal, intraurethral The base, for example, may comprise one or more of the injection or infusion techniques. The pharmaceutical com following: petrolatum, lanolin, polyethylene glycols, bees position is formulated So as to allow the active ingredients wax, mineral oil, diluents Such as water and alcohol, and contained therein to be bioavailable upon administration of emulsifiers and Stabilizers. Thickening agents may be the composition to a patient. Compositions that will be present in a pharmaceutical composition for topical admin administered to a patient take the form of one or more istration. If intended for transdermal administration, the dosage units, where for example, a tablet may be a single composition may include a transdermal patch or iontophore dosage unit, and a container of one or more compounds of sis device. Topical formulations may contain a concentration the invention in aeroSol form may hold a plurality of dosage of the agent(s) for treating a disease associated with oxida tive modification of a protein of from about 0.1 to about 10% units. W/V (weight per unit volume). 0138 For oral administration, an excipient and/or binder 0143. The composition may be intended for rectal admin may be present. Examples are Sucrose, kaolin, glycerin, istration, in the form, e.g., of a Suppository which will melt Starch dextrins, Sodium alginate, carboxymethylcellulose in the rectum and release the drug. The composition for and ethyl cellulose. Coloring and/or flavoring agents may be rectal administration may contain an oleaginous base as a present. A coating shell may be employed. Suitable nonirritating excipient. Such bases include, without 0.139. The composition may be in the form of a liquid, limitation, lanolin, cocoa butter and polyethylene glycol. e.g., an elixir, Syrup, Solution, emulsion or Suspension. The 0144. In the methods of the invention, the agent(s) for liquid may be for oral administration or for delivery by treating a disease associated with oxidative modification of injection, as two examples. When intended for oral admin a protein may be administered through use of insert(s), istration, preferred compositions contain, in addition to one bead(s), timed-release formulation(s), patch(es) or fast-re or more agents for treating a disease associated with Oxida lease formulation(s). tive modification of a protein, one or more of a Sweetening 0145. It will be evident to those of ordinary skill in the art agent, preservatives, dye/colorant and flavor enhancer. In a that the optimal dosage of the agent(s) for treating a disease composition intended to be administered by injection, one or asSociated with Oxidative modification of a protein may more of a Surfactant, preservative, wetting agent, dispersing depend on the weight and physical condition of the patient; agent, Suspending agent, buffer, Stabilizer and isotonic agent on the Severity and longevity of the physical condition being may be included. treated; on the particular form of the active ingredient, the 0140. A liquid pharmaceutical composition as used manner of administration and the composition employed. It herein, whether in the form of a Solution, Suspension or other is to be understood that use of an agent for treating a disease like form, may include one or more of the following asSociated with Oxidative modification of a protein in a adjuvants: Sterile diluents Such as water for injection, Saline chemotherapy can involve Such a compound being bound to Solution, preferably physiological Saline, Ringer's Solution, an agent, for example, a monoclonal or polyclonal antibody, isotonic Sodium chloride, fixed oils Such as Synthetic mono a protein or a liposome, which assist the delivery of Said or digylcerides which may serve as the Solvent or Suspend compound. ing medium, polyethylene glycols, glycerin, propylene gly 0146 These and related advantages will be appreciated col or other Solvents, antibacterial agents Such as benzyl by those familiar with the art. The following Examples are alcohol or methyl paraben; antioxidants Such as ascorbic offered by way of illustration and not limitation. US 2004/0101874 A1 May 27, 2004 22

EXAMPLES analysis using the cocktail of antibodies directed against components of the electron transport chain. Quantification Example 1 of the electron transport chain complexes across the gradient was performed on images captured on a Fluor-S Multil PREPARATION OF HUMAN HEART mager (BioPad, Hercules, Calif.) and analyzed using Quan MITOCHONDRIA tity One software (BioPad). 0147 Human heart mitochondria were obtained from 0149 Immediately prior to processing and analysis by Analytical Biological Services (Wilmington, Del.) and were mass spectrometry (see below), the concentrated gradient further purified by metrizamide gradient centrifugation (see, fractions and the Solubilized pellet were Successively Sub e.g., Rosenthal, R. E., et al., 1987, J. Cereb. Blood Flow jected to electrophoresis on NuPAGE gels using ultraclean Metab. 7:752-8). Mitochondria (40 mg) were resuspended in reagents. Buffers were made using HPLC grade water, and MSHE (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, 1 a gel rig and Staining box were Set aside for these samples. mM EGTA plus a Complete protease inhibitor cocktail tablet Aliquots (25 ug) of each concentrated gradient fraction were (Roche, Indianapolis, Ind.)) and loaded onto a 35%/17% loaded on a 4-12% NuPage gel and run at 25 mA for 1 h, metrizamide gradient in 6%. Percoll. Gradients were centri then 35 mA for another 1 h 20 min. Gels were fixed for 10 fuged for 45 min at 19000 rpm, 4° C. in a SW40 rotor. The min (40% methanol, 10% acetic acid), washed three times heavy mitochondrial fraction was collected from the 35/17% for 5 min in HPLC grade water, stained with colloidal interface, diluted in MSHE before pelleting at 12000 g for Coomassie for 10-15 Sec, and then partially destained in 10 min, and resuspended in MSHE. Protein concentrations Water. were determined using the BioRad DC protein assay (Bio Rad Laboratories, Hercules, Calif.). The purity of the mito Example 3 chondria was assessed by Western analysis using antisera directed against actin (Abcam, Cambridge, UK), dynamin II GEL PROCESSING AND MASS (Transduction Labs, Lexington, Ky.), KDEL, and LAMP1 SPECTROMETRICANALYSIS OF (Stressgen, Victoria, BC Canada) to detect contamination POLYPEPTIDES due to cytoplasm, plasma membrane, ER, and lySOSomes, respectively. The integrity of the mitochondria was assessed 0150. The lightly Coomassie-stained electrophoretic gels by Western analysis using a cocktail of antibodies directed from Example 2 were imaged placed on a light box in a against components of the electron transport chain; laminar flow hood on a plastic cutting mat with a 6.5x1 mm NDUFS2, 70 kD subunit of complex II, core I of complex grid placed underneath. To avoid keratin contamination all III, cox 4, and ATP synthase alpha; all from Molecular manipulations were performed wearing latex gloves, shower Probes (Eugene, Oreg.). A representative example of west caps and lab coats. Starting at the bottom the gel, approxi ern immunoblot analysis of mitochondrial fractions pre mately 1 mm slices were excised across the entire width of pared essentially as described here is shown in FIG. 1. a gel lane with a clean razor, further cut into approximately 1 mm cubes and transferred to 500 it microcentrifuge tubes Example 2 that had been prewashed with 50:50 water:acetonitrile. This procedure was progressively continued to the top the gel to SUCROSE DENSITY GRADIENT ensure comprehensive coverage of all proteins in the gel FRACTIONATION OF SOLUBILIZED lane. Although most gel Slices were 1 mm thick, when MITOCHONDRIA discrete bands were encountered they were Selectively 0148 Metrizamide purified mitochondria (13 mg) were excised, while near the top of the gel Slightly thicker Slices resuspended in MSHE plus protease inhibitors and solubi were taken where the protein concentration was lower. This lized with 1% lauryl maltoside for 25 min on ice with resulted in 50-64 slices for each of the 12 lanes processed frequent Vortexing. Samples were centrifuged at 14000 rpm, (corresponding to Sucrose fractions 1-10, combined 11/12 4 C. for 20 min. The pellet was frozen by immersion in and the pellet). liquid nitrogen and stored at -80 C. The Supernatant was 0151. The gel pieces were incubated with 200 till destain Subjected to Sucrose gradient centrifugation (Hanson, B.J. et solution (25 mM ammonium bicarbonate, 25% acetonitrile) al., 2001, Electrophoresis 22:950-959). The gradient con at 37 C. for 45 min. The destain solution was decanted and sisted of 1 mL step-fractions of 35, 32.5, 30, 27.5, 25, 22.5, another cycle of destaining performed if there was residual 20, 17.5, 15 and 10% sucrose in 10 mM Tris, pH 7.5/1 mM coloration. The gel pieces were then dried on a Genevac EDTA/0.05% lauryl maltoside, plus protease inhibitors). concentrator using the “cool heat Setting (about 30 min). The solubilized mitochondria were loaded onto the gradient The dried gel pieces were slightly moistened with 5 till 50 in 5% sucrose and centrifuged at 38000 rpm, 4 C. for 16.5 mM ammonium bicarbonate, 5% acetonitrile and 5 lull of h in a SW40 rotor. The gradient was collected from the freshly prepared ice cold Promega modified trypsin (0.1 bottom in 1 mL fractions. The gradient fractions were mg/mL in 50 mM ammonium bicarbonate, 5% acetonitrile) concentrated in Microcon YM-3 centrifugal concentrators added. The gel pieces were allowed to Soak up the trypsin (Millipore, Bedford, Mass.). The concentrated samples were solution for 10 min, and then were fully reswelled with a 65 quantitated using the BioRad DC protein reagent, Snap till aliquot of 50 mM ammonium bicarbonate, 5% acetoni frozen by immersion in liquid nitrogen and stored at -80 C. trile. After an overnight incubation at 37 C., the digestion Separation of proteins acroSS the gradient was initially was terminated by addition of 7.5 till 10% acetic acid assessed by Subjecting 1 DL aliquots of the concentrated followed by brief Vortexing and light centrifugation in a fractions to electrophoresis on precast 4-12% NuPAGE gels microcentrifuge. The digest Supernatants were Subsequently in Mes buffer (Invitrogen, Carlsbad, Calif.) followed by transferred to Secondary prewashed 500 till microcentrifuge staining with SimplyBlue Safe Stain (Invitrogen) or Western tubes and carefully concentrated using the Genevac to final US 2004/0101874 A1 May 27, 2004

Volumes of 10-20 it. At no stage were the digests taken to excised were also taken into account. The remaining por dryness, in order to avoid irreversible adsorption of low tions of the digests were subjected to automated LC/MS/MS abundance peptides to the walls of the tubes. analysis. The microtiter plate containing the remaining pep 0152 The concentrated digests were then carefully tide digest mixture were transferred to an Endurance decanted to avoid particulates and transferred to the Wells of autosampler connected to a MicroTech Ultimate LC system. a V-bottom 220 u, polypropylene microtiter 96 well plate. The digest (10 uD) was transferred to a capillary trapping This plate was directly placed in a Symbiot (Applied Bio column containing C18 reversed phase resin at 20 u/min systems, Foster City, Calif.) robotic MALDI target spotter using a third pump containing solvent A (95% water, 5% and 0.5 till aliquots were spotted on a 2x96 well PS1 acetonitrile, 0.5% acetic acid) and washed for 3 min. A MALDI target along with a 0.3 till aliquot of alpha-hydroxy gradient of solvent A to solvent B (80% acetonitrile, 20% cinnamic acid matrix in 50% ACN, 0.1% TFA. Between water, 0.5% acetic acid) 20% to 80% over 40 min was used each row of Sample spots, calibrant (Des Arg1 Bradykinin, to elute peptides through a 4.5 cm 75 uC-18 packed Picofrit M. 904.4681; angiotensin 1, 1296.6853; Glul-Fibrinopep column (New Objectives Inc., Woburn, Mass.) at a flow rate tide B, 1570.6774; Neurotensin, 1672.91.75) was spotted for of 200-500 mL/min directly into the heated capillary orifice close external calibration between each Successive MALDI of a Finnigan LCQ Ion Trap Mass spectrometer equipped Spectrum. with a Finnigan dynamic nanospray Source (Thermo Finni gan, San Jose, Calif.). 0153. MALDI spectra were acquired on a Voyager DE STR under the following conditions: positive reflectron 0155 Mass spectra were acquired in the m/z 400-2000 mode with delayed extraction, accelerating Voltage 20 kV, range under the following conditions: positive polarity, grid Voltage 65%, mirror Voltage ratio 1.12, extraction delay capillary temperature 148 C., Source Voltage 2.4 kV, Source time 125 nSec and low mass gate 500 Da. Spectral acqui current 80 uA, capillary voltage 29 V and tube lens offset 0 Sition was automated using a spiral Search pattern with Saved V. After one full scan MS of the column effluent was Spectra being the average of 3 Successful acquisitions from recorded, two MS/MS spectra of the most intense and 400 laser shots at 20 HZ repetition rate in the m/z. 850-3000 Second most intense MS peaks were recorded over the m/z. range with a minimum intensity of 750 counts in the m/z. 100-2000 range with an isolation width of 2.5 and normal 1000-3000 range. Peptide mass fingerprints were analyzed ized collision energy 35. Dynamic exclusion was employed using the program Protein Prospector (Clauser, K. R. et al., to Select the maximum number of unique peptide peaks from 1999, Analytical Chemistry 71, 14:2871). Peaks from base the chromatograms. After replicate MS/MS spectra were line corrected, noise filtered deisotoped spectra were filtered acquired for a precursor ion, the m/z value of ion was placed to remove autolytic trypsin and most keratin peaks and then on an exclusion list with a t1.5 u window for 3 min. Each subjected to two modes of analysis. The first involved chromatogram was Subsequently analyzed with the program tolerant matching of 4 or 5 peaks to proteins in the database SEQUEST (Ducret et al., 1998, Protein Sci. 7: 706-719). within a 100 ppm window. In general, proteins matching The minimum requirement for a hit were at least 2 peptides with MOWSE scores (see Pappin, D. J. C. et al., 1993, for a particular protein having an X >1.7 for a +1 ion, X >2 for a +2 ion or X >3. In all cases A. must be Current Biology 3: 327-332 for an explanation of MOWSE co co co scores) in excess of 10000 were considered hits. The second greater than 0.1. analysis involved using the program “intellical” (Applied 0156) A set of 3025 polypeptides SEQ ID NOS:1-3025 BioSystems) which demands high precision. As a first pass, was identified in the GENBANK database on the basis of the 25 proteins would be selected from the database with 3 above-described selection criteria for hits from the mito matches with in 150 ppm mass accuracy. The program chondrial protein preparations recovered according to the would then look for a uniform deviation between the procedures detailed above. Table 1 presents the numbers observed and calculated peptide masses and recalibrate the SEQ ID NOS:1-3025 corresponding to the Sequence List Spectrum against the best fits. In general, a protein was ing Submitted herewith for all 3025 polypeptides identified considered a hit that had 4 peptides matching within 15 ppm herein as mitochondrial components, along with the GEN of the recalibrated spectrum and MOWSE scores over 1000 BANK accession numbers for these sequences and (if using these more rigorous parameters. These analyses were known) a brief description of each protein based on its fully automated using PS1 software (Applied Biosystems). Sequence characteristics and database annotation. Additional FIG. 2 shows a representative example of a MALDI mass polypeptides that were identified included those having Spectrum generated from polypeptides derived from a single amino acid sequences as set forth in NCBI/Genbank Acc. one-dimensional gel Slice. Nos. 35655 and 1421609, and reference herein to any one of 0154 As well as these selection criteria, the relative SEQ ID NOS:1-3025 may according to certain embodiments intensity of the matching peaks and the molecular weight of be understood to include NCBI/Genbank Acc. Nos. 35655 the identified protein relative to the band from which it was and 142160.

TABLE 1.

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

1. 13013 ND 4 2 28590 reading frame HSA US 2004/0101874 A1 May 27, 2004 24

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 287 anion transport protein 3010 type I collagen 3147 follicle stimulating hormone receptor 3164 glyceraldehyde 3-phosphate dehydrogenase 3174 glutathione-insulin transhydrogenase (216 AA) 3467 hexokinase 1 347 myeloperoxidase 7214 vitronectin precursor - human 7222 heat shock protein 90-beta - human 8675 carrier ANT3 - human (fragment) 8752 dnaK-type molecular chaperone HSPA5 precursor - human 885 protein-L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77) splice form II - human 15 8865 O succinate dehydrogenase (ubiquinone) (EC 1.3.5.1) 27K iron-sulfur protein precursor, mitochondrial - human (fragment) 16 8874 1. T-cell receptor beta chain V region - human (fragment) 17 8897 2 undulin 1 18 O529 4 alternative splicing factor ASF-2 19 O547 5 myosin-binding protein C, skeletal muscle - human 2O O559 5 cell adhesion protein SQM1 21 O614 O glycophorin A 22 O618 5 GTP-binding protein Rab2 23 O690 6 lipopolysaccharide-binding protein 24 O697 O mcf2 protein 25 O755 4 pyruvate kinase isozyme M2 26 O763 1. ryanodine receptor type 1, skeletal muscle - human 27 O791 2 transcription factor E3 28 1396 2 annexin VI 29 1431 2 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (Calcium pump 2) (SERCA2) (SR Ca(2+)-ATPase 2) (Calcium-transporting ATPase sarcoplasmic reticulum type, slow twitch skeletal muscle isoform) (Endoplasmic reticulum class 1/2 Ca(2+) ATPase) 3O 1437 4 Na, K-ATPase subunit alpha 1 31 1437 4 Sodium/potassium-transporting ATPase alpha-1 chain precursor (Sodium pump 1) (Na+/K+ ATPase 1) 32 1454 ATPase beta F1 33 1520 C-1-TETRAHYDROFOLATE SYNTHASE, CYTOPLASMIC (C1-THF SYNTHASE) 34 1710 3 cox 5b 35 1775 9 UCR 4 CYTOCHROME C1 36 1786 3 UCR cyt b 37 2O64 3 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE, MUSCLE 38 2074 9 MAJOR GASTROINTESTINALTUMOR-ASSOCATED PROTEIN GAF33-2 39 2166 5 Glutathione peroxidase 1 (GSHPx-1) (Cellular glutathione peroxidase) 40 2327 7 PROTEIN HOX-C6(HHO.C8) 41 2357 1. heat shock 27 KD protein 42 2367 8 heat shock 90 kD protein HSP 90-ALPHA (HSP 86) 43 2367 8 Heat shock protein HSP 90-alpha (HSP 86) 44 25.48 4 HEPATOCYTE GROWTH FACTOR RECEPTOR PRECURSOR(C- MET)(HGF-SF RECEPTOR) 45 2907 O E 1-beta 46 2937 9 heat shock 60 kDa protein, mitochondrial precursor (Hsp60) (60 kDa chaperonin) (CPN60) (Heat shock protein 60) (HSP-60) (Mitochondrial matrix protein P1) (P60 lymphocyte protein) (HuCHA60) 47 2990 2 Phosphoglycerate kinase 1 (Primer recognition protein 2) (PRP 2) 48 3074 9 ALKALINE PHOSPHATASE, TISSUE-NONSPECIFIC ISOZYME PRECURSOR 49 3216 4 RETINOBLASTOMA-ASSOCIATED PROTEIN(P105-RB) 50 3606 6 TRIOSEPHOSPHATE ISOMERASE 51 3609 O TROPOMYOSIN BETA CHAIN, SKELETAL MUSCLE 52 3621 3 Troponin I, cardiac muscle 53 41.68 6 FINGER PROTEIN 8 54 7783 6 alpha-1-antitrypsin precursor 55 7834 5 aloalbumin Venezia 56 7839 O aldehyde dehydrogenase 57 7842 6 alpha-fodrin 58 7873 6 apolipoprotein B100 59 7889 6 beta-3-adrenergic receptor 60 7927 9 ATPase beta subunit 61 8052 9 chromogranin. A US 2004/0101874 A1 May 27, 2004 25

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 62 181238 cytochrome c1 63 184477 retinoic acid receptor 64 188590 myosin light chain 3 65 1886.72 mannose 6-phosphate receptor 66 1894.22 proliferating cell nuclear protein P120 67 189514 p80-coilin 68 1902O1 porin 69 190474 salivary proline-rich protein 1 70 190804 ubiquinone-binding protein 71 190804 UCR 6 ubiquinone-binding protein 72 223374 isomerase, triosephosphate 73 223582 histone H4 74 223632 dismutase, Cu/Zn superoxide 75 224309 protein delta T3, glyco 76 225897 glycogen phosphorylase 77 225985 amyloid related serum protein SAA 78 226007 ventricular myosin L1 79 226021 growth regulated nuclear 68 protein 8O 226209 cox. 8 81 227297 ND FeS NADH dehydrogenase FeS protein 82 227448 phosphofructokinase 83 228.097 receptor-like Tyr phosphatase 84 229149 hemoglobin beta 85 2294.79 lipoprotein Gln I 86 2294.79 lipoproteinGln I 87 23OOO4 Human Neutrophil Elastase (HNE) (E.C.3.4.21.37) (Also Referred To As Human Leucocyte Elastase (HLE)) Complex With Methoxysuccinyl-Ala Ala-Pro-Ala Chloromethyl Ketone (MSACK) 88 231743 G1/S-SPECIFIC CYCLIN D3 89 232472 nucleotide diphosphate kinase subunit A, p19/nm23-H1 human, Peptide Partial, 12 aa, segment 1 of 3 90 238427 Porin 31 HM human, skeletal muscle membranes, Peptide, 282 aa 91 251.188 protein phosphatase from PCR fragment H9 92 283950 Oxoglutarate dehydrogenase (lipoamide) (EC 1.2.4.2) precursor - human 93 284,319 mucin-associated antigen - human (fragment) 94 285975 rab GDI 95 292.793 T-cell receptor beta 96 306926 insulin-like growth factor binding protein 2 97 307021 mu-immunoglobulin 98 3121.37 aldolase C 99 337758 pre-serum amyloid P component OO 338O17 SEF2-1D protein O1 339647 hormone binding protein precursor O2 346275 myelin transcription factor 1 - human (fragment) O3 352335 reductase, NADH cytochrome b5 O4 385.479 N-methyl-D-aspartate glutamate receptor channel; NMDA GluR channel 05 386,745 guanine nucleotide-binding protein G-s-alpha-3 O6 386872 myoglobin O7 387,010 pyruvate dehydrogenase E1-beta subunit precursor O8 387O11 pyruvate dehydrogenase E1-alpha O9 387O11 pyruvate dehydrogenase E1-alpha precursor 1O 387O16 phosphoglycerate mutase 11 393124 Unknown 12 41.6776 CD27 LIGAND(CD70 ANTIGEN) 13 434755 rat general mitochondrial matrix processing protease mRNA (RATMPP)., similar to 14 43.6222 Unknown 15 4386SO paired box protein 16 448295 TLS protein 17 458862 heart fatty acid binding protein; hFABP 18 469045 h-contactin 2 precursor 19 47678O Ras guanine nucleotide exchange factor son-of-sevenless (sos) 1 - human 481,043 MHC class III histocompatibility antigen HLA-B-associated protein 2 similarity - human 21 483239 homeotic protein engrailed 2 - human 22 499158 acetoacetyl-CoA thiolase mitochondrial 23 516764 motor protein 24 516768 motor protein 25 533.538 diamine oxidase, copper/topa quinone containing 26 551604 pregnancy-specific beta-1 glycoprotein US 2004/0101874 A1 May 27, 2004 26

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 27 5.53254. cytochrome b5 reductase (EC 1.6.2.2) 28 5.53597 myosin heavy chain beta-subunit 29 553734 putative 3O 553734 Unknown 31 577307 The ha3662 gene product is related to mouse glycerophosphate dehydrogenase. 32 595.267 gastrin-binding protein 78 kDa 33 606609 GBP 34 627364 adenovirus E1A-associated 130k protein - human 35 627367 desmoyokin - human (fragments) 36 631OFO AHNAK-related protein - human (fragment) 37 687714 dynein heavy chain, isotype 1B 38 7O3O83 cytochrome b5 39 704.445 ATPase subunit 8 40 728834 Alu subfamily SB2 sequence contamination warning entry 41 802.150 pancreatic peptidylglycine alpha-amidating monooxygenase; PA 42 903598 Krueppel-type protein 43 992629 orf 44 OOO865 This CDS feature is included to show the translation of the corresponding V region. Presently translation qualifiers on V region features are illegal 45 OO1941 dihydropyridine receptor alpha 1 subunit 46 O33182 Y- RNA recognition motif protein 47 O53O81 calpastatin 48 O65362 Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length. Non Myristoylated 49 O70477 insulin receptor precursor - human 50 O71834 dihydrolipoamide S-succinyltransferase 51 O82355 epidermal autoantigen 450K (clone pE450-B) - human (fragment) 52 O82428 GTPase-activating protein rhoGAP 53 O82553 JC-kappa protein 54 O82567 laminin A3 55 O82692 phospholipase C beta 3 56 O82723 propionyl Coenzyme A carboxylase, beta polypeptide 57 O82723 propionyl-CoA carboxylase (EC 6.4.1.3) beta chain precursor - human 58 O85294 cell-cycle-dependent 350K nuclear protein - human (fragment) 59 O85373 protein disulfide-isomeraseER60 precursor 60 O91688 heat shock protein 61 O96O24 isoAsp protein carboxyl methyltransferase 62 O96067 tat-associated protein 63 O3677 myosin-light-chain kinase 64 24876 Krueppel-related DNA-binding protein 65 30694 erythrocyte adducin alpha subunit 66 36416 mitosis-specific chromosome segregation protein SMC1 of S. cerevisiae., similar to 67 36741 predicted protein of 548 amino acids 68 51113 PDE1C3 69 60932 DRAL gene product gi7209525db|BAA92253.1 (AB038794) DRALSlim3/FHL2 70 68719 C6.1.A PROTEIN 71 68781 EXTRACELLULAR CALCIUM-SENSING RECEPTOR PRECURSOR 72 69072 APOPAIN PRECURSOR ( PROTEASE CPP32) (YAMA PROTEIN) (CPP-32) (CASPASE-3) 73 69204 dodecenoyl-CoA Delta-isomerase 74 7O654 ANTIGENK-67 75 72554 VDAC-2 76 74572 Thromboxane A2 receptor (TXA2-R) (Prostanoid TP receptor) 77 772.30 Zinc finger 78 77438 broa2 79 84699 tyrosyl-tRNA synthetase 8O 96.398 Unknown 81 96433 Unknown 82 22O311 elongation factor-1 alpha 83 235848 HMG CoA synthase 84 235902 FRAP-related protein 85 2374O6 Cu/Zn-superoxide dismutase 86 245894 cardiac myosin binding protein-C 87 245985 beta 2-adrenergic receptor, beta 2AR Y354A human, Peptide Partial Mutagenesis, 24 aa 88 246236 ptp-IV1b, PTP-IV1 gene product 89 262579 ND 1 90 26258O ND 2 US 2004/0101874 A1 May 27, 2004 27

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

191 2625 81 cox 1 192 2625 82 ATPase 6 193 2929 41 hydroxymethylglutaryl-CoA lyase 194 2935 61 Diff40 gene product 195 3350 64 fibrillin 196 3350 72 G34 (big gastrin) 197 3352 12 medullasin N-term. 198 335250 Rod coMP phosphodiesterase 199 335277 Unknown 2OO 34O1 42 alpha1-antichymotrypsin 2O1 3463 17 heat shock 70 kD protein 7 2O2 3519 OO NEUROBLAST DIFFERENTATIONASSOCATED PROTEIN 2O3 3519 OO Segment 1 of 2 Neuroblast differentiation associated protein AHNAK (Desmoyokin) 2O4 3519 O1 NEUROBLAST DIFFERENTATIONASSOCATED PROTEIN 205 354222 aldehyde dehydrogenase E3 2O6 3597 15 Na+, K+ ATPase 2O7 3597 15 Na+, K+ ATPase 208 359759 histamine H2 receptor 209 362755 endopeptidase La homolog (EC 3.4.21.-) precursor, mitochondrial (version 1) 210 3818 14 skeletal muscle LIM-protein SLIM 211 3991 05 phosphatidylinositol (4,5)bisphosphate 5-phosphatase homolog 212 3998 O1 p167 213 4081 88 desmin 214 5040 2O Yeast translation activator GCN1 (P1: A48126), similar to 215 5178 99 RAGE-1 ORF5; one of 3 possible coding regions 216 5826 92 TATA box-binding protein 217 5871 38 sorcin 218 587477 TCOF1 gene 219 588292 Ca channel: SUBUNIT = alpha: ISOTYPE = L 22O 6555 94 HES1 221 6572 66 S10 GTP-binding protein 222 665723 RPD3 protein 223 6882 67 polo like kinase 224 7O66 11 ELONGATION FACTOR TU, MITOCHONDRIAL PRECURSOR 225 708O 98 Histone H1t 226 7091 23 DNA MISMATCH REPAIR PROTEIN MSH6 (MUTS-ALPHA 160 KDA SUBUNIT 227 7099 47 PYRUVATE CARBOXYLASE PRECURSOR 228 7102 79 dihyrolipoamide acetyl transferase 229 7185 O2 aconitase mitochondrial 230 7185 O2 aconitase, mitochondrial 231 73OO 78 130 KDA LEUCINE-RICH PROTEIN(GP130) 232 7314 14 ZINC FINGER PROTEIN 138 233 7625 33 carnitine palmitoyltransferase I 234 7632 38 lysosomal trafficking regulator LYST 235 7733 81 APXL 236 7784 1O unknown 237 7784 32 Treacher Collins syndrome 238 8052 8O alpha II spectrin 239 8698 O3 fatty acid binding protein 3 240 930 1O GM-CSF receptor alpha subunit soluble 3 241 942 87 Lactoferrin, H253m N Terminal Lobe Of Human 242 9435 32 Profilin I Crystallized In High Salt Actin-Binding Protein, Human Platelet 243 3-hydroxyacyl-CoA dehydrogenase, isoform 2 244 Putative gene. Genscan predictions confirmed by EST splicing.; coded for by human cDNAs AA122029 (NID:g1678048), D31562 (NID: g644442), AA158721 (NID: g1733515), R59640 (NID: g830335) and F13082 (NID: g709111) 245 RNA editase 246 Zinc finger 5 protein 247 leukocyte antigen, HLA-A2 variant 248 21177 dihydrolipoamide S-(2-methylpropanoyl)transferase (EC 2.3.1.-) precursor - human 249 21178 73 pyruvate kinase (EC 2.7.1.40), muscle splice form M1 - human 250 211.83 44 arginine - tRNA ligase (EC 6.1.1.19) - human 251 211.89 70 histone H1 - human (fragment) 252 21192. 68 alpha-tubulin - human (fragment) 253 211.93 90 proapo-A-I protein - human 254 21195 33 giantin US 2004/0101874 A1 May 27, 2004 28

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 255 2119712 dnaK-type molecular chaperone HSPA1L heat shock protein 256 21199.18 P43 - human 257 2134903 CG1 protein, kinectin 1 258 2135068 enhancer protein 259 21356.11 melanoma ubiquitous mutated protein - human (fragment) 260 2135819 neuropolypeptide h;3, brain 261 2135911 3',5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) 4A, cAMP specific, long splice form - human 262 21362O7 succinate-semialdehyde dehydrogenase (EC 1.2.1.24) - human (fragment) 263 2136282 TOG protein 264 2144337 pyruvate dehydrogenase (lipoamide) (EC 1.2.4.1) beta chain precursor, long splice form - human 265 2145O11 putative collagen homolog protein-b 266 2146960 methyl CpG binding protein 2 - human (fragment) 267 2217.933 PKU-beta 268 2224,581 Unknown 269 2224.583 Unknown 270 2224621 Unknown 271 2224663 Unknown 272 2243110 Unknown 273 2244.654 274 2270925 beta4-integrin 275 2286,145 caspase-like apoptosis regulatory protein 276 229.3556 Ran binding protein 2 277 2306809 X-linked nuclear protein 278 2317769 probable zinc finger protein H101 279 239,3734 C. elegans F11A10.5: 80% similarity to Z68297 (Pl 28O 2393,763 NAD (H)-specific isocitrate dehydrogenase gamma subunit 281 245.4586 reverse transcriptase 282 24651.78 283 2498864 RRPS PROTEIN HOMOLOG 284 2499753 PROTEIN-TYROSINE PHOSPHATASE KAPPA PRECURSOR 285 2506118 MULTIDRUG RESISTANCE PROTEIN 1 286 2507187 PROTEIN-L-ISOASPARTATE(D-ASPARTATE) O METHYLTRANSFERASE (PROTEIN-BETA-ASPARTATE METHYLTRANSFERASE) (PIMT) 287 2511440 calcium/calmodulin-dependent protein kinase II; CaM kinase II 288 25,11779 beta III spectrin 289 2565032 transcription activator/ protein delta/YY1; similar 290 2624694 Single-Stranded Dna Binding Protein, Human Mitochondrial 291 2653817 lipopolysaccharide binding protein 292 266121 Oxidative 3 alpha hydroxysteroid dehydrogenase 293 2662397 HADHB 294 2665782 voltage-gated sodium channel, subtype III 295 26955.74 leukocyte function-associated molecule-1 alpha subunit 296 2769254 NIPSNAP2 protein 297 2769254 NIPSNAP2 protein 298 2811135 retinal rod Na+/Ca+, K+ exchanger 299 2822143 R30217 1 3OO 2852604 Unknown 3O1 2865252 Unknown 3O2 2873.377 exportin t 3O3 298.173 Cypa Complexed With Hagpia 3O4 F22329 1 305 Zinc finger protein 306 kappa 1 immunoglobulin light chain variable region 307 3O43584 Unknown 3O8 3O43646 Unknown 309 3O4688O LIM-homeodomain protein LMX1B/LMX1.2 310 3114510 T State Human Hemoglobin alpha V96w, Alpha Aquomet, Beta Deoxy 311 312372 ND 24 K NADH dehydrogenase 24-kDa subunit of complex I 312 3153859 thioredoxin delta 3 313 31686O4 proline and rich nuclear 314 3211975 putative glialblastoma cell differentiation-related protein 315 3211977 sarco-fendoplasmic reticulum Ca-ATPase 3 316 32.12539 Isovaleryl-Coa Dehydrogenase. At 2.6 Angstroms Resolution: Structural Basis For Substrate Specificity 317 3252827 Unknown 318 3252827 Unknown 319 32.56.185 target of myb1 homolog) US 2004/0101874 A1 May 27, 2004 29

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 32O 3273228 acyl-CoA dehydrogenase very-long-chain 321 3273.386 plasmallemmal porin 322 3294.17O dJ232K4.1 (hypothetical 141.7 kD protein JUMONJI) 323 3299887 ES/130-related protein 324 3327040 Unknown 325 3327O54 Unknown 326 3327O54 Unknown 327 3360457 cul-3 328 3402141 Lysozymes. At Constant Positions 329 3402145 Lysozyme 330 354O239 ND Fe-S2 NADH dehydrogenase-ubiquinone Fe-S protein 2 precursor 331 3599521 musculin 332 3641621 gp180-carboxypeptidase D-like enzyme 333 3641621 gp180-carboxypeptidase D-like enzyme 334 366OO40 Fkbp Mutant F36v Complexed With Remodeled Synthetic Ligand 335 3660556 hdkk-4 336 369.4663 Unknown 337 3717965 DA-12C 338 3766197 succinyl-CoA synthetase beta subunit, ATP-specific 339 3766197 succinyl-CoA synthetase beta subunit, ATP-specific 340 3766,199 succinyl-CoA synthetase beta subunit GTP-specific 341 3766451 CHRNB2 342 38821.47 Unknown 343 38823O1 Unknown 344 3885362 sepiapterin reductase 345 3891975 Cathepsin G 346 3982589 SOX-28 protein 347 3986482 translation initiation factor elF3 p40 subunit; elF3p40 348 40O8131 chaperonin 10 349 O96860 fibronectin 350 O974.09 PAX-9 351 103446 NAD+-specific isocitrate dehydrogenase beta subunit isoform A 352 127947 guanine nucleotide-exchange factor 353 13972O Chymase 354 151929 PCAF-associated factor 400 355 153874 single-stranded mitochondrial DNA-binding protein precursor 356 2O4963 MUC-1/X mucin short variant 357 2O6175 ubiquitin-specific protease 358 210351 novel protein 359 24O227 Unknown 360 240243 Unknown 361 240305 Unknown 362 261577 CD8 beta chain 363 262430 CMP-NeuAc: lactosylceramide alpha-2,3-sialyltransferase 364 26.3556 Unknown 365 406346 guanylate cyclase activating protein 3 366 406564 succinyl-CoA synthetase beta subunit GTP-specific 367 4O6651 h-sco1 368 416457 mitotic checkpoint protein 369 495.063 yeast suppressor protein SRP40) d108K11.3 (similar to 370 SO1869 acyl-Coenzyme A oxidase 2, branched chain 371 5O1967 alpha-2C-adrenergic receptor; alpha-2C-1 adrenergic receptor; alpha-2C 1 adrenoceptor; alpha-2-adrenergic receptor, renal type; alpha2-AR-C4 372 4 SO2O11 adenylate kinase 1 373 SO2O13 adenylate kinase 2 isoform a; Adenylate kinase-2, mitochondrial 374 502097 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4: adenine nucleotide translocator 1 (skeletal muscle) 375 SO2101 annexin I 376 502107 annexin V 377 SO2111 annexin VII isoform 1 378 SO22O1 ADP-ribosylation factor 1 379 502273 ATPase, Na+/K+ transporting, alpha 3 polypeptide 38O 502297 ATPase delta F1 381 SO23O3 ATPase OSCP F1 382 5O2327 AU RNA-binding protein?enoyl-Coenzyme A hydratase precursor 383 SO2331 arginine vasopressin receptor 1A; V1a vasopressin receptor; vascular?hepatic-type arginine vasopressin receptor; antidiuretic hormone receptor 1A 384 4 502379 BCL10 385 4 SO2419 biliverdin reductase B (flavin reductase (NADPH)) US 2004/0101874 A1 May 27, 2004 30

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

386 4 5.02457 ATP-binding cassette, sub-family B (MDR/TAP), member 11; ABC member 16, MDR/TAP subfamily 387 5.02459 basigin; collagenase stimulatory factor; M6 antigen 388 502509 complement component 5 receptor 1 (C5a ligand); complement component-5 receptor-2 (C5a ligand) 389 502517 carbonic anhydrase I 390 502563 calpain 2, large subunit 391 SO26O1 carbonyl reductase 3; carbonyl reductase3 Homo sap 392 SO2603 chromobox homolog 4 (Pc class homolog, Drosophila); chromobox homolog 4 (Drosophila Pe class) 393 4 502703 CDC6 homolog; CDC6 (cell division cycle 6, S. cerevisiae) homolog: CDC18 (cell division cycle 18, S. pombe, homolog)-like; CDC6-related protein 394 502719 cadherin 13 preproprotein; H-cadherin; heart-cadherin; T-cad 395 SO2841 carbohydratesulfotransferase 1 396 502855 sarcomeric mitochondrial creatine kinase precursor; creatine kinase, mitochondrial 2; basic-type mitochondrial creatine kinase 397 502985 cox 6b 398 502987 cox 7a muscle 399 502989 cox. 7a liver 502991 cox 7b 502993 cox 7c 503O15 copine III SO3O21 liver carnitine palmitoyltransferase I: L-CPT1 SO3O49 cysteine-rich protein 2: Cystein-rich intestinal protein 503057 crystallin, alpha B; crystallin, alpha-2; Rosenthal fiber component; heat shock 20 kD like-protein O6 SO3143 cathepsin D O7 503177 chromosome X open reading frame 2 O8 SO3269 deoxycytidine kinase gi11436224refXP 00347 O9 SO33O1 2,4-dienoyl CoA reductase 1 precursor 1O 503375 dihydropyrimidinase 11 SO3431 dysferlin; dystrophy-associated fer-1-like 1 12 SO3443 endothelin converting enzyme 1 13 SO3447 peroxisomal enoyl-coenzyme A hydratase-like protein; delta3,5-delta2,4- dienoyl-CoA isomerase; peroxisomal enoyl-CoA hydratase 1; dienoyl-CoA isomerase 14 503475 eukaryotic translation elongation factor 1 alpha 2 15 50.3507 eukaryotic translation initiation factor 2, subunit 3 16 503537 eukaryotic translation initiation factor 4E binding protein 3 17 5036O7 electron transfer flavoprotein alpha polypeptide 18 SO3609 electron transfer flavoprotein beta polypeptide 19 SO3613 envoplakin 2O SO3651 fatty-acid-Coenzyme Aligase, long-chain 1 21 SO3667 fibrillin 2 + F422 22 503731 FK506-binding protein 6 23 SO3835 frizzled 9 24 SO3843 adaptor-related protein complex 1, gamma 2 subunit; gamma2-a 25 503899 N-acetylgalactosamine-6-sulfatase precursor 26 503937 glioblastoma amplified sequence 27 SO4041 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2: Guanine nucleotide-binding protein (G protein), alpha inhibiting 28 SO4049 guanine nucleotide binding proteintransducin alpha-chain 29 SO4O67 aspartate aminotransferase 1; glutamic-Oxaloacetic transamin 3O 504071 platelet glycoprotein lb alpha polypeptide precursor 31 SO41.69 glutathione synthetase 32 SO41.89 glutathione transferase Zeta 1 (maleylacetoacetate isomerase); glutathione transferase Zeta 1 SO4.483 hypoxanthine phosphoribosyltransferase 1 SO4487 -rich calcium-binding protein precursor SARCOPLASMIC RETICULUM 35 504517 heat shock 27 kD protein 1 36 SO4521 heat shock 60 kD protein 1 (chaperonin) 37 SO4523 heat shock 10 kD protein 1 (chaperonin 10) 38 SO4523 heat shock 10 kD protein 1 (chaperonin 10) 39 SO4665 interleukin 2 receptor, beta; Interleukin-2 receptor, beta polypeptide 40 SO4689 IMP (inosine monophosphate) dehydrogenase 2 41 SO4733 insulin receptor substrate 4 42 504795 inositol 1,4,5-triphosphate receptor, type 3 43 SO4867 ring finger protein (C3HC4 type) 8; C3HC4-type zinc finger protein; zinc US 2004/0101874 A1 May 27, 2004 31

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS finger protein 44 504975 low density lipoprotein receptor precursor; LDLR precursor; LDL receptor 45 SO4991 leukemia inhibitory factor (cholinergic differentiation factor); cholinergic differentiation factor 46 505071 MAP-kinase activating death domain protein 47 505093 monoamine oxidase B 48 505093 monoamine oxidase B 49 5051.45 malic enzyme 2, NAD(+)-dependent, mitochondrial 50 5051.45 malic enzyme 2, NAD(+)-dependent, mitochondrial; Malic enzyme, mitochondrial; malic enzyme 2, mitochondrial; pyruvic-malic carboxylase; malate dehydrogenase 51 505153 MAP/ERK kinase kinase 3 52 505249 mutS homolog 3 (E. coli); mutS (E. coli) homolog 3 53 505257 moesin 54 505257 moesin 55 505355 ND B8 56 505.357 ND 9k NDUFA4 57 505359 ND B14 58 SO5361 ND B12 59 SO5363 ND 16k, SGDH 60 505365 ND B17 61 505367 ND 6k 62 505369 ND 18K NADH dehydrogenase (ubiquinone) Fe-S protein 4 (18 kD) (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) Fe S protein 4, 18 kD (NADH-coenzyme Q; mitochondrial respiratory chain complex I (18-KD subunit) 4 63 4 505371 ND 23K NADH dehydrogenase (ubiquinone) Fe-S protein 8 (23 kD) (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) Fe S protein 8 (23 kD) (NADH-coenzyme Q 64 505.375 neogenin homolog 1 (chicken); neogenin (chicken) homolog 1 65 505.399 NIPSNAP homolog 1: 4-nitrophenylphosphatase domain and non neuronal SNAP25-like 1 66 5054.05 glycoprotein (transmembrane) nmb; transmembrane glycoprotein 67 505591 peroxiredoxin 1: Proliferation-associated gene A; proliferation-associated gene A (natural killer-enhancing factor A) 68 505621 prostatic binding protein; phosphatidylethanolamine binding protein 69 505685 pyruvate dehydrogenase (lipoamide) alpha 1: Pyruvate dehydrogenase, E1-alpha polypeptide-1 70 4 505687 pyruvate dehydrogenase (lipoamide) beta; Pyruvate dehydrogenase, E1 beta polypeptide 71 505693 pyruvate dehydrogenase kinase, isoenzyme 4 72 505717 peroxisomal biogenesis factor 11A 73 505773 Prohibitin 74 505,775 carrier phosphate isoform B 75 505,775 phosphate carrier precursor isoform 1b; phosphate carrier, mitochondrial; phosphate carrier, mitochondrial precursor 76 5058O1 phosphoinositide-3-kinase, class 3 77 505869 phospholipase C, gamma 1 (formerly subtype 148) 78 505887 phospholamban 79 505893 proteolipid protein 2 8O 505909 peripheral myelin protein 2: M-FABP 81 505911 postmeiotic segregation 1: Postmeiotic segregation increased (S. cerevisiae)-like 1 82 505925 putative neurotransmitter receptor 83 505965 POU domain, class 4, transcription factor 3 84 506O77 protein kinase C substrate 80 KD-H 85 506091 mitogen-activated protein kinase 6 86 SO6189 proteasome (prosome, macropain) subunit, alpha type, 7 87 506197 proteasome (prosome, macropain) subunit, beta type, 3; Proteasome subunit, beta type, 3 88 4 SO6291 protein tyrosine phosphatase, non-receptor type 2, isoform 1: T-cell protein tyrosine phosphatase 89 506371 RAB5B, member RAS oncogene family 90 SO64O1 raf proto-oncogene serine/threonine protein kinase 91 SO6413 RAP1A, member of RAS oncogene family; RAS-related protein RAP1A 92 5064.45 RNA binding motif protein 4 93 506517 regulator of G-protein signalling 2, 24 kD 94 506787 IQ motif containing GTPase activating protein 1; rasGAP-like with IQ motifs 4 95 4 506959 TAL1 (SCL) interrupting locus; SCL interrupting locus 4 96 4 506975 carrier family 12 (sodium/potassium?chloride transporters), member 2 US 2004/0101874 A1 May 27, 2004 32

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 497 506977 carrier family 12 (sodium/chloride transporters), member 3 498 506997 solute carrier family 25 (mitochondrial carrier; Oxoglutarate carrier), member 11; solute carrier family 20 (Oxoglutarate carrier), member 4 499 507007 carrier family 25 (mitochondrial carrier, Aralar), member 12; calcium binding mitochondrial carrier superfamily member Aralar 500 507021 solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane protein band 3, Diego blood group) 444 5071.85 sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase); Sepiapterin reductase 502 507215 signal recognition particle 54 kD 503 507299 SudD suppressor of bim D6 homolog (A. nidulans); human homolog of Aspergillus nidulans sudD gene product; sudD (suppressor of bim D6, Aspergillus nidulans) homolog SO4 507389 elongin A; transcription elongation factor B (SIII) 505 5074O1 transcription factor 6-like 1 SO6 5074O1 transcription factor 6-like 1 (mitochondrial transcription factor 1-like) 507 SO7431 thyrotrophic embryonic factor; Thyrotroph embryonic factor 508 SO7443 transcription factor AP-2 beta (activating enhancer binding protein 2 beta); transcription factor AP-2 beta (activating enhancer-binding protein 2 beta) 509 5076.09 tumor necrosis factor (ligand) superfamily, member 9 510 SO7643 tumor protein D52-like 2; hD54 511 507645 triosephosphate isomerase 1 512 507645 triosephosphate isomerase 1 513 507665 tyrosylprotein sulfotransferase 1 514 507677 tumor rejection antigen (gp96) 1: Tumor rejection antigen-1 (gp96) 515 507713 tetratricopeptide repeat domain 2 516 507733 Tu translation elongation factor, mitochondrial 517 507783 ubiquitin-conjugating enzyme E2H (homologous to yeast UBC8) 518 507789 ubiquitin-conjugating enzyme E2L 3 519 507793 ubiquitin-conjugating enzyme E2N 52O SO7841 ubiquinol-cytochrome c reductase core protein I 521 SO7843 ubiquinol-cytochrome c reductase core protein II 522 507853 ubiquitin specific protease, proto-oncogene: Unph 523 507857 ubiquitin specific protease 7 (herpes virus-associated) 524 507879 voltage-dependent anion channel 1 525 507.913 WAS protein family, member 1: WASP family Verprolin-homologous protein; scar, dictyostelium, homology of 1 526 4 507953 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, Zeta polypeptide; Tyrosine 3-monooxygenase/tryptophan 5 monooxygenase activation 527 507963 Zinc finger protein homologous to Zfp37 in mouse 528 507979 Zinc finger protein 132 529 522O26 Bassoon protein; match to PID: g3043642; similar to PID: g3413810, C terminus matches KIAAO559, N-terminus similar to 530 529887 NG35 531 557032 lactate dehydrogenase B 532 557.036 microseminoprotein, beta 533 557044 propionyl Coenzyme A carboxylase, beta polypeptide 534 557235 acyl-CoA dehydrogenase very long chain 535 557247 acylphosphatase 2, muscle type 536 557265 beta-1-adrenergic receptor gi15298.066 refixP 537 557305 aldolase. A protein 538 557311 adenosine monophosphate deaminase 1 (isoform M) 539 557317 annexin XI 540 557365 Bloom syndrome protein 541 557403 carnitine/acylcarnitine translocase; Carnitine-acylcarnitine translocase; carnitine-acylcarnitine carrier; solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 542 557403 carrier carnitine-acylcarnitine translocase 543 5574.09 cardiac calsequestrin 2 544 557439 cyclin-dependent kinase 3 545 557451 chromodomain helicase DNA binding protein 3; Mi-2a; zinc-finger helicase (Snf2-like) 546 4 557565 excision repair cross-complementing rodent repair deficiency, complementation group 6 547 557579 fatty acid binding protein 4, adipocyte; A-FABP 548 557657 immature colon carcinoma transcript 1 549 557735 monoamine oxidase A 550 557759 myeloperoxidase 551 557765 5-methyltetrahydrofolate-homocysteine methyltransferase; 5 methyltetrahydrofolate-homocysteine methyltransferase 1 US 2004/0101874 A1 May 27, 2004 33

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 552. 557767 methylmalonyl Coenzyme A mutase precursor 553 557769 mevalonate kinase 554 557771 protein C, cardiac; myosin-binding protein C, cardiac 555 557775 myosin light chain 2 556 557817 Succinyl CoA: 3-oxoacid CoA transferase 557 557817 Succinyl CoA: 3-oxoacid CoA transferase; succinyl-CoA: 3-ketoacid-CoA transferase precursor 558 557833 Propionyl-Coenzyme A carboxylase, alpha polypeptide 559 557845 ribonucleotide reductase M2 polypeptide 560 557867 sulfite oxidase 561 557867 sulfite oxidase, mitochondrial 562 557876 ATP-binding cassette, sub-family A member 4: ATP binding cassette transporter; ATP-binding transporter, retina-specific; rim protein 563 587083 564 5895.04 Unknown 565 589644 Unknown 566 678807 Unknown 567 68O705 CGI-33 protein 568 68O721 thyroid peroxidase 569 6891.04 NDASH 570 730927 associated PD1 571 757732 programmed cell death 8 (apoptosis-inducing factor) 572 757762 ring finger protein 14; androgen receptor associated protein 573 757786 N-acylsphingosine amidohydrolase (acid ceramidase) 574 757852 BCS1 (yeast homolog)-like 575 758024 coilin; coilin p80 576 758O3O coatomer protein complex, subunit alpha; alpha coat protein; Xenin 577 758038 cox 5a 578 758040 coX 6c 579 758118 mitochondrial ribosomal protein S29, 28S death associated protein 3: 58O 758118 mitochondrial ribosomal protein S29, 28S death associated protein 3; 581 75812O death-associated protein 1 582 7581.56 diacylglycerol kinase, iota 583 758.192 serine/threonine kinase 17a (apoptosis-inducing) 584 758242 early development regulator 2; homolog of polyhomeotic 2 585 758312 electron-transferring-flavoprotein dehydrogenase 586 758352 ferredoxin 1 precursor; adrenodoxin 587 7.58490 GTP binding protein 1 588 7.58498 hexose-6-phosphate dehydrogenase precursor 589 758504 hydroxyacyl-Coenzyme A dehydrogenase, type II 590 75852O hect domain and RLD 2 591 75852O hect domain and RLD 2 592 758570 heat shock 70 kD protein 9B (mortalin-2); heat shock 70 kD protein 9 (mortalin); Heat-shock 70 kD protein-9 (mortalin); mot-2; mthsp75 593 758582 isocitrate dehydrogenase 3 (NAD+) gamma 594 7586O4 interleukin enhancer binding factor 3, 90 kD: M-phase phosphoprotein 4; nuclear factor associated with dsRNA 595 758664 acetylglucosaminyltransferase-like protein 596 758682 protease, serine, 15; Lon protease-like protein 597 758714 microsomal glutathione S-transferase 3 598 75875O myosin DXB 599 758768 ND 42k 6OO 75.8772 NDB9 6O1 758774 ND 22k, PDSW 6O2 758776 ND 7k 603 758778 ND 8k, AGGG 604 7587.84 ND B14.5 605 758786 ND 49k 606 758788 ND 3Ok 6O7 758790 ND 15k 608 758792 ND 13 k-A 609 758818 Notch homolog 4 (Drosophila); Notch, drosophila, homolog of, 4: Notch (Drosophila) homolog 4 610 4 758832 neuregulin 2 isoform 1: neural- and thymus-derived activator for ErbB kinases 611 758852 organic cation transporter like 3 612 75894O chromosome 14 open reading frame 2: mitochondrial proteolipid 68 MP homolog 613 4 75894O mitochondrial proteolipid 68 MP homolog 614 4 75902O RAB5C, member RAS oncogene family; RAB, member of RAS oncogene family-like; RAB5C, member of RAS oncogene family US 2004/0101874 A1 May 27, 2004 34

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

615 4 759068 cytochrome oxidase deficient homolog 1 616 4 7.5908O succinate dehydrogenase complex, subunit A, flavoprotein precursor; succinate dehydrogenase complex flavoprotein subunit precursor 617 4 7.5908O succinate dehydrogenase, subunit A, flavoprotein (Fp) 618 7.59082 serum deprivation response (phosphatidylserine-binding protein) 619 : 759112 solute carrier family 16 (monocarboxylic acid transporters), member 3: monocarboxylate transporter 3 62O 7591.44 carrier family 9 (sodium?hydrogen exchanger), isoform 5 621 759146 slit homolog 2 (Drosophila); slit (Drosophila) homolog 2 622 75916O small nuclear ribonucleoprotein D3 polypeptide 623 7591.96 symplekin 624 760549 IDN3 625 76.1539 voltage-dependent calcium channel alpha 1G subunit bisoform 626 826643 annexin A3 627 826649 mitochondrial ribosomal protein L49 628 826649 mitochondrial ribosomal protein L49; chromosome 11 open reading frame 4 629 826655 calbindin 1 630 826661 nuclear receptor subfamily 1, group I, member 3 631 826661 nuclear receptor subfamily 1, group I, member 3; constitutive androstane receptor-beta; orphan nuclear hormone receptor 632 4 826772 insulin-like growth factor binding protein, acid labile subunit 633 826848 ND B13 634 826850 ND B14.5a NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7 (14.5 kD, B14.5a) 635 4 826852 ND 8k 636 4 826856 ND 75K NADH dehydrogenase (ubiquinone) Fe-S protein 1 (75 kD) (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone), Fe S protein-1 (75 kD); NADH-ubiquinone oxidoreductase 75 kD subunit precursor 637 826898 profilin 1 638 826914 phospholipase A2, group IVB 639 82695O kallikrein 7 640 827O65 Zinc finger protein 147 641 877291 receptor for Advanced Glycation End Products 642 885281 glutamate dehydrogenase 1 643 8853.31 G protein-coupled receptor 42 644 885389 hydroxyacyl glutathione hydrolase; glyoxalase 2 645 885389 hydroxyacyl glutathione hydrolase; hydroxyacyl glutathione hydrolase; glyoxalase 2; Hydroxyacyl glutathione hydrolase; glyoxalase II; hydroxyacylglutathione hydroxylase 646 8854O1 cytochrome cheme lyase 647 885533 peptidylglycine alpha-amidating monooxygenase COOH-terminal 648 8855.53 postmeiotic segregation increased 2-like 9 649 885565 peroxisomal acyl-CoA thioesterase 650 885615 signal transducer and activator of transcription 2, 113 kD 651 885,665 achaete-scute complex homolog-like 2; achaete-scute complex (Drosophila) homolog-like 2 652 887552 653 89.4370 ND B22 654 9146O1 Unknown 655 4929697 CGI-114 protein 656 SO31609 branched chain alpha-ketoacid dehydrogenase kinase 657 SO31631 CD36 antigen 658 SO31691 chromosome 21 open reading frame 33; human HES1 protein, homolog o E. coli and zebrafish ES1 protein 659 5031707 glycoprotein. A repetitions predominant precursor; garpin 660 5031777 isocitrate dehydrogenase 3 (NAD+) alpha 661 5031777 isocitrate dehydrogenase 3 alpha 662 5031875 amin AfC 663 SO31881 eucyl/cystinyl aminopeptidase; leucyl/cystinyl aminopeptidase (Oxytocinase) 664 SO31943 ranscription factor NSCL-1 helix-loop-helix protein 665 5031987 peptidylprolyl isomerase F MITOCHONDRIAL PRECURSOR( 666 5032O17 RAD50 (S. cerevisiae) homolog 667 5032051 ribosomal protein S1440S 668 5032095 carrier family 21 (prostaglandin transporter), member 2 669 SO321.81 ranslocase of inner mitochondrial membrane Tim17b 670 50.32215 ranslational inhibitor protein 671 SO51381 FK506 binding protein 12-rapamycin associated protein 1 672 5059062 pilin-like transcription factor 673 5114261 voltage-dependent anion channel isoform 2 US 2004/0101874 A1 May 27, 2004 35

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 674 38999 NADH-Ubiquinone reductase 675 74539 malate dehydrogenase 1, NAD (soluble) 676 74539 malate dehydrogenase 1, NAD (soluble); Malate dehydrogenase, soluble 677 74541 malate dehydrogenase 2, NAD (mitochondrial); Malate dehydrogenase, mitochondrial 678 74563 MHC binding factor, beta 679 74627 plasma glutamate carboxypeptidase; aminopeptidase 68O 74.739 tubulin, beta, 5 681 5 74743 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 682 NY-REN-6 antigen 683 54 53549 thioredoxin peroxidase; thioredoxin peroxidase (antioxidant enzyme) 684 54 53559 ATPase d FO 685 54 53670 golgi transport complex 1 (90 kD subunit); golgi transport complex 1 (90 kDa subunit) 686 54 53750 brain acid-soluble protein 1; neuronal tissue-enriched acidic protein 687 54 53890 PIBF1 gene product 688 54 53902 NIMA-interacting, 4 (parvulin) peptidyl-prolyl cis-trans isomerase EPVH 689 54 53990 proteasome (prosome, macropain) activator subunit 1 (PA28 alpha) 690 54 54O28 related RAS viral (r-ras) oncogene homolog; Oncogene RRAS 691 54 54122 translocase of inner mitochondrial membrane Tim23 692 54 54148 UNC13 693 54 54152 ubiquinol-cytochrome c reductase binding protein 694 54 5418O zinc finger protein 193 695 5578989 Unknown 696 5689405 Unknown 697 5689555 Unknown 698 57O1717 UDP-N-acetylglucosamine:alpha-1,3-D-mannoside beta-1,4-N- acetylglucosaminyltransferase IV-homologue 699 5725250 G7 protein 7OO 5725370 involved in chromosomal translocation 701 5729802 Unknown 702 5729875 progesterone binding protein 703 5729877 heat shock 70 kD protein 8; heat shock 70 kD protein 8 (HSP73); heat shock cognate protein, 71-kDa; heat shock 70 kd protein 10 (HSC71) 704 5729887 IQ motif containing GTPase activating protein 2, RasGAP-related protein 705 5729937 metaxin 2 7O6 5729937 metaxin 2 707 572.9966 MHC class I region ORF 708 573OO27 GAP-associated tyrosine phosphoprotein p62 (Samó8) 709 573OO33 sodium channel, voltage-gated, type X, alpha polypeptide 710 573O110 ubiquitin specific protease 3 gi1072034Osp|Q9Y6I4UBP3 HUMAN UBIOUITIN CARBOXYL-TERMINAL HYDROLASE 3 711 succinate dehydrogenase flavoprotein subunit 712 PPARgamma coactivator-1 713 Gag-Pro-Pol-Env protein 71.4 AFG3 (ATPase family gene 3, yeast)-like 2 715 mitofilin inner membrane protein, mitochondrial (mitofilin); motor protein 716 RAB35, member RAS oncogene family; ras-related protein rab-1 717 coated vesicle membrane protein 718 sex comb on midleg (Drosophila)-like 1 719 transmembrane trafficking protein 720 U2 small nuclear RNA auxillary factor 1; U2 snRNP auxiliary factor small subunit; splicing factor U2AF 35 kDa subunit 721 5821952 Rotamer Strain. As A Determinant Of Protein Structural Specificity 722 5882259 genethonin 3 723 5901896 ATPase epsilon F1 724 5901926 cleavage and specific factor 5, 25 kD subunit 725 590.1982 isocitrate dehydrogenase 3 (NAD+) beta 726 5902106 SRY (sex determining region Y)-box 20 727 5902110 SRY (sex determining region Y)-box 22; SPY (sex-determining region Y)- box 22 728 5924.409 tight junction protein ZO-2 isoform C 729 6005717 ATPase e FO 730 6005772 putative G protein coupled receptor 731 6OO5938 utrophin; dystrophin-related protein 732 6OO5938 utrophin; dystrophin-related protein 733 6OO5948 WW domain-containing binding protein 4; formin binding protein 21 734 6O10711 hereditary haemochromatosis protein precursor 735 6031192. phosphate carrier precursor isoform 1a; phosphate carrier, mitochondrial; phosphate carrier, mitochondrial precursor 736 ND B15 US 2004/0101874 A1 May 27, 2004 36

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 737 6094658 truncated form of cytochrome Bc1 J chain; similar to 1BGY 738 617SO38 Son of sevenless protein homolog 2 (SOS-2) 739 6176530 alanine-glyoxylate aminotransferase homolog 740 6249687 R31155 1 741 6273778 trabeculin-alpha 742 6274550 ND B22 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9 (22 kD, B22) 743 6288790 beta-ureidopropionase 744 633O385 Unknown 745 6331429 Unknown 746 6382O58 V-abl Abelson murine leukemia viral oncogene homolog 1 isoform b: Abelson murine leukemia viral (v-abl) oncogene homolog 1 747 diaphanous 2 isoform 12C; Diaphanous, Drosophila, homolog of, 2; diaphanous (Drosophila, homolog) 2 748 643,3936 acZonin 749 6456828 phosphoglycerate kinase 1 750 6523797 adrenal gland protein AD-002 751. 6572219 UCR ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide like 1) dj37OM22.2 ( 752 6580492 cN28H9.1 (novel protein) 753 6594629 pRGR2 754. 6598323 GDP dissociation inhibitor 2; rab GDP-dissociation inhibitor, beta 755 6624,122 3-hydroxyisobutyrate dehydrogenase 756 66311OO natural killer-tumor recognition sequence 757 6649914 growth/differentiation factor-11 758 667.8455 transcription termination factor, RNA polymerase I 759 6681764 ND 39k NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 (39 kD); NADH dehydrogenase (ubiquinone) Fe-S protein 2-like (NADH coenzyme Q reductase) 760 6683124 Unknown 761 6686262 ZINC FINGER PROTEIN 36 762 6688130 poly-(ADP-ribose) polymerase II 763 67298O3 Heat-Shock 70 kd Protein 42 kd Atpase N-Terminal Domain 764 67395OO LDLR-FUT fusion protein 765 684.1066 calcium-binding transporter 766 684.1110 Unknown 767 6841194 HSPC272 768 684.1440 HSPC108 769 684.1930 receptor beta chain 770 6912238 peroxiredoxin 5; antioxidant enzyme B166 771 691.2322 crumbs homolog 1; crumbs (Drosophila) homolog 1 772 6912396 glyoxylate reductase/hydroxypyruvate reductase 773 6912440 double-stranded RNA-binding zinc finger protein JAZ 774 6912482 LETM1 leucine zipper-EF-hand containing transmembrane protein 1 775 6912482 leucine zipper-EF-hand containing transmembrane protein 1 776 691.2536 nicotinamide nucleotide transhydrogenase 777 691.2536 nicotinamide nucleotide transhydrogenase 778 6912538 neurotensin receptor 2; neurotensin receptor, type 2 779 691.2664 sirtuin 5, isoform 1; sir2-like 5; sirtuin type 5; sirtuin (silent mating type information regulation 2, S. cerevisiae, homolog) 5; silent mating type information regulation 2, S. cerevisiae, homolog 5 691271.4 translocase of inner mitochondrial membrane 9 homolog (yeast); translocase of inner mitochondrial membrane 9 (yeast) homolog 781 691271.4 translocase of inner mitochondrial membrane Tim 9a 782 6996429 acetyl-coenzyme A synthethase (acetate-coA ligase)) d568C11.3 (novel AMP-binding enzyme similar to 783 6996429 novel AMP-binding enzyme similar to acetyl-coenzyme A synthethase (acetate-coA ligase) 784 7O18398 hemopoietic cell kinase 785 7019351 cardiotrophin-like cytokine; neurotrophin-1/B-cell stimulating factor-3 786 701.9545 secreted protein of unknown function 787 702O216 Unknown 788 7020807 mitochondrial ribosomal protein L22, similar to 789 7022241 Unknown 790 7022343 Unknown 791 7022728 Unknown 792 7022751 Unknown 793 7242949 Unknown 794 7242979 Unknown 795 7243141 Unknown 796 7243219 Unknown US 2004/0101874 A1 May 27, 2004 37

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

797 7243272 Unknown 798 724328O Unknown 799 7245352 Hexokinase I With Glucose And Adip In The Active Site, Mutant Monomer Of Recombinant Human 7329718 Unknown 743.0427 ionizing radiation resistance conferring protein - human 743.1153 malate dehydrogenase (EC 1.1.1.37), cytosolic - human 743.1833 NAD(P)+ transhydrogenase (B-specific) (EC 1.6.1.1) precursor, mitochondrial - human 804 7436377 plasma membrane Ca2+-ATPase variant 4a PMCA4a - human (fragment) 805 743.9346 protein-tyrosine-phosphatase 806 7441369 tubulin beta chain - human 807 74470.71 syntaxin 808 7447698 UDPglucuronosyltransferase (EC 2.4.1.-) 1A10 precursor - human 809 7452946 X-like 1 protein 810 7459551 Unknown 811 74878O1 Unknown 812 75 1895 Unknown 813 75 2435 filamin, muscle 814 75 2482 helicase II - human 815 75 2482 helicase II - human gi606833gbAAC50069.1(UO9820) helicase II 816 75 2513 Unknown 817 75 2598 Unknown 818 75 2628 Unknown 819 75 2754 Unknown 82O 75 2754 Unknown 821 75 2776 Unknown 822 75 2977 Unknown 823 75 3005 Unknown 824 75 3021 Unknown 825 75 3022 Unknown 826 75 3O76 Unknown 827 75 3172 N-chimerin homolog F25965 3 - human 828 75 3177 ND 14.1K NADH dehydrogenase (ubiquinone) (EC 1.6.5.3) 14.1K chain - human 829 75 31.78 ND acyl carrier NADH dehydrogenase (ubiquinone) (EC 1.6.5.3) acyl carrier chain, mitochondrial - human (fragment) 830 75 3274 probable thyroid receptor interactor - human (fragment) 831 75 3374 thrombospondin-p50 - human (fragment) 832 7S24346 adenylate kinase 2 isoform b: Adenylate kinase-2, mitochondrial 833 7527760 Unknown 834 7582306 ALEX3 protein 835 759.5299 opioid growth factor receptor 836 7643782 837 7656.959 calpain 7; calpain like protease; 838 7656999 catenin 839 7657039 death receptor 6 840 7657050 hypothetical protein, estradiol-induced 841 7657257 translocase of Outer mitochondrial membrane 20 (yeast) homolog 842 7657257 translocase of outer mitochondrial membrane 20homolog (TOM20) 843 7657343 metalloprotease 1 (pitrilysin family) 844 7657347 mitochondrial carrier homolog 2 845 7657347 mitochondrial carrier homolog 2 846 7657369 ND 19k NDUFA8 847 7657469 rat integral membrane glycoprotein POM121, similar to 848 765 7486 low molecular mass ubiquinone-binding protein 849 7657534 spastic ataxia of Charlevoix-Saguenay 850 7657554 soggy-1 gene; dickkopf-like 1 (soggy) 851 7657562 SH3-domain binding protein 4 852 7657581 solute carrier family 25, member 13 (citrin) 853 7657615 podocin 854 76616O2 DKFZP564B167 protein 855 76616O2 Unknown 856 7661678 RAS-related protein RAP1B: K-REV DKFZP586HO723 protein; 857 7661720 HIRA interacting protein 5; HIRIP5 protein; HIRA-interacting protein 5: HIRA-interacting protein 5 858 7661732 HSPC009 protein 859 7661732 Unknown 860 7661800 HSPC141 protein 861 7661872 leucyl-tRNA synthetase, mitochondrial US 2004/0101874 A1 May 27, 2004 38

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 862 7661872 leucyl-tRNA synthetase, mitochondrial; KIAA0028 protein 863 7661960 Rough Deal homolog, centromere?kinetochore protein; Rough Deal (Drosophila) homolog, centromere?kinetochore protein 864 7661996 Unknown 865 7662O42 Rho guanine nucleotide exchange factor 10 866 7662O46 Unknown 867 7662O92 Unknown 868 76621.68 Unknown 869 76621.90 Unknown 870 76621.90 Unknown 871 766228O histone deacetylase 7B isoform HDRP; histone deacetylase 7: MEF-2 interacting transcription repressor (MITR) protein; histone deacetylase 7B 872 7662284 Unknown 873 7662314 Unknown 874 7662452 Unknown 875 7662470 neuroligin. 1 876 766248O Unknown 877 7662639 PTD011 protein 878 7662645 mitochondrial ribosomal protein S18B; mitochondrial ribosomal protein S18-2; mitochondrial 28S ribosomal protein S18-2 879 7662673 translocase of Outer mitochondrial membrane 70 homolog A (yeast); translocase of Outer mitochondrial membrane 70 (yeast) homolog A; KIAAO719 gene product 88O 7662673 translocase of Outer mitochondrial membrane 7Ohomolog A 881 7669477 RNA-specific adenosine deaminase B1, isoform DRABA2b; RNA editase; human dsRNA adenosine deaminase DRADA2b 882 76694.92 glyceraldehyde-3-phosphate dehydrogenase 883 766952O neuregulin 1 isoform indfa3; heregulin, alpha (45 kD, ERBB2 p 185 activator); glial growth factor 884 7671629 KRAB box containing C2H2 type zinc finger protein 885 7671653 Unknown 886 767-7070 silent information regulator 2 homolog 887 7678804 mitochondrial isoleucine tRNA synthetase 888 77O5485 Unknown 889 77055O1 Unknown 890 770.5594 CGI-10 protein 891 77.05616 CGI-112 protein 892 77.05626 mitochondrial ribosomal protein S16 893 77.05626 mitochondrial ribosomal protein S16; 28S ribosomal protein S16, mitochondrial 894 77.05646 CGI-150 protein 895 77.05704 glutathione S-transferase subunit 13 homolog mitochondrial 896 77.05738 mitochondrial ribosomal protein S7; 30S ribosomal protein S7 homolog 897 77.05797 CGI-87 protein 898 770.5805 mitochondrial ribosomal protein S2 899 770.5805 mitochondrial ribosomal protein S2 900 77.05889 NEU1 protein 901 7705987 glycolipid transfer protein 902 77O6057 mitochondrial ribosomal protein L27 903 77O6073 GS15 904 77O6117 peptide transporter 3; likely ortholog of rat peptide/histidine transporter 2 905 77O6121 testicular haploid expressed gene 906 77O6146 hBOIT for potent brain type organic ion transporter 907 77O6154 908 77O6314 CGI-77 protein 909 77O6349 mitochondrial ribosomal protein S33 910 77O6449 fatty-acid-Coenzyme Aligase, long-chain 5; long-chain acyl-CoA synthetase 5; long-chain fatty acid coenzyme Aligase 5; FACL5 for fatty acid coenzyme Aligase 5 911 77O6481 MO25 protein 912 77O6549 CDC2-related protein kinase 7 913 7710129 LIM domain only 6 914 777O231 Unknown 915 7799988 large-conductance calcium-activated potassium channel beta 916 795.97O6 Unknown 917 79.59889 Unknown 918 795.9907 PRO2472 919 7981263 Unknown 92O 8051579 adenylate kinase 3: Adenylate kinase-3, mitochondrial; GTP: AMP phosphotransferase 921 8131894 mitofilin US 2004/0101874 A1 May 27, 2004 39

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 922 8216989 putative cell cycle control protein 923 8217423 bA108L7.7 (novel protein similar to C. elegans C25A1.13 (Tr: O02220)) 924 8394.499 ubiquitin associated protein 925 8488995 ND 20K NADH-ubiquinone oxidoreductase 20 kDa subunit, mitochondrial precursor (Complex I-20 KD) (CI-20 KD) (PSST subunit) 926 857.0444 Contains similarity to an unnamed protein from Homo Sapiens 927 8574O3O diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein))) d1013A10.3 (related to DBI ( 928 8574O70 NFKB1 929 86,71846 RNA adenosine deaminase gene, exon 15, Contains similarity to 930 891964.5 T-cell receptor beta chain 931 8922O81 Unknown 932 8922O81 Unknown 933 8922.275 Unknown 934 8922285 Unknown 935 8922307 Unknown 936 892242O neuropilin and tolloid like-2 937 892.2465 Unknown 938 8922511 mitochondrial ribosomal protein S18A 939 8922517 Unknown 940 8922.569 Unknown 941 892.2629 Unknown 942 892.2665 Unknown 943 89227O1 putative lipid kinase 944 8922742 Unknown 945 8922787 Unknown 946 892.2804 Unknown 947 892.2838 Unknown 948 8923OO1 Unknown 949 892.3221 Unknown 950 8923.291 Unknown 951 8923390 Unknown 952 8923390 Unknown 953 8923415 Unknown 954 8923417 Unknown 955 8923528 Unknown 956 892387O hOAT4 957 892393O uncharacterized hematopoietic stem/progenitor cells protein 958 892393O uncharacterized hematopoietic stem/progenitor cells protein MDSO 959 8927581 testes-specific heterogenous nuclear ribonucleoprotein G-T 96.O 8928O67 Malonyl-CoA decarboxylase, mitochondrial precursor (MCD) 961 9049352 3-methylcrotonyl-CoA carboxylase biotin-containing subunit 962 92.5661O protocadherin beta 15 precursor 963 92.57242 succinate dehydrogenase complex, subunit B, iron Sulfur (Ip); iron-sulfur subunit 964 9296943 Cyclin T2 965 9297078 UBIOUINOL-CYTOCHROME CREDUCTASE COMPLEX 7.2 KDA PROTEIN 966 9367862 Unknown 967 9438229 phospholipase C beta 1 968 95O1146 meiotic DNA transesterase/topoisomerase homolog isoform 2 969 9506437 FAPP1-associated protein 1 970 9506611 Unknown 971 9506611 Unknown 972 9506637 rab11-binding protein gi7023581dbiBAA92015.1 (AKOO1978) unnamed protein product, similar to 973 9506697 Unknown 974. 9506713 nucleolar protein family A, member 1; H/ACA small nucleolar RNPs protein 1 975 9506785 homeo box (H6 family) 1 976 9622528 NSAID-activated protein 1 NAG-1 977 988.4738 AP-2 beta transcription factor 978 99.10184 DC13 protein 979 991O244 mitochondrial ribosomal protein S22; gibt protein; chromosome 3 open reading frame 5; mitochondrial 28S ribosomal protein S22 98O 991O28O UDP-glucose ceramide glucosyltransferase-like 1 981 99.10382 mitochondrial import receptor Tom 22 982 99.10382 mitochondrial import receptor Tom 22 983 991.1130 protein phosphatase 984 993O803 A kinase (PRKA) anchor protein 7 985 995.5433 Unknown US 2004/0101874 A1 May 27, 2004 40

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 986 996.6799 disrupter of silencing 10 987 996.6893 CGI-203 protein 988 OO47106 Ca boxypeptidase A3 989 OO47118 G-protein gamma-12 subunit 990 OO4712O insulin receptor substrate 991 OO47167 KOW 992 OO47177 KOW 993 OO471.83 KOW 994 OO47187 KOW 995 OO471.99 KOW 996 OO47213 KOW 997 OO47231 KOW 998 OO47239 KOW 999 OO47243 KOW OOO OO47247 KOW OO1 OO47249 KOW OO2 OO47277 Sarcollemmal-associated protein OO3 OO47277 Un KOW OO4 OO47279 Un KOW O05 OO47281 Un KOW OO6 OO47283 Un KOW OO7 OO47317 L-periaxin O08 OO47329 Un KOW O09 OO47335 Zinc finger protein O10 OO47341 Un KOW O11 OO47341 Un KOW O12 OO47347 Un KOW O13 OO47361 Un KOW O14 OO926O4 HUG1 gene O15 OO92623 hematopoietic PBX-interacting protein gi9930 O16 OO92657 13 kDa differentiation-associated protein; NADH:ubiquinone oxidoreductase O17 ND B17.2 O18 L-3-Hydroxyacyl-Coa Dehydrogenase Complexed With Acetoacetyl-Coa And Nad+ O1795.99 ND NDUFS2 O17988O muscle-specific protein O1812O6 GABA(A) receptor-associated protein like 1 O190653 sphingosine-1-phosphate lyase 1 O190692 junctophilin 3; junctophilin type3 giQ886738 O241702 putative ZIC3 Binding protein from Xenopus laevis, similar to O2417O6 Un KOW O2574.09 natural resistance-associated macrophage protein 1 O25.7494 N-ethylmaleimide-sensitive factor O334442 hyd roxysteroid (17-beta) dehydrogenase 7 O334443 Un KOW O334466 Un KOW O337605 peroxisomal short-chain alcohol dehydrogenase 32782 testin 32971 Un KOW 33147 poly(A) polymerase gamma; SRP RNA 3’ adenylating enzyme?pap2 3332O huntingtin-associated protein 33905 KOW 33929 KOW 34O23 KOW 34055 KOW 34106 Fanconi anemia complementation group D2 protein 341.51 KOW 34167 KOW 341.83 KOW 34243 KOW 34293 KOW 34345 KOW 34521 KOW 34757 Un KOW 34.850 Zinc finger protein 226 34904 KOW 34988 KOW 35007 KOW 3524.4 KOW 35551 KOW US 2004/0101874 A1 May 27, 2004 41

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS O55 0435,767 Unknown O56 0435899 Unknown O57 0435947 Unknown O58 O436OO7 Unknown 059 0.436258 Unknown O60 O4362.63 Unknown O61 043.6325 Unknown O62 O436604 Unknown O63 O437144 Smac O64 O437144 Unknown O65 0437.178 mitochondrial ribosomal protein L1 O66 O437.189 Unknown O67 0437384 M-phase phosphoprotein 1 O68 O437960 Unknown O69 O437984 Unknown O70 O438291 Unknown O71 0438353 McKusick-Kaufman syndrome protein O72 0438441. Unknown O73 O4387O2 Unknown O74 0438857 Unknown O75 0438928 mitochondrial ribosomal protein S11 O76 O4.38968 Unknown O77 O439079 Unknown O78 O4392.44 Unknown O79 O439312 Unknown O8O O440252 bromodomain PHD finger transcription factor O81 O440347 Unknown O82 O440357 Unknown O83 O440367 Unknown O84 O440389 Unknown O85 O440402 Unknown O86 O440484 Unknown O87 O44-1879 Unknown O88 O44-19304 Unknown O89 0443472 Rhesus blood group-associated glycoprotein (RH5OA) O90 0503988 Unknown O91 0518340 muscleblind (Drosophila)-like O92 0567164 gene amplified in squamous cell carcinoma-1 O93 O639097 solute carrier family 24 (sodium/potassium/calcium exchanger), member 3) dJ122P22.1 ( O94 O645199 ADAM-TS disintegrin and metalloprotease with thrombospondin motifs-7 preproprotein; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 7 O95 O716563 calnexin O96 O719935 CELL DIVISION CYCLE 2-LIKE PROTEIN KINASE 5(CDC2-RELATED PROTEIN KINASE 5) O97 O72O29O SORTING NEXIN 1.4 O98 O720297 SYNAPTOJANIN 2 (SYNAPTIC INOSITOL-1,4,5-TRISPHOSPHATE 5 PHOSPHATASE 2) O99 O720409 Zinc finger protein 294 OO O764847 ND B18 O1 O798812 MLTK-alpha O2 O834587 fer-1 like protein 3 O3 O834762 PNAS-102 O4 O834.786 PNAS-117 05 O834968 mannosidase, alpha B, lysosomal O6 0835000 pancreatic lipase O7 0835.002 Rho GDP dissociation inhibitor (GDI) beta O8 0835023 inositol 1,4,5-triphosphate receptor, type 1 1O 0835045 retinaldehyde dehydrogenase 2 11 0835057 N-acetyltransferase, homolog of S. cerevisiae ARD1; N-acetyltransferase ARD1, human homolog of 12 0835059 farnesyltransferase, CAAXbox, beta 13 0835063 nucleophosmin (nucleolar phosphoprotein B23, numatrin) 14 O835087 ND 1 Ok 15 0835089 neurofilament, heavy polypeptide (200 kD); Neurofilament, heavy polypeptide 16 0835109 myotubularin related protein 3; FYVE (Fab1 YGLO23 Vsp27 EEA1 domain) dual-specificity protein phosphatase 17 0835155 tumor necrosis factor (cachectin) US 2004/0101874 A1 May 27, 2004 42

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 18 O8351.65 CD59 antigen p18-20 19 O835.173 nitric oxide synthase 1 2O O8351.89 glutathione reductase 21 O83522O ATPase, Ca++ transporting, fast twitch 1 22 O863907 associated protein; breast cancer 23 O863927 peptidylprolyl isomerase A 24 O8639.45 ATP-dependant DNA helicase II 25 O863985 G4 protein 26 O864O11 CGI-44 protein; sulfide dehydrogenase like (yeast) 27 O864O43 kidney and liver proline oxidase 1 28 O864O77 calcium channel, voltage-dependent, alpha 1H subunit 29 O945,428 membrane-associated guanylate kinase MAGI3 3O O2471O Unknown 31 O24714 ubiquitin B 32 O34.855 Unknown 33 O38674 CD79B antigen, isoform 1 precursor: B-cell-specific glycoprotein B29 34 O55998 guanine nucleotide binding protein beta subunit 4 Homo sapi 35 O56030 protocadherin gamma subfamily A, 2, isoform 1 precursor 36 O66958 mutant beta-globin 37 O66968 EH domain-containing protein FKSG7 38 095436 valosin-containing protein 39 O96171 RNA polymerase III transcription initiation factor B 40 121497 Trp4-associated protein TAP1, similar to 41 127695 SYTISSX4 fusion protein 42 128O19 cytochrome c 43 128O31 protocadherin gamma subfamily B, 5, isoform 1 precursor 44 139093 GrpE-like protein cochaperone 45 141885 carrier family 5 (choline transporter), member 7 46 141891 ERGL protein 47 177148 mitochondrial ribosomal protein L12 48 1771.48 mitoribosomal protein L12 49 225260 DNATOPOISOMERASE I 50 225266 transient receptor potential cation channel, subfamily M, member 5: MLSN1- and TRP-related; MLSN1 and TRP-related 51 245229 ninein-Lim isoform 52 252721 glutaryl-CoA dehydrogenase 53 252721 glutaryl-CoA dehydrogenase (EC 1.3.99.7) imported - human 54 267525 probable RNA helicase 55 27.5568 mucin 5B 56 275986 glycerol-3-phosphate dehydrogenase 3 57 276083 fatty-acid-Coenzyme Aligase, long-chain 2 58 276083 long-chain fatty-acid-Coenzyme Aligase 2; acyl-activating enzyme; acyl CoA synthetase; fatty acid thiokinase (long-chain); lignoceroyl-CoA synthase; long-chain acyl-CoA synthetase 2 59 276655 ribosomal protein S26 imported - human 60 276938 willin 2 61 277141 heat shock 90 kD protein beta 62 280538 Unknown 63 280677 Unknown 64 281511 Unknown 65 321341 MondoA 66 321569 olfactory receptor, family 3, subfamily A, member 2 67 321571 slit homolog 3 (Drosophila); slit (Drosophila) homolog 3; slit (Drosophila) homolog 2; slit2 68 321579 myosin, heavy polypeptide 13, skeletal muscle; extraocular muscle myosin heavy chain 69 321581 succinyl-CoA synthetase alpha subunit 70 321.583 succinate-CoA ligase, ADP-forming, beta subunit 71 321613 epilepsy, progressive myoclonus type 2, Lafora disease (laforin) 72 321615 T-box 3 protein; T-box 3; T-box transcription factor TBX3 73 32332O ubiquitin-conjugating enzyme E2 variant 1 (isoform 2, similar to variant 2 (UBE2V2, MMS2) 74 342.570 metalloproteinase 24 (membrane-inserted), matrix 75 342672 myosin, heavy polypeptide 3, skeletal muscle, embryonic 76 345448 lipopolysaccharide-binding protein 77 345.456 fibroblast growth factor receptor-like 1 precursor 78 345478 Unknown 79 345539 novel Helicase C-terminal domain 8O 359874 GTP-binding protein 2 81 359883 Unknown 82 3599.46 leucine zipper-EF-hand containing transmembrane protein 1 US 2004/0101874 A1 May 27, 2004 43

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

83 35.9985 Unknown 84 359986 Unknown 85 36OOO9 Bcl-Rambo 86 36OOO9 Unknown 87 36OO63 matrilin 2 precursor 88 36OO67 Unknown 89 36OO79 Unknown 90 36O112 Unknown 91 36O155 Unknown 92 36O155 Unknown 93 36O156 Unknown 94 36O162 Unknown 95 360185 Unknown 96 36O188 Unknown 97 360228 Unknown 98 36O2SO Unknown 99 36O251 Unknown 2OO 36O294 Unknown 2O1 36O310 myosin Vlla, long form - human 2O2 36O321 properdin 2O3 374664 isocitrate dehydrogenase (EC 1.1.1.42), cytosolic 204 385354 polybromo 1 205 385,644 CTCL tumor antigen se2-1 2O6 385,664 CTCL tumor antigen se89-1 2O7 386147 prosaposin 208 3994.66 D-2-hydroxy-acid dehydrogenase 209 4 15O24 diacylglycerol kinase, alpha (80 kD) 210 16393 mitochondrial ribosomal protein L22 211 16669 nicotinamide nucleotide transhydrogenase 212 17363 low molecular mass ubiquinone-binding protein 213 17363 low molecular mass ubiquinone-binding protein 214 18549 eyes absent (Drosophila) homolog 4 215 1871.4 Unknown 216 19832 phosphorylase kinase, alpha 1 217 21027 Unknown 218 22272 ribosomal protein S6 kinase, 90 kD 219 23142 basic leucine zipper nuclear factor 1 22O 2388O alpha-SNAP 221 24.404 mitochondrial ribosomal protein S23 222 24724 neurofilament 3 223 255.65 Unknown 224 25836 low density lipoprotein receptor-related protein 3 225 27613 Unknown 226 2.7636 GTPase Rab14 227 2823O aldehyde dehydrogenase 1 family, member B1 228 298O3 Unknown 229 3O299 hexokinase 1 230 31667 multiple inositol polyphosphate phosphatase 2 231 32O18 Unknown 232 32441 Unknown 233 32489 general transcription factor IIE, polypeptide 1 (alpha subunit, 56 kD) 234 33OO7 peroxisomal enoyl-coenzyme A hydratase-like protein 235 33596 tryptophanyl-tRNA synthetase 236 34O79 Unknown 237 34447 Unknown 238 34986 COO6 HUMAN PUTATIVE UBIOUINONE BIOSYNTHESIS MONOOXGENASE COO 239 35257 Unknown 240 35724. mannosidase, beta A, lysosomal 241 36135 RAS-RELATED PROTEINR-RAS2 242 36533 aldehyde dehydrogenase 2 family (mitochondrial) 243 36778 inositol polyphosphate-4-phosphatase, type II, 105 kD 244 37205 Unknown 245 4OOO3 transgelin 246 41230 skeletal muscle specific actinin, alpha 3 247 93459 PRO2619 248 93489 PRO262O 249 93,522 Unknown 250 93552 Unknown 251 96.882 ELK4 protein, isoform b: ETS-domain protein; SRF accessory protein 1 252 976O1 metallaproteinase-disintegrin US 2004/0101874 A1 May 27, 2004 44

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

253 5261.49 ATPase CF6 FO 254 526456 frataxin 255 526471 tripartite motif protein TRIM14 isoform alpha 256 526573 heat shock cognate protein 54 257 526789 inorganic pyrophosphatase 2 258 545761 potassium channel, subfamily K, member 12; tandem pore domain potassium channel THIK-2 259 545847 basic-helix-loop-helix-PAS protein 260 545863 methylcrotonoyl-Coenzyme A carboxylase 2 261 545869 popeye protein 2 262 545894 RFamide-related peptide precursor 263 559927 mitochondrial ribosomal protein S14 264 5964O2 MAGE-D4 265 596859 mitochondrial ribosomal protein L17 266 6O2741 complement component 8, alpha polypeptide 267 602963 heparan sulfate proteoglycan perlecan 268 611734 GREB1a 269 612659 FXYD domain-containing ion transport regulator 7 270 612670 phospholemman, isoform b precursor; FXYD domain-containing 271 640566 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolasefenoyl-Coenzyme A hydratase beta 272 640578 glyoxylate reductase/hydroxypyruvate reductase 273 641249 protein kinase Nimu-R1 274 641283 LIM homeobox protein 5 275 641413 cell division cycle 25B, isoform 3: CDC25B 276 76.1696 bHLHZip transcription factor BIGMAX gamma 277 863673 guanine nucleotide binding protein (G protein), alpha stimulating activity polypeptide 1) d309F20.1.5 (isoform 5 of 278 890755 RNA helicase II/Gu protein 279 907570 mutant desmin 28O 908171 Fas-binding protein Daxx 281 935053 sarcolemmal associated protein 1 282 968OO3 5-azacytidine induced gene 2, similar to 283 968152 somatostatin receptor-interacting protein 284 990879 phosphoglycerate kinase 2 285 991867 odorant receptor HOR3'beta5 286 2OO1946 My003 protein 287 2OO1986 My022 protein 288 2OO1992 brain myO25 289 2002O38 brain myO45 protein 290 2002042 brain myO48 protein 291 2OO22O1 serine/threonine protein kinase PFTAIRE-1 292 2OO3293 organic anion transporter 2 293 2005493 NPDO11 294 2005918 GRM19 295 2OO6049 EF1a-like protein 296 2006205 TNFIP-so 297 2038977 Unknown 298 2043738 thioredoxin reductase, mitochondrial 299 2O52810 Unknown 3OO 2O5282O COQ7 protein; timing protein; ubiquinone biosynthesis protein 301 2O52826 RAB-8b protein, small GTP-binding protein 3O2 2O52828 Unknown 303 2O52872 Unknown 304 2O52908 Unknown 305 2052971 methyltransferase COQ3 306 2O52989 Unknown 307 2O52991 Unknown 3O8 2053107 Unknown 309 2053245 Unknown 310 2053255 Unknown 311 2060822 serologically defined breast cancer antigen NY-BR-16 312 2O60832 serologically defined breast cancer antigen NY-BR-40 313 2O611.85 ASC-1 complex subunit P200 314 2O81909 semaphorin Y 315 2214171 putative small GTP-binding protein (rab1b) 316 2214288 dJ402H5.2 (novel protein similar to worm and fly proteins) 317 223OO15 CYTOCHROME B5 OUTERMITOCHONDRIAL MEMBRANE ISOFORM PRECURSOR 31.8 2230075 GLYCEROL KINASE, TESTIS SPECIFIC 1 319 2232373 rab6 GTPase activating protein (GAP and centrosome-associated) US 2004/0101874 A1 May 27, 2004 45

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 32O 2232421 tricarboxylate carrier protein 321 2232477 Unknown 322 2239360 LYST-interacting protein LIP6 323 2246901 tumor protein D52-like 2 324 2248755 mono ATP-binding cassette protein 325 2314005 Unknown 326 2314O16 transcription factor TFIIS, similar to 327 2314O29 proteasome subunit 7 328 2314O62 Unknown 329 231.4123 uncharacterized hematopoietic stem/progenitor cells protein MDSO30 (8923932) 330 2314190 dJ445H2.2 (novel protein) 331 2314195 Unknown 332 23284.45 NPAS3 333 2382773 caspase recruitment domain protein 11 334 2382789 OSBP-related protein 7: ORP7 335 2383,092 Unknown 336 24O7403 tripartite motif protein TRIM9 isoform alpha 337 24O8656 calpain. 1, large subunit 338 2597655 kinetochore protein 339 262O194 Unknown 340 262O246 CD36 341 262O2S2 CD36 342 262O871 phosphoinositide-3-kinase gamma catalytic subunit 343 26.21903 cathepsin S 344 264 3.256 pilin-like transcription factor 345 264 3326 CIP1-INTERACTING ZINC FINGER PROTEIN (NUCLEAR PROTEIN NP94) 346 264 3329 CGI-51 347 264 3417 Pyruvate dehydrogenase protein X component, mitochondrial precursor (Dihydrolipoamide dehydrogenase-binding protein of pyruvate dehydrogenase complex) (E3-binding protein) (E3BP) (proX) 348 264 3637 ADAM-TS 4 PRECURSOR (A DISINTEGRIN AND METALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS 4) 349 264 3716 PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 13 350 264 3796 RETINOBLASTOMA-BINDING PROTEIN 8 351 264 3896 Zinc finger protein 236 352 264 4.018 AF-6 PROTEIN 353 264 4O90 T-BOXTRANSCRIPTION FACTORTBX18 354 264 4310 COATOMER BETA SUBUNIT(BETA-COP) 355 264 437O Zinc finger X-linked protein ZXDB 356 2652715 nucleolar GTPase 357 2652761 Unknown 358 2652763 Unknown 359 2652773 Unknown 360 2652981 glycogen synthase kinase 3 beta 361 2652989 Unknown 362 2653017 LRP16 protein 363 2653371 phosphoglycerate mutase 1 364 2653507 aspartate transaminase 2 365 2653549 mitochondrial ribosomal protein S6 366 2653687 Unknown 367 265.3775 helicase-like protein NHL 368 2653827 mitochondrial carrier homolog 1 or presenilin-associated protein 369 2653855 dynamitin 370 2654O77 NICE-5 protein 371 2654149 Unknown 372 2654285 peptide N-glycanase homolog 373 2654289 transcription termination factor, mitochondrial 374 2654333 HCDI protein 375 2654.407 N-Acetylglucosamine kinase 376 2654521 Unknown 377 2654627 metalloprotease 1 378 2654.675 transcobalamin II; macrocytic anemia 379 26551.33 CGI-63 protein, similar to 38O 2655157 centrosomal protein 2 381 2655195 heat shock 75 protein 382 2656979 antigen, T-cell receptor 383 2657106 Unknown 384 2659007 protein kinase D2 385 2669909 long-chain fatty-acid-Coenzyme Aligase 4, isoform 2; long-chain acyl US 2004/0101874 A1 May 27, 2004 46

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS CoA synthetase 4: acyl-activating enzyme 386 2697.31 2 putative chromatin modulator 387 2697482 novel zinc finger protein similar to rat RIN ZF) 388 2697776 polyadenylation specificity factor 389 2697899 Unknown 390 2697903 Unknown 391 2697947 Unknown 392 269795 1. Unknown 393 269795 7 Unknown 394 2697983 Unknown 395 269799 1. Unknown 396 2697995 Unknown 397 2698O37 Unknown 398 2698043 Unknown 399 2698OS likley ortholog of rat CPG2 protein OO 269806 Unknown O1 2698O75 Unknown O2 2700223 recombination activating protein 1 O3 2707570 enoyl Coenzyme A hydratase, short chain, 1, mitochondrial O4 271166O protein kinase, lysine deficient 1 05 2711664 Unknown O6 2711674 yeast Upf3, variant B, similar to O7 272581 3 poly(ADP-ribosyl)transferase O8 272965 2 cell adhesion molecule with homology to L1CAM (close homologue of L1) O9 2733033 caldesmon 1 or) NAG22 protein 1O 273309 1. replication initiation region protein (60 kD) 11 2734392 annexin A13 12 273481 6 PRP4/STK/WD splicing factor 13 273521 7 surfeit 5 14 273.5226 adenylate kinase 3 alpha 15 273.543 O PKCa-interacting protein PICOT 16 273.8042 klotho 17 2738974 Unknown 18 27408O8 A kinase anchor protein 10 19 27412O2 UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase 2O 274186 6 protein expressed in thyroid, similar to 21 2742OO8 chondroitin sulfate proteoglycan 3 22 27424 5 complement component C1q receptor 23 27511 PNAS-140 24 27511 PNAS-141 25 2751.45 PDZ domain-containing protein AIPC 26 28O324 Unknown 27 28O328 VDAC-3 28 28O334 transcription factor 19, similar to 29 28O338 antiquitin 1 3O 28O356 transgelin 2 31 28O384 protein kinase, cAMP-dependent, regulatory, type II, alpha, similar to 32 28O385 metastasis suppressor protein 33 28O39 glucosidase I, similar to 34 280.404 nuclear protein E3-3 orf1 35 2804O6 FK506-binding protein 4 (59 kD), similar to 36 280418 colon cancer-associated protein Mic1, similar to 37 280422 Unknown 38 280.43 expressed sequence 2 embryonic lethal, similar to 39 280.43 carbonyl reductase 40 28O466 Unknown 41 280474 3 Unknown 42 280475 5 NPD002 protein, similar to 43 2804821 Unknown 44 2804897 branched chain aminotransferase 2, mitochondrial, similar to 45 280,490 1. isocitrate dehydrogenase 3 gamma 46 280SO21 acyl-Coenzyme A dehydrogenase family, member 8 47 1. roundabouth 48 7 serine/threonine kinase 33 49 286232O WDC146 50 296.335 3 fenestrated-endothelial linked structure protein 51 3027604 mitochondrial ribosomal protein S34 52 3027608 Unknown 53 3027640 lysine-ketoglutarate reductase/saccharopine dehydrogenase 54 309505 4 ovarian immunoreactive antigen 55 3096727 Smac Bound To Xiap-Bir3 Domain US 2004/0101874 A1 May 27, 2004 47

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 56 3096755 Ras G12v - Pi 3-Kinase Gamma Complex 57 30971.56 ND 39 k 58 3O97243 Unknown 59 3097693 Unknown 60 11705 Carnitine O-acetyltransferase (Carnitine acetylase) (CAT) 61 11762 solute carrier family 19 (folate transporter), member 1, similar to 62 12O23 coenzyme Q, 7homolog 63 23976 ARGININE-TRNA-PROTEIN TRANSFERASE 1 64 24237 F-box only protein 10 65 24,883 HsKin17 protein 66 2.8992 Unknown 67 28998 Unknown 68 29014 Unknown 69 2908O Unknown 70 29092 Unknown 71 2.9144 Unknown 72 61081 testis protein 73 77634 surfactant protein B-binding protein 74 77648 EGF factor 8 protein 75 777OO Unknown 76 84052 butyrophilin, subfamily 2, member A3 77 941.97 kinesin family member 13B; guanylate kinase associated kinesin 78 3194522 PMF-1 binding protein 79 3236495 quinone oxidoreductase; NADPH 8O 3236559 Unknown 81 3242069 nuclear transcription factor NFX2 82 3242172 potassium voltage-gated channel, Shab-related subfamily, member 2 83 3242739 myelin P2 protein 84 324.9985 Lowe oculocerebrorenal syndrome protein 85 325.9127 cullin CULAB 86 3259497 retinoblastoma-binding protein 1, isoform I 87 3272567 ND 5 88 3272568 ND 6 89 3272595 ND 5 NADH dehydrogenase subunit 5 90 3272697 ND 1 NADH dehydrogenase subunit 1 91 3272855 ATPase 8 92 32.73190 cox 2 93 3274124 Unknown 94 3276227 chromogramin B(isoform 2) 95 3276598 Unknown 96 3.276617 Unknown 97 3278690 Unknown 98 3.32471O interleukin 3 receptor, alpha (low affinity); Interleukin-3 499 3325066 cadherin EGF LAG seven-pass G-type receptor 3: EGF-like-domain 500 3.325.162 Unknown 5O1 3325394 phosphatidylserine synthase 1, similar to 502 33592O1 Unknown 503 3375614 peroxisomal long-chain acyl-coA thioesterase SO4 3375634 human immunodeficiency virus type I enhancer-binding protein 505 3375744 Unknown SO6 3375809 Unknown 507 3375817 Unknown 508 3375838 Unknown 509 3375872 Unknown 510 3375932 Unknown 511 33.75940 Unknown 512 33.75942 Unknown 513 3376007 Unknown 514 3376O11 engulfment and cell motility 3; ced-12 homolog 3 515 3376021 Unknown 516 3376038 Unknown 517 3376052 Unknown 518 3376093 Unknown 519 3376107 Unknown 52O 3376144 Unknown 521 3376,284 Unknown 522 3376331 Unknown 523 3376437 Unknown 524 3376445 Unknown 525 33764.90 Unknown 526 337658O Unknown US 2004/0101874 A1 May 27, 2004 48

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

527 33766.17 Unknown 528 3376640 putative N-acetyltransferase 529 3.376.662 Unknown 530 3376717 Unknown 531 3376741 Unknown 532 3376747 Unknown 533 3376749 Unknown 534 3376776 Unknown 535 3376812 type 1 protein phosphatase inhibitor 536 3376826 UL16-binding protein 1 537 3376854 UBX domain-containing 1; UBX domain-containing 2 538 3376991 voltage-dependent calcium channel beta 2 subunit 539 3386494 Unknown 540 3399777 Macrophage Migration Inhibitory Factor (Mif) Complexed With Inhibitor. 541 34 31759 PARAPLEGIN 542 34 31763 Pre-mRNA cleavage complex II protein Pcf11 543 34 351.31 WW domain-containing binding protein 4 544 34 35350 ferredoxin reductase isoform 1 545 34 3608O cleft lip and palate associated transmembrane protein 1 546 34 36.188 mitochondrial ribosomal protein S2 547 34 361.97 Unknown 548 34 36275 LON PROTEASE HOMOLOG, MITOCHONDRIAL PRECURSOR 549 34 36.296 Unknown 550 34 363O8 Unknown 551 34 36,335 IF-1 ATPase inhibitor precursor 552. 34 36.395 Unknown 553 34 36413 glucose phosphate isomerase 554. 34 45577 EDAG 555 34 49263 Unknown 556 34 49269 Unknown 557 34 69731 breast cancer antigen NY-BR-1.1 558 34 7OO94 apolipoprotein L, 5 559 34 77253 Unknown 560 34 87904 Unknown 561 34 89087 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; protease inhibitor 2 (anti-elastase) 562 34 89095 sialoadhesin precursor; sialic acid-binding immunoglobulin-like lectin 1 563 34 91972 liver nuclear protein 564 3507059 ubiquitin protein ligase 565 3509322 suppression of tumorigenicity 5 566 3514831 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10, ATP-dependent RNA helicase 567 3516379 aldehyde Oxidase 1 568 3518228 methylcrotonoyl-Coenzyme A carboxylase 569 3528660 ribosomal protein L4, similar to 570 3528960 ND 18k 571 3529047 transforming growth factor, alpha 572 3529221 PTD017 protein 573 3529257 aldo-keto reductase family 1, member B1 574. 3537192 SCCA1b 575 354.0475 serum amyloid A2 576 354.0477 wingless-type MMTV integration site family, member 3 precursor 57.7 354,0574 Unknown 578 354,0576 Unknown 579 3540590 C/EBP-induced protein 58O 354O606 suppressor of potassium transport defect 3 g 581 3543342 Unknown 582 35434.46 Unknown 583 3543618 ATPase B FO 584 35437O6 Unknown 585 3543933 Unknown 586 3544OO7 Unknown 587 3544O72 glycerol-3-phosphate dehydrogenase 1 (soluble), similarity to 588 3559241 Unknown 589 3559363 mitochondrial ribosomal protein L9 590 35594.04 mitochondrial ribosomal protein L43 591 356O110 Unknown 592 3569848 cell cycle progression 2 protein 593 35699.13 exonuclease NEF-sp 594 3569930 toll-like receptor 10 595 3569948 Unknown US 2004/0101874 A1 May 27, 2004 49

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 596 3569962 small GTP-binding protein 597 3591536 Unknown 598 3606056 DNA dependent protein kinase catalytic subunit 599 362O885 mitochondrial ribosomal protein S6 6OO 36.23251 transcription factor EB, similar to 6O1 3623369 Unknown 6O2 362.3465 peroxisomal long-chain acyl-coA thioesterase 603 36.23483 lysosomal-associated membrane protein 1 604 362.3595 DNA segment on chromosome 191177 expressed sequence 605 3623615 Unknown 606 3623617 Unknown 6O7 3623635 Unknown 608 3623689 Unknown 609 3623693 Unknown 610 3626.125 ADAM-TS-9 precursor (A disintegrin and metalloproteinase with thrombospondin motifs 9) (ADAM-TS9) (ADAM-TS9) 611 36,27233 aldo-keto reductase family 7, member A3 612 36.27252 Oxoglutarate dehydrogenase 613 36.27389 elongation factor-2 kinase 614 36.27804 acyl-Coenzyme A dehydrogenase, short?branched chain precursor 615 3628.614 Na, K-ATPase subunit alpha 2 616 36.28881 Unknown 617 36.29150 coX 4 618 363O128 faciogenital dysplasia protein 619 363O492 Unknown 62O 3630567 Unknown 621 363O862 aldehyde dehydrogenase 5 family, member A1 622 363O871 Unknown 623 363O873 protein kinase, cAMP-dependent, regulatory, type II, beta 624 3631242 reelin 625 3631440 PEROXREDOXIN 2 626 3631521 mitochondrial ribosomal protein S7 627 3631678 UCR 5 628 3631907 mitogen-activated protein kinase kinase kinase kinase 1 629 3632179 myosin, heavy polypeptide 13, skeletal muscle 630 3632266 thyroid hormone receptor interactor 2; PPARG binding protein 631 3632616 carrier ANT2 632 36.32896 phosphoglucomutase 1 633 36331.68 plastin 3 precursor 634 3633370 Notchhomolog 3 635 3635754 CTCL tumor antigen se1-1 636 3635919 Unknown (now 4507953) 637 3636O42 Unknown 638 3636O47 3-hydroxyisobutyryl-Coenzyme A hydrolase 639 3636157 Unknown 640 3636168 eukaryotic translation elongation factor 1 beta 2 641 3636504 interferon-induced protein 75, 52 kD 642 3636598 Unknown 643 36,37083 Unknown 644 3637529 Unknown 645 363.7537 ETAA16 protein 646 3637608 ND 75 K 647 3637631 VDAC-2 voltage-dependent anion channel 2 (H. Sapiens), similar to 648 3637711 glycine cleavage system protein H (aminomethyl carrier) (H. Sapiens), similar to 649 3637735 Unknown 650 3637796 Unknown 651 3637833 cox 7a like, COX7RP (cytochrome c oxidase subunit VII-related protein), estrogen receptor binding CpG island 652 3637948 glutathione S-transferase M5 653 3638591 Unknown 654 3638758 Unknown 655 3639105 Unknown 656 3639114 succinate dehydrogenase, Ip 657 36391.87 Unknown 658 3639470 Unknown 659 36396.28 acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme A thiolase), mitochondrial 660 3639817 malic enzyme 3, NADP(+)-dependent, mitochondrial 661 364O712 phosphoinositide-3-kinase, class 2, alpha polypeptide 662 364O950 interleukin 11 receptor, alpha US 2004/0101874 A1 May 27, 2004 50

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

663 3641918 sirtuin 3 664 3643.253 kinesin family member 3A 665 3643321 Unknown 666 3643514 Unknown 667 3643534 ribosomal protein L12; 60S ribosomal protein L12 (H. Sapiens), similar to 668 3643564 eXOstoses 1 669 3643652 flavohemoprotein b5 + b5R 670 3643704 protein tyrosine phosphatase, receptor type 671 3644108 proteasome 26S subunit, non-ATPase, 1 672 3644418 Unknown 673 3644786 butyrophilin, subfamily 1, member A1 674 3645381 HLA-B associated transcript 2 (H. Sapiens), similar to 675 3645.492 heat shock 70 kD protein-like 1 676 3645618 dihydropyrimidinase related protein-3 677 36.46385 creatine kinase, sarcomeric mitochondrial 678 3646,774 quinoid dihydropteridine reductase 679 3647276 L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain 68O 3647558 carrier ANT1 681 364,792O gamma-glutamyltransferase 1 682 364,7960 tumor necrosis factor, alpha-induced protein 2 683 3648234 Unknown 684 3648426 cox assembly protein isoform 2 685 3648611 serine/threonine kinase 2 686 3648964 alanyl-tRNA synthetase 687 364901O odzhomolog 1 688 364.9058 Unknown 689 3649119 SEX gene 690 364.9217 VDAC-1 691 36494.75 Unknown 692 3649658 UCR ubiquinol-cytochrome c reductase binding protein 693 3650446 heat shock 70 kD protein 2 694 3650574 glutamate dehydrogenase 2 mitochondrial precursor 695 3650639 melanoma antigen, family B, 1 696 3650785 spectrin, beta, non-erythrocytic 5 697 3650793 elongation factor SIII p15 subunit 698 3650874 putative receptor protein 699 3650942 Unknown 700 3650992 Unknown 701 3651O38 leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4 702 3651229 Rho GTPase activating protein 6 isoform 4 703 3651413 Fc fragment of IgG binding protein (H. Sapiens), similar to 704 3651526 androgen-induced prostate proliferative shutoff associated protein 705 3651706 golgin-like protein 7O6 3651985 type 1 RNA helicase pNORF1 or nonsense-mediated mRNA decay trans-acting factor 707 3652204 Unknown 708 3652240 ribosomal protein S7 709 3652246 ARF protein 710 3652324 ras-related small GTPasehypothetical protein X 711 36528O1 Rap1 guanine-nucleotide-exchange factor directly activated by cA 712 3653O49 acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor 713 3653910 carboxypeptidase D precursor 71.4 3654274 Unknown 715 3654278 Unknown 716 3654294 Unknown 717 3654678 Unknown 718 3654685 ATP-binding cassette, sub-family C, member 1, isoform 6 719 36551.45 UCR ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide like 1 720 3655148 EH-domain containing 2: EH domain containing 2, similar to 721 3655297 Unknown 722 3676336 Unknown 723 3676857 heat shock 70 kD protein 2: Heat-shock 70 kD protein-2 724 3699811 WHSC1L1 protein isoform long; Wolf-Hirschhorn syndrome candidate 1 like 1 protein 725 3751974 Unknown 726 3774961 autoimmune infertility-related protein 727 3775.158 Unknown 728 3775166 Unknown 729 37751.86 ring finger protein 17 isoform long US 2004/0101874 A1 May 27, 2004 51

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

730 37752O8 Unknown 731 3775210 Unknown 732 3775216 Unknown 733 3775232 Unknown 734 378.4938 Unknown 735 3786129 RAS-RELATED PROTEINRAB-33B 736 3786847 L-Lactate Dehydrogenase H Chain, Ternary Complex With Nadh And Oxamate 737 37871.97 DEAD/Hbox polypeptide 11 738 3.787215 sirtuin 5, isoform 2 739 3.787217 FAT tumor suppressor 2 precursor; multiple epidermal growth factor-like domains 1; cadherin family member 8 740 3794.267 RAB7, member RAS oncogene family; Ras-associated protein RAB 741 3872241 ligand of numb-protein X 742 3874.437 cerebral protein-11 743 3876386 epiplakin 1 744 3.8992.31 mitochondrial ribosomal protein L9 745 3.899275 Unknown 746 392.9460 PTH-responsive Osteosarcoma B1 protein 747 39294.67 chemokine binding protein 2 748 39374O1 Unknown 749 3937769 RIKEN cDNA 1200013F24 gene, similar to 750 3937888 heterogeneous nuclear ribonucleoprotein C 751. 393817O Unknown 752 3938215 taxol resistant associated protein 753 3938297 heat shock cognate 71-kd protein, similar to 754. 3938.442 neuronal protein, mitochondrial Complex I subunit 755 39.38539 cyclin D binding Myb-like transcription factor 1 756 39385.71 Unknown 757 3938593 Unknown 758 3938619 creatine kinase, muscle 759 3994164 Charcot-Marie-Tooth duplicated region transcript 1 760 3994.188 AKAP-associated sperm protein 761 399.4259 mitochondrial ribosomal protein S5 762 399428O complement-c1q tumor necrosis factor-related protein 7 + F792 763 3994325 putative b,b-carotene-9', 10'-dioxygenase 764 4 O17783 Unknown 765 4 O17783 Unknown 766 4 O17807 Unknown 767 4 O17833 Unknown 768 4 O17865 Unknown 769 4 O17899 Unknown 770 4 O17903 Unknown 771 4 O17903 Unknown 772 4 O17923 Unknown 773 4 O17941 Unknown 774 4 O17943 Unknown 775 4 O17949 Unknown 776 4 O17957 Unknown 777 4 O17971 Unknown 778 4 O283.89 mitochondrial ribosomal protein L41 779 4 O2.8403 mitochondrial ribosomal protein S28 78O 4 O284.05 mitochondrial ribosomal protein S29 781 4 O28875 UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter; KIAA0260 protein; UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter 782 4 O28877 mitochondrial ribosomal protein S25; mitochondrial 28S ribosomal protein S25 783 O41699 ESTRADIOL. 17 BETA-DEHYDROGENASE 8 784 O41874 MAPKK like protein kinase/PDZ-binding kinase 785 O41889 Unknown 786 O41976 Unknown 787 O41978 CDAO2 protein 788 O41989 Unknown 789 O42O18 Unknown 790 O42O66 Unknown 791 O42110 Unknown 792 O42216 Unknown 793 O42323 Unknown 794 O42336 Unknown 795 O42441 Unknown US 2004/0101874 A1 May 27, 2004 52

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

796 4 O42814 Unknown 797 4 O42822 Unknown 798 4 O428SO Unknown 799 4 O42923 chromosome 9 open reading frame 5 8OO 4 O431.87 aldehyde dehydrogenase 4 A1 8O1 4 O43217 plectin 1, intermediate filament bindi 8O2 4 043281 leucine-rich neuronal protein 803 4 O43412 Unknown 804 4 O43451 succinyl-CoA synthetase beta subunit GTP-specific 805 4 043654 phosphofructokinase, muscle, similar to 806 4 O436.66 Unknown 807 4 O43738 Unknown 808 4 24.942 ribophorin I, similar to 809 4 24976 kinesin family member C3 810 4 3.3213 Unknown 811 4 3.3215 Unknown 812 4 3.3217 Unknown 813 4 3.3235 Unknown 814 4 41157 heterogeneous nuclear ribonucleoprotein H3, isoform a 815 4 496O7 chloride channel 7: CIC-7 816 4 496.25 817 4 496.49 siah binding protein 1; FBP interacting repressor; pyrimidine tract binding splicing factor; Ro ribonucleoprotein-binding protein 1 81.8 4.9677 lectomedin-3 819 49686 Unknown 82O 49690 Unknown 821 49769 GAJ protein 822 49789 Unknown 823 49904 tumor endothelial marker 8, isoform 1 precursor; anthrax toxin receptor 824 49971 Unknown 825 SOOO1 Unknown 826 5OO17 Unknown 827 50O39 Unknown 828 SOO62 Unknown 829 5OO72 Unknown 830 5OO72 Unknown 831 SOO8O Unknown 832 SO116 Unknown 833 5O128 phosphodiesterase 5A 834 SO134 Unknown 835 5O155 Unknown 836 65260 Unknown 837 65270 mitochondrial ribosomal protein L13 838 929.43 MEGF10 protein 839 94461 A kinase anchor protein 9 840 96.457 protocadherin gamma subfamily A, 12, isoform 2 precursor; cadherin 21; fibroblast cadherin FIB3 841 96465 protocadherin gamma subfamily A, 3, isoform 2 precursor 842 98.176 ND 51 k 843 98272 Bcl-XL-binding protein v68, similar to 844 983O3 Unknown 845 1536 neurexin 2; neurexin II 846 1570 conserved ERA-like GTPase 847 1720 desmuslin 848 1857 Unknown 849 1903 ubiquitin specific protease 850 1907 Zinc finger protein 347; zinc finger 1111 851 1923 PKCI-1-related HIT protein 852 1939 methylmalonyl-CoA epimerase 853 248761 cAMP-specific cyclic nucleotide phosphodiesterase 854 24.9144 RAB11B, member RAS oncogene family 855 249338 Unknown 856 2493.42 internexin neuronal intermediate filament protein, alpha; neurofilament 5 (66 kD); neurofilament-66, tax-binding protein 857 2493.76 Unknown 858 24.9428 Unknown 859 249446 Unknown 860 249.454 Unknown 861 249474 Unknown 862 2495O6 Unknown 863 249588 lactamase, beta US 2004/0101874 A1 May 27, 2004 53

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

864 4 249596 Unknown 865 4 24962O Unknown 866 4 249967 staufenhomolog 2 867 4 2SOO63 peroxiredoxin 3 868 4 25O110 Unknown 869 4 2SO319 Unknown 870 4 2SO458 stromal cell derived factor 5, similar to 871 4 250628 Unknown 872 4 2SO744 Unknown 873 4 25.1209 chloride intracellular channel 1 874 4 269578 metallothionein IV 875 4 277739 Erythrocyte Band-3 Protein, Crystal Structure Of The Cytoplasmic Domain Of Human 876 28OOSO Vps39/Vam6-like protein 877 285.174. elongation factor G 878 286.186 ZINC FINGER PROTEIN 185(P1-A) g 879 286.294 Unknown 88O 289.323 LIP isoform of BLIP 881 318622 Unknown 882 3.29511 bA43OM15.1 (novel protein (ortholog of rat four repeation channel)) 883 3.29531 Unknown 884 336727 Unknown 885 336768 Unknown 886 336775 ND PDSW 887 349,362 major histocompatibility complex, class I, F 888 424O13 WNT-5B protein precursor 889 424776 Unknown 890 485049 T-cell receptor V delta 1 891 48868O Phosphoglucose IsomeraseNEUROLEUKINAUTOCRINE MOTILITY FACTORMATURATION Factor 892 53.0763 citrate lyase, similar to 893 5491.63 Matrilin-2 precursor 894 571713 tonicity-responsive enhancer binding protein 895 575679 hemicentin 896 6O2477 DNA-BINDING PROTEINA 897 6O2507 Unknown 898 6O2841 cysteine string protein 1 899 602856 Unknown 900 6O2907 Unknown 901 602977 Unknown 902 6O3O84 putative DNA binding protein 903 603309 heat shock 60 kD MITOCHONDRIAL 904 603403 stomatin-like 2 905 67.0360 zinc finger protein 278, long C isoform; POZ-AT hook-zinc finger protein 906 714447 sorting nexin 7 907 714514 DIHYDROLIPOAMIDE DEHYDROGENASE-BINDING PROTEIN OF PYRUVATE DEHYDROGENASE COMPLEX 908 714528 Unknown 909 715.007 Unknown 910 719392 cofilin 2 911 72O172 Unknown 912 72O558 succinate dehydrogenase, flavoprotein subunit 913 721241 low density lipoprotein-related protein-associated protein 1 914 721350 testicular protein kinase 2 915 721365 hypothetical protein, estradiol-induced 916 721507 serine/threonine kinase 18 917 721966 Unknown 918 722003 cadherin 12, type 2 919 722193 3-hydroxybutyrate dehydrogenase 92O 722.283 Unknown 921 722554 Unknown 922 722589 mitochondrial ribosomal protein L22 923 722898 mitochondrial ribosomal protein S27 924 7231.45 acid phosphatase 1 isoform b 925 7234O7 Unknown 926 723451 mitochondrial ribosomal protein L20 927 723531 p25 928 724O42 ASB-3 protein 929 7242O6 Unknown 930 724379 Unknown 931 724557 phosphatidylinositol glycan, class K US 2004/0101874 A1 May 27, 2004 54

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

932 4 724575 Unknown 933 4 724751 phosphorylase, glycogen; brain 934 4 724.805 Unknown 935 4 725181. lymphocyte antigen 75 936 4 725399 TNF-induced protein 937 4 72542O syntaxin 12 938 4 7255.45 RNA-binding protein regulatory subunit 939 4 725791 Unknown 940 4 725848 acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain 941 4 726372 Unknown 942 4 726632 Unknown 943 4 726693 Unknown 944 4 726725 Unknown 945 4 726866 Unknown 946 4 7271 74 leucine-rich PPR-motif containing 947 4 727486 succinate dehydrogenase, subunit D 948 4 727827 Unknown 949 4 728O81 excision repair cross-complementing rodent repair deficiency 950 4 728229 phosphoinositide-3-kinase, regulatory subunit 4, p150 951 4 728316 natural killer cell receptor 2B4 952 4 728439 Unknown 953 4 728817 Unknown 954 4 728839 Unknown 955 4 728858 sterol carrier protein 2 956 4 728945 DMRT-like family B with proline-rich C-terminal, 1 957 4 729172 elastin microfibril interface located protein 958 4 7294.75 959 4 7294.87 mast cell carboxypeptidase A3 precursor 96.O 4 7297.83 dihydrolipoamide branched chain transacylase 961 4 73O158 TATA element modulatory factor 1 962 4 73O499 Unknown 963 4 730569 adenylate cyclase 3 964 4 7306OO Unknown 965 4 730775 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolasefenoyl-Coenzyme A hydratase alpha 966 730782 kinesin heavy chain member 2 967 732O14 Unknown 968 73248 calcium channel, voltage-dependent, alpha 1E subunit 969 732525 selective LIM binding factor, rat homolog 970 73272 adenomatosis polyposis coli 971 732789 mitofilin 972 732886 thyroid hormone receptor-associated protein, 150 kDa subunit 973 7331.83 adaptor-related protein complex 2, mu 1 subunit 974. 73345 enkephalinase 975 Unknown 976 myofibrillogenesis regulator MR-1 977 chondroitin sulfate proteoglycan 2 978 serine/threonine kinase 16 979 Unknown 98O lymphoid enhancer binding factor-1 981 Unknown 982 Unknown 983 Unknown 984 DEAD/Hbox polypeptide 1 985 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a-like 1 986 mitochondrial isoleucine tRNA synthetase 987 Ste-20 related kinase 988 989 Unknown 990 nuclear factor, interleukin 3 regulated 991 Unknown 992 Unknown 993 735899 cytochrome b5 reductase 1 994 736223 UCR 1 995 736227 Rho-associated, coiled-coil containing protein kinase 2 996 736267 protein disulfide isomerase-related protein 997 736397 Unknown 998 736.560 Unknown 999 736612 Unknown 2OOO 736678 lactotransferrin US 2004/0101874 A1 May 27, 2004 55

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

2001 4 736760 voltage-dependent anion channel 2 2002 4 736866 DnaJhomolog, subfamily B, member 12 2003 4 737445 sema domain, immunoglobulin domain (1g), short basic domain, 2004 4 737746 myeloid differentiation primary response gene 2005 4 737907 Unknown 2006 4 738004 Unknown 2007 4 738099 Apobec-1 complementation factor; APOBEC-1 stimulating protein 2008 4 738103 annexin IV 2009 4 7383.06 putative, similar to 2010 4 738689 Unknown 2011 4 73895.0 Unknown 2012 4 739002 Unknown 2013 4 739106 Unknown 2014 4 739392 Unknown 2015 4 739472 potassium voltage-gated channel, shaker-related subfamily 2O16 4 73988O Unknown 2017 4 74 HEAT SHOCK 27 KDA PROTEIN (HSP 27) (STRESS-RESPONSIVE PROTEIN 27) (SRP27) (ESTROGEN-REGULATED 24 KDA PROTEIN) (28 KDA HEAT SHOCK PROTEIN), similar to 74 A kinase anchor protein 2 74 thioredoxin 74 TAF2 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 150 kD 2021 FUMARATE HYDRATASE, MITOCHONDRIAL PRECURSOR (FUMARASE) 2O22 74 O792 v-ral simian leukemia viral oncogene homolog A (ras related) 2O23 74 O886 Unknown 2O24 74 1177 Unknown 2025 1234 Unknown 2O26 74 1376 Fas-activated serine/threonine kinase, isoform 2 2027 74 1510 Unknown 2O28 74 1555 Unknown 2O29 74 1636 Unknown 2O3O 1782 uncharacterized hematopoietic stem/progenitor cells protein MDSO 2O31 74 2266 RNA helicase 2032 74 2273 Unknown 2O33 2317 Unknown 2O34 74 26OO vimentin 2035 74 2688 diphthamide biosynthesis-like protein 2 2O36 74 2977 inter-alphainhibitor, H2 polypeptide 2037 74 3O31 Unknown 2O38 3873 TAR (HIV) RNA binding protein 1 2O39 74 4O78 gamma filamin 2040 74 4132 heat shock 70 kD protein 5 (glucose-regulated protein, 78 kD) 2041 74 4234 nuclear receptor subfamily 6, group A, member 1, isoform 1 2042 74 429O Hermansky-Pudlak syndrome protein 2043 4642 Unknown 2044 74 47O2 rat myomegalin, similar to 2045 74 5217 lipocalin 2 (oncogene 24-p3) 2O46 74 5424 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) 2047 74 5489 wingless-type MMTV integration site family, member 3A 2O48 74 58O8 guanine nucleotide binding proteinalpha 12 2049 74 5853 Z-band alternatively spliced PDZ-motif 2050 74 5861 Z-band alternatively spliced PDZ-motif 2051 5865 Unknown 2052 74 6475 Unknown 2053 74 6487 ACYL-COA DEHYDROGENASE, VERY-LONG-CHAIN SPECIFIC + F36, similar to 2O54 74 6491 Unknown 2055 6535 RAB7, member RAS oncogene family 2056 74 6585 yeast adenylate cyclase, similar to 2057 74 7216 carrier aralar 2O58 74 7249 CGI-135 protein 2059 74 7260 serologically defined colon cancer antigen 1 2060 7375 lysophospholipase I 2O61 74 797O Unknown 2O62 74 8292 Unknown 2O63 84OO Unknown 2O64 74 8439 Unknown 2O65 74 88.31 Unknown 2O66 74 8858 transformation/transcription domain-associated protein US 2004/0101874 A1 May 27, 2004 56

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2O67 749079 vacuolar protein sorting protein 18 2O68 74.9154 Unknown 2O69 7492.13 serine-threonine kinase/MAD3-like protein kinase 2070 749294 GCN2 elF2alpha kinase 2O71 749.361 Unknown 2O72 749419 Unknown 2O73 749523 Unknown 2074 749588 Unknown 2O75 749765 A kinase anchor protein 6 2O76 749776 Unknown 207 7 75O136 Unknown 2O78 7SO148 Unknown 2O79 75O186 LAMIN A/C (70 KDA LAMIN) 208O 750222 Unknown 2081 750259 Rho/Rac guanine nucleotide exchange factor 2 2O82 750405 pyruvate kinase, muscle (H. Sapiens), similar to 2083 7512O3 Unknown 2084 751493 N-acylsphingosine amidohydrolase 2085 75.1551 Unknown 2O86 751705 Unknown 2087 751808 purine nucleoside phosphorylase 2088 751866 IGF-II mRNA-binding protein 3 2089 752O24 carrier aralar2 2090 752229 dihydrolipoamide dehydrogenase 2091 752236 Unknown 2092 752239 laminin, beta 1 precursor 2093 752249 spectrin, beta, erythrocytic (includes spherocytosis, clinical type I) 2094 752728 guanine nucleotide exchange factor Lbc or A-kinase anchoring protein 2095 753117 Unknown 2096 753239 kinectin 1 2097 7S3384 A kinase (PRKA) anchor protein (gravin) 12 2098 753693 adaptor-related protein complex 4, sigma 1 subunit, similar to 2099 753915 Ras protein-specific guanine nucleotide-releasing factor 1 OO 754.222 farnesol receptor HRR-1 O1 754627 Unknown O2 754848 Unknown O3 754867 Unknown O4 755.192 Unknown 05 755316 Zinc finger protein 91 O6 755336 tumor rejection antigen 1 O7 755347 Unknown O8 755357 mitochondrial ribosomal protein L18 O9 7554.36 Superoxide dismutase 2, mitochondrial 1O 755456 Zinc finger protein 256 11 755952 lysophospholipase I, similar to 12 756295 Na, K-ATPase subunit alpha 3 13 756299 pot. ORF (1013 AA), similar to 14 756626 DNA (cytosine-5)-methyltransferase 15 7566.30 mitochondrial ribosomal protein L4 16 756895 dUTP pyrophosphatase 17 756939 Unknown 18 756944 Unknown 19 7571.47 Unknown 2O 757210 FSH primary responsehomolog 1 21 757677 phosphoglycerate kinase 1 22 757711 Unknown 23 758OO1 ND 24K NADH dehydrogenase (ubiquinone) flavoprotein 2 (24 kD) (H. Sapiens), similar to 24 75852O ATPase, Cu++ transporting, beta polypeptide (Wilson disease) 25 7593O2 golgi autoantigen, golgin subfamily a, 3 26 759459 hook2 protein 27 759609 Unknown 28 759903 transcription factor 29 759981 Unknown 3O 760649 inositol 1,4,5-triphosphate receptor, type 2 31 761208 glyceraldehyde 3-phosphate dehydrogenase like 32 761398 tubulin beta 5, similar to 33 76.1496 programmed cell death 8 (apoptosis-inducing factor) 34 76.1689 calcium channel, voltage-dependent, beta 3 subunit 35 762250 protein tyrosine phosphatase, receptor type, B 36 762650 Unknown US 2004/0101874 A1 May 27, 2004 57

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

37 4 762696 granzyme M precursor 38 4 763105 Unknown 39 4 763304 Src homology 2 domain-containing transforming protein D, similar to 40 4 763427 death-associated protein kinase 3, ZIP-kinase 41 4 763491 NY-REN-58 antigen 42 4 763709 Unknown 43 4 763948 FERM, RhoGEF, and pleckstrin domain protein 1; chondrocyte-derived eZrin-like protein, similar to 2 44 764159 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl CoenzymeA thiolase) 7642O2 hydroxyacyl-Coenzyme A dehydrogenase, type II 764412 D-amino-acid oxidase 764.458 male-specific lethal-3 (Drosophila)-like 1 764705 Unknown 764874 Unknown 764936 G protein-coupled receptor 19 765579 Unknown 76.5581 peroxiredoxin 5 765684 kinesin family member 4 766197 Unknown 766,265 Unknown 766346 glutathione S-transferase P1-1 766373 regulatory factor X, 4 766393 transmembrane protein (63 kD), endoplasmic reticulum/Golgi 766635 prohibitin, B-cell associated protein 766937 DRIM protein or Key-1A6 protein 767036 Unknown 767224 protein kinase C and casein kinase substrate 767305 protein C, cardiac 767738 CALCIUM ATPASE 2(SERCA2) 767795 Unknown 768227 purinergic receptor P2X, ligand-gated ion channel, 7 768743 thioredoxin peroxidase 769051 ND B14.5a 769064 Unknown 769085 Unknown 7 1. 769089 Unknown 769268 GalNAc alpha-2, 6-sialyltransferase I, long form 769776 peripheral benzodiazepine receptor-associated protein 1 77OO42 Unknown 77OO70 Unknown 77O170 Unknown 770383 Unknown 77.0569 Unknown 77O608 small fragment nuclease 77O670 Unknown 770915 Unknown 77O940 angiotensin I converting enzyme 77.0968 Unknown 771355 beta-2-glycoprotein I precursor 771369 brain-immunoglobulin-like molecule with tyrosine-based activation motifs 771396 isocitrate dehydrogenase 3 beta (NAD+) 771416 murine retrovirus integration site 1 homolog 771689 myosin, heavy polypeptide 1, skeletal muscle, adult 772O46 Unknown 772333 phosphorylase, glycogen; brain (H. Sapiens), similar to 772.527 Unknown 772555 Unknown 772672 calpain 5 772954 copine I 773.504 tyrosine kinase, non-receptor, 1 773592 AHNAK nucleoprotein (desmoyokin) 773948 Unknown 774045 Unknown 774139 ATPase g 774236 Unknown 774282 apolipoprotein A-I precursor 774359 ionotropic ATP receptor P2X5b 774503 phospholipase D2 774525 carrier oxoglutarate 774778 Unknown US 2004/0101874 A1 May 27, 2004 58

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

22O6 4 774.78O karyopherin (importin) beta 1 22O7 4 774844 succinate dehydrogenase, subunit C 2208 4 775218 Unknown 2209 4 775320 Unknown 2210 4 775363 baculoviral IAP repeat-containing protein 5 2211 4 775444 carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5, similar to 2212 4 775476 endocytic receptor (macrophage mannose receptor family) 2213 4 77.5546 malonyl-CoA decarboxylase 2214 4 775827 ubiquinol-cytochrome c reductase core protein II 2215 4 775827 UCR 2 2216 4 776296 Unknown 2217 4 776.472 nuclear receptor co-repressor 1 2218 4 776681 Unknown 2219 4 776736 Unknown 2220 4 776778 ATP-binding cassette, sub-family A member 3 2221 4 776800 cat eye syndrome chromosome region, candidate 5, isoform 1 2222 4 776960 Unknown 2223 4 77698O carrier citrate transporter 2224 4 77-7215 protein disulfide isomerase, pancreatic; protein disulfide isomerase, similar to 2225 777313 ND 13 k-B 2226 777483 general transcription factor IIIC, polypeptide 1 (alpha subunit, 220 kD) 2227 777522 Unknown 2228 777630 AT-binding transcription factor 1 2229 777716 Unknown 223O 777813 Unknown 2231 777901 Unknown 2232 778O35 Unknown 2233 778104. adaptor-related protein complex 1, beta 1 subunit 2234 77.8235 Unknown 2235 778381 eIF4E-transporter 2236 77.8431 ret finger protein-like 2 2237 778654 THIOSULFATE SULFURTRANSFERASE (RHODANESE) 2238 779326 Unknown 2239 7796.86 Unknown 2240 779867 N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminida 2241 779881 periplakin 2242 779964 Unknown 2243 780055 protease, serine, 7 2244 78O117 Unknown 2.245 78O193 synaptojanin 1 2246 780272 intersectin 1 (SH3 domain protein) 2247 780668 ES1 protein (KNP-I protein 22 (ThiJ/Pfpl family motif) 2248 780705 phosphofructokinase, liver 2249 78O857 Unknown 2250 781094 huntingtin 2251 781125 quinoid dihydropteridine reductase (H. Sapiens), similar to 2252 781.245 fatty-acid-Coenzyme Aligase, long-chain 6 2253 781533 Unknown 2254 781826 receptor (TNFRSF)-interacting serine-threonine kinase 1 2255 781890 Unknown 2256 781979 Unknown 2257 781989 putative transcription factor/GTF2I repeat domain-containing 1, isoform 2 2258 782O63 malate dehydrogenase 2, NAD (mitochondrial) 2259 782332 HLA-B associated transcript-3, similar to 2260 782751 Unknown 2261 782921 protein kinase C and casein kinase substrate in neurons 1 2262 782973 tubby like protein 1 2263 783O11 p38 mitogen-activated protein kinase 2264 78.3112 Unknown 2265 78.3333 Supervillin, isoform 1 2266 783455 Unknown 2267 783.504 Unknown 2268 78.3675 small GTP binding protein RAB6 isoform 2269 783738 inositol polyphosphate phosphatase-like 1 2270 784O11 Unknown 2271 784O64 mitogen-activated protein kinase kinase kinase 11 2272 784-122 atrophin-1 2273 784.162 Ubiquitin isopeptidase T 2274. 784612 Unknown 2275 78.4913 EH-domain containing 4 US 2004/0101874 A1 May 27, 2004 59

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

2276 4785008 Unknown 2277 4785181 microfibrillar-associated protein 1 2278 4785356 Unknown 2279 47854.05 polo (Drosophia)-like kinase 228O 4785865 Unknown 2281 4785919 copper containing amine oxidase 3 precursor; amine oxidase (copper containing); copper amine oxidase precursor; vascular adhesion protein 1: vascular adhesion protein 1, similar to 2282 4786231 Unknown 2283 478.6366 PAR-6 beta 2284 4.786394 cytochrome P450, subfamily XXIV precursor 2285 4.786884 Unknown 2286 4787.181 CUB and sushi multiple domains protein 1 short form 2287 479O190 SMART/HDAC1 associated repressor protein 2288 5O12003 Unknown 2289 5O1204.8 HERV-H LTR-associating 3, similar to 2290 5O2O655 ATP/GTP-binding protein 2291 5026974 obscurin 2292 SO296.19 fracture callus 1 homolog 2293 5029922 Unknown 2294 SO3O240 ATPase alpha, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle, similar to 2295 SO41811 Hermansky-Pudlak syndrome type-3 protein 2296 5076827 Pcph proto-oncogene protein 2297 50793.48 angiotensinogen proteinase inhibitor, 2298 5079392 replication control protein 1 2299 50794.08 Unknown 23OO 5079735 Unknown 2301 508O291 dipeptidyl peptidase 7+F206, similar to 23O2 5080429 Unknown 23O3 5080454 Unknown 2304 5080499 serineproteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1, similar to 2305 5126735 heat shock 27 kD protein 1, similar to 2306 514724.8 putative breast epithelial stromal interaction protein 2307 5147337 progestin induced protein; ubiquitin-protein ligase Homo sa 2308 51494 76 arginyl-tRNA synthetase 2309 5150811 mitochondrial ribosomal protein S36 2310 52O8648 central cannabinoid receptor, isoform b; CB1 receptor; brain cannabinoid receptor 1 2311 52134 79 putative DNA polymerase delta p38 subunit 2312 5213542 2313 52144 23 Unknown 2314 52144 86 Unknown 2315 52147O6 Unknown 2316 5215308 dystroglycan 1, similar to 2317 52274 56 ch-TOG protein from Homo sapiens Arabidopsis tha 2318 52.77229 Homologue to Drosophila photoreceptor protein calphotin 2319 52.774 15 scavenger receptor cysteine-rich type 1 protein M160 precursor 232O 5277514 Unknown 2321 5278 88 Unknown 2322 5281 50 unkempt (Drosophila)-like 2323 5281837 PX domain-containing protein kinase 2324 5294558 RAS-RELATED PROTEINRAB-5A 2325 5294560 RAB5A, member RAS oncogene family 2326 52946.67 bassoon (presynaptic cytomatrix protein) 2327 5294817 GalNAc-4-sulfotransferase 2 (H. Sapiens), similar to 2328 5295270 MADhomolog 5 2329 5295.351 VDAC-1 233O 5295412 Unknown 2331 52955.74 laminin receptor1 2332 5295842 Unknown 2333 5296,104 optic atrophy 1 2334 5296351 splicing factor 3b, subunit 1, 155 kD 2335 5296,762 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 2336 5296824 lipin 1 2337 5297926 transforming growth factor, alpha 2338 5298022 mitochondrial ribosomal protein L53 2339 52991.36 Unknown 2340 5299287 Unknown 2341 5299581 Unknown US 2004/0101874 A1 May 27, 2004 60

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2342 52997.84 glutamate receptor, metabotropic 1 2343 5299942 Unknown 2344 53OO149 modulator of transcription factor GATA-4 in cardiomyocytes 2345 53O1488 SERINE/THREONINE PROTEIN KINASE 24(MST-3) 2346 53O2O83 CD2-associated protein 2347 5302719 Unknown 2348 53O2936 citrate synthase precursor 2349 53O388O Glutamate receptor interacting protein 2350 53O4843 Unknown 2351 53O4935 destrin (actin depolymerizing factor) 2352 53054.04 Unknown 2353 5305472 troponin I, cardiac 2354 53O5838 ReA-associated inhibitor 2355 53.06072 transcriptional repressor NAC1 2356 5306753 Unknown 2357 5307117 rho guanine nucleotide exchange factor 12 2358 5307634 ND 23 k 2359 531.4651 Oxygen regulated protein 2360 5318843 aconitase 2, mitochondrial 2361 5318933 cytochrome b5 reductase 2362 5321298 Unknown 2363 532138O v-erb-a avian erythroblastic leukemia viral oncogene homolog-like 4 2364 5321446 Unknown 2365 534.1707 Unknown 2366 53.75094. RSK-like protein 2367 54.51842 ADAM-TS disintegrin and metalloproteinase domain 19, isoform 1 preproprotein; meltrin beta; metalloprotease-disintegrin meltrin beta 2368 5451854 midline 1, isoform beta; midline-1; zinc finger X and Y 2369 5451916 bone morphogenetic protein receptor, type II, isoform 1 precursor; type II activin receptor-like kinase; serine/threonine kinase 2370 5451923 serologically defined colon cancer antigen 33 2371 552.9996 son of sevenless homolog 1 (Drosophila); son of sevenless (Drosophila) homolog 1 2372 553O243 villin 2 (eZrin), similar to 2373 553O305 Unknown 2374 5553127 hexokinase 2; hexokinase-2, muscle 2375 555.3137 2376 5559225 Unknown 2377 55593O3 Unknown 2378 5559516 Unknown 2379 5559753 Unknown 238O 562O821 Unknown 2381 562O841 Unknown 2.382 562O853 Unknown 2383 562O867 Unknown 2384 562O879 Unknown 2385 56.20927 Unknown 2386 56.20933 Unknown 2387 568OOO4 H2B histone family, member Q, similar to 2388 568O171 semaF cytoplasmic domain associated protein 3 2389 5718530 POM121 membrane glycoprotein (rat homolog)-like 2 2390 577.8991 Unknown 2391 577908O Unknown 2392 5779126 guanine nucleotide binding protein (G protein), a 2393 57791.56 Unknown 2394 5795410 Unknown 2395 58O8373 erythroid membrane-associated protein 2396 58O86O7 ATPase f FO 2397 5826629 Peroxiredoxin 5 2398 5928.608 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5, similar to 2399 5928907 Unknown 24OO 5929O3O Unknown 24O1 5929352 mitochondrial ribosomal protein L1 24O2 5929856 Unknown 2403 592.9892 Unknown 2404 5988268 Myb-Domain. Of Human Rap1 2405 598.8350 Lysozyme 24O6 5990494 Unknown 24O7 5991827 hexokinase 1, isoform HKI-R; brain form 24.08 5991829 hexokinase 1, isoform HK-ta?tb; brain form hexokinase US 2004/0101874 A1 May 27, 2004 61

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

24 O9 5991859 Unknown 24 1O 6033591 SH2 domain-containing phosphatase anchor protein 2b 24 11 6041807 Unknown 24 12 6 56815 Sec23-interacting protein p125 24 13 56952 Unknown 24 14 57047 succinate dehydrogenase complex, subunit A, flavoprotein precursor 24 15 57111 progesterone membrane binding protein 24 16 57253 uridine 5 monophosphate hydrolase 1; pyrimidine 5-nucleotidase, similar tO 24 17 57453 Unknown 24 18 57682 IDN3 protein 24 19 580.05 RNA-binding protein regulatory subunit 24 2O 58O38 putative, similar to 24 21 58324 heat shock 70 kD protein (Mortalin-2) 24 22 58747 CLIP-associating protein 2 24 23 59170 Unknown 24 24 593O2 Unknown 24 25 59416 Unknown 24 26 59569 Unknown 24 27 59594 carnitine palmitoyltransferase II 24 28 597O1 ribosomal protein S7 (H. Sapiens), similar to 24 29 59788 S100 calcium-binding protein A6 24 3O 59874 Unknown 24 31 6O276 spectrin, beta, non-erythrocytic 1 (H. Sapiens), similar to 24 32 6O441 putative, similar to 24 33 6O793 glycosyltransferase AD-017 24 34 60823 phosphatidylinositol-4-phosphate 5-kinase, type I, beta 24 35 60929 retinoblastoma-binding protein 5 24 36 61569 ryanodine receptor 2 24 37 61583 endoplasmic reticulum oxidoreductin 1-Lbeta 24 38 61627 Rho guanine nucleotide exchange factor 10 24 39 61681 Unknown 24 40 61727 stromal cell derived factor receptor 1 isoform a 24 41 62032 PEPTIDYL-PROLYL CIS-TRANS ISOMERASEB PRECURSOR (PPIASE) (ROTAMASE) (CYCLOPHILIN B) 24 42 63.057 Unknown 24 43 63065 RIKEN cDNA 2410008H17 gene, similar to 24 44 63124 TTF-I interacting peptide 20 24 45 63817 Bc 1 24 46 6471O Unknown 24 47 64895 rabaptin-5 24 48 6498O Unknown 24 49 65190 Unknown 24 50 65554 Unknown 24 51 65872 accessory proteins BAP31/BAP29 (H. Sapiens), similar to 24 52 6.6325 Unknown 24 53 66513 pericentrin B 24 54 68619 Unknown 24 55 714.86 Unknown 24 56 71987 monoamine oxidase A 24 57 72349 triadin 24 58 7.4655 Unknown 24 59 75846 atrophin-1 interacting protein 1; activin receptor interacting protein 24 60 76937 excision repair protein 1 24 61 77368 putative, similar to 24 62 77559 MLL2 protein 24 63 78062 Unknown 24 64 78117 Unknown 24 65 78214 GTP-rho binding protein 1, similar to 24 66 81084 G protein-coupled receptor 51 24 67 92.638 isocitrate dehydrogenase 2 (NADP+), mitochondrial 24 68 96598 coX 6a 24 69 98361 Unknown 24 70 6 98481 Unknown 24 71 6306537 cadherin 20, type 2 preproprotein 24 72 6306954 Unknown 24 73 6306978 annexin A2 24 74 6307164 CGI-90 protein 24 75 6307227 Unknown 24 76 6307270 Unknown 24 77 6307468 Unknown US 2004/0101874 A1 May 27, 2004 62

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2478 6307475 neuroepithelial cell transforming gene 1 2479 63591O2 Unknown 248O 63591.95 Unknown 2481 6416451 tRNA-nucleotidyltransferase 2482 6418373 Unknown 2483 64-18423 guanylate binding protein 4 2484 65O7813 tumor necrosis factor receptor Superfamily, member 21, similar to 2485 654912S Unknown 2486 65491.99 Unknown 2487 6549271 Unknown 2488 6549294 Unknown 2489 654962O Unknown 2490 654988O Unknown 2491 6549918 Unknown 2492 65SO394 Unknown 2493 6550518 Unknown 24.94 6550576 Unknown 2495 6550810 Unknown 2496 6550845 Unknown 2497 6551173 Unknown 2498 6551429 Unknown 2499 6551580 Unknown 2500 6551610 Unknown 25O1 6551739 myosin light chain kinase 25O2 6551769 Unknown 2503 6551917 Unknown 2SO4 6551953 Unknown 2505 6551957 Unknown 2506 6552104 Unknown 2507 6552271 Unknown 2508 6552547 Unknown 2509 6552885 Unknown 2510 655.2927 Unknown 2511 6552957 Unknown 2512 6552.988 Unknown 2513 6553O31 Unknown 2514 6553.078 Unknown 2515 655.3235 Unknown 2516 6553285 Unknown 2517 6553362 Unknown 2518 6554014 Unknown 2519 655.4275 Unknown 252O 6554.604 mitochondrial ribosomal protein S23 2521 655.46O7 mitochondrial ribosomal protein S10; NB4 apoptosis/differentiation related protein; mitochondrial 28S ribosomal protein S10 2522 67.41033 protease 26S subunit, ATPase 1 2523 6753264 Unknown 2524 6876860 Unknown 2525 6877O71 ATPase gamma F1 2526 6877127 synaptophysin-like protein, similar to 2527 6877285 duodenal cytochrome b, similar to 2528 6877328 Unknown 2529 6877328 Unknown 25.30 6877459 Unknown 2531 6877964 isovaleryl Coenzyme A dehydrogenase 2532 6878101 Unknown 2533 6924265 Unknown 2534 6924269 Unknown 2535 695O603 mitochondrial ribosomal protein S35; mitochondrial 28S ribosomal protein S28 2536 mitochondrial ribosomal protein S27; mitochondrial 28S ribosomal protein S27 2537 697.4753 sodium-potassium-chloride cotransporter 2.538 7O16315 olfactory receptor-like protein JCG4 2539 7028367 gelsolin (amyloidosis, Finnish type), similar to 2540 7028379 Unknown 2541 7375734 Cyclin G-associated kinase 2542 73785.99 Gamma-interferon-inducible protein If-16 (Interferon-inducible myeloid differentiation transcriptional activator) (IFI 16) 2543 7380287 Mitochondrial 39S ribosomal protein L56 (MRP-L56) (Serine beta lactamase-like protein LACTB) US 2004/0101874 A1 May 27, 2004 63

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2544 738.0426 Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA (Processing alpha 1.2-mannosidase IA) (Alpha-1,2-mannosidase IA) (Mannosidase alpha class 1A member 1) (Man(9)-alpha-mannosidase) (Man9-mannosidase) 2545 7389971 Unknown 2546 74 O2865 thiosulfate sulfurtransferase (rhodanese) 2547 74 32231 MSTPO22 2548 74 34O94 putative, similar to 2549 74 34314 Unknown 2550 74 34411 Unknown 2551 74 34.458 Unknown 2552 74 34554 Unknown 2553 74 34671 Unknown 2554 74 35264 INNER EAR-SPECIFIC COLLAGEN PRECURSOR (SACCULAR COLLAGEN), similar to 2555 74 35299 Unknown 2556 74 35748 phosphorylase, glycogen; brain 2557 74 36,258 ND 13 K-B NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5; hypothetical protein FLJ12147; Complex I-13 KD-B; ubiquinone reductase; type I dehydrogenase, similar to 2558 74 36498 Unknown 2.559 74 36513 VDAC-1 VOLTAGE-DEPENDENT ANON-SELECTIVE CHANNEL PROTEIN 1 (VDAC-1) (RVDAC1) (OUTER MITOCHONDRIAL MEMBRANE PROTEIN PORIN 1), similar to 2560 74 36561 Unknown 2561 74 36979 Unknown 2562 74 37312 Unknown 2563 74 38284 Unknown 2564 74 39551 REGULATOR OF G-PROTEIN SIGNALING 12 (RGS12), similar to 2565 74 4O287 anaplastic lymphoma kinase Ki-1, similar to 2566 74 2134 one twenty two protein; hypothetical protein FLJ12479, similar to 2567 74 2500 Molybdenum cofactor synthesis protein cinnamon, similar to 2568 74 2568 Unknown 2569 74 3010 hematological and neurological expressed sequence 1, similar to 2570 74 3439 Unknown 2571. 74 3833 glyceraldehyde-3-phosphate dehydrogenase, similar to 2572 74 4O67 RIKEN cDNA 0610011N22, similar to 2573 74 46OO Unknown 2574 74 4969 solute carrier family 4, anion exchanger, member 3 2575 74 5877 xylulokinase homolog (H. influenzae) 2576 74 6038 Unknown 2577 74 6807 plastin 1 2578 74 7126 Unknown 2579 74 7383 Unknown 258O 74 47877 Unknown 2581 74 50O39 Unknown 2582 74 SO491 factor V. similar to 2583 74 51676 putative, similar to 2584 74 51748 Unknown 2585 74 518O1 Unknown 2586 74 52377 Unknown 2587 74 54350 putative protein, similar to 2588 74 54582 phosphoglycerate mutase 1 (brain); Phosphoglycerate mutase A, nonmuscle form, similar to 2589 74 55099 putative, similar to 2590 74 55439 heat shock 60 kD protein 1 (chaperonin) (H. Sapiens), similar to 2591 74 55445 Mitochondrial Complex I protein, now 21754001 2592 74 55927 Unknown 2593 74 56092 Unknown 2594 74 56384 non-specific cross reacting antigen, similar to 2595 74 57389 Unknown 2596 74 58483 Unknown 2597 74 58911 Unknown 2598 74 59115 Melanoma-associated antigen 11 (MAGE-11 antigen), similar to 2599 74 59319 putative, similar to 26OO 74 594.08 small Rho-like GTPase RhoA, similar to 26O1 74 594.79 Unknown 26O2 74 59746 VOLTAGE-DEPENDENT ANON-SELECTIVE CHANNEL PROTEIN 2 (OUTER MITOCHONDRIAL MEMBRANE PROTEIN PORIN 2), similar to 2603 74 Unknown 2604 74 Unknown 2605 74 Unknown US 2004/0101874 A1 May 27, 2004 64

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2606 74 60836 testis expressed sequence 13A, similar to 26O7 74 61025 Unknown 2608 74 61670 RIKEN cDNA 94.30083G14, similar to 2609 74 62761 Unknown 2610 74 63437 Unknown 2611 74 64527 match: multiple proteins; match: Q08151 P28.185 Q01111 Q43554; match: Q08150 Q4O195 P2O340 Q39222; match: Q40368 P36412 P40393 Q40723; match: CEO1798 Q38923 Q40191 Q41022; match: Q39433 Q40177 Q40218 Q08146; match: P10949 P11023 Q, similar to 2612 74 64573 Unknown 2613 74 64724 eukaryotic translation elongation factor 1 alpha 1, similar to 2614 74 64807 phosphoglycerate mutase 2 (muscle) 2615 74 64864 Unknown 2616 74 65135 v-raf murine sarcoma viral oncogene homolog B1 26.17 74 652.13 Unknown 2618 74 65562 Unknown 2619 74 66.365 Unknown 262O 74 66818 Unknown 2621 74 68096 prohibitin, similar to 2622 74 68798 Unknown 26.23 74 69624 Unknown 2624 74 70256 Unknown 2625 74 70269 chromosome 15 open reading frame 2, similar to 2626 74 70290 Unknown 2627 74 71316 Unknown 2628 74 71893 Unknown 2629 74 72555 Unknown 2630 74 72883 ND 51K NADH dehydrogenase (ubiquinone) flavoprotein 1 (51 kD) 2631 74 74293 midline 1: Finger on X and Y (in rat only on X), similar to 2632 74 74785 VDAC-1 voltage-dependent anion channel 1, similar to 2633 74 751.84 Y39B6A. pp.p, similar to 2634 74 76245 Unknown 2635 74 76469 Unknown 2636 74 764.71 Unknown 2637 74 78738 Unknown 2638 74 81443 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4 hydroxylase), beta polypeptide (protein disulfide isomerase; thyroid hormone binding protein p55) 2639 74 81778 Unknown 2640 74 82O59 Unknown 2641 74 82696 Kruppel-type zinc finger (C2H2), similar to 2642 74 82.910 Unknown 2643 74 82953 putative methyl-binding domain protein MBD105, similar to 2644 74 83121 rhophilin-like protein (H. Sapiens), similar to 2645 74 831.87 Unknown 2646 74 83.399 RAB11B, member RAS oncogene family 2647 74 83482 Unknown 2648 74 8482O acetyl-Coenzyme A synthetase 2 (AMP forming)-like 2649 74 84.835 Unknown 26SO 74 85036 Unknown 2651 74 85099 Unknown 2652 74 85128 Unknown 2653 74 85337 Unknown 2654 74 857OO Unknown 2655 74 85787 Mitochondrial Acyl-CoA Thioesterase 2656 74 86O71 DKFZP434OO47 protein, similar to 2657 74 86087 Unknown 2658 74 86456 Unknown 2659 74 864.63 Unknown 2660 74 86622 Unknown 2661 74 86915 Unknown 2662 74 87175 dentin phosphoryn, similar to 2663 74 87390 Unknown 2664 74 87672 Unknown 2665 74 87733 F40G9.9.p, similar to 2666 74 87809 glyceraldehyde-3-phosphate dehydrogenase, similar to 2667 74 87981 F4N2.10, similar to 2668 74 88153 Unknown 2669 74 89631 Unknown 2670 74 91107 Unknown 2671 7511874 Unknown US 2004/0101874 A1 May 27, 2004 65

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

2672 75.11976 Un KOW 2673 75.1208O WAS protein family, member 1 2674 7512147 Un KOW 2675 7736731 mixed lineage kinase 4-beta 2676 783408O haymaker protein 2677 7865554 mitochondrial ribosomal protein L9, 60S mitochondrial precursor (L9 mt) 2678 7939563 Un KOW 2679 7943068 Tcf-4 BETA-Catenin Complex 268O 79434O7 Au h Protein, An Rna-Binding Homologue Of Enoyl-Coa Hydratase 2681 7981,863 ND 5 2682 798.5539 ND 4 2683 8044194 Un KOW 2684 8O87815 Un KOW 2685 8O88572 RIKEN cDNA 493.0553CO5 gene, similar to 2686 8147097 CG1800 gene product Drosophila melanogaster homolog 2687 8157651 bul ous pemphigoid antigen 1 eA 2688 8158416 chromosome 20 open reading frame 188 protein; likely ortholog of mouse transient receptor protein 4, associated protein 2689 82O1886 chromosome 20 open reading frame 175 2690 82O1913 winged-helix nude 2691 82O4214 Un KOW 2692 82O4272 Un KOW 2693 8252315 pro pionyl-CoA carboxylase alpha subunit 2694 8252778 ankyrin repeat-containing protein ASB-2 2695 849O293 ephrin B3, similar to 2696 8490363 calsequestrin 2 (cardiac muscle) 2697 8490389 KOW 2698 8490639 KOW 2699 854.3654 KOW 27OO 8543672 KOW 2701 8544062 Un KOW 2702 854.4103 transcription factor Dp-1, similar to 2703 85445O2 Un KOW 2704 85451.49 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily f, member 1 (H. Sapiens), similar to 2705 85451.97 Un KOW 27O6 8545286 Un KOW 2707 85455.25 Un KOW 2708 8545711 trithorax-related, similar to 2709 8545867 forkhead box D2 2710 8546,369 Un KOW 2711 8546495 N-acetylglucosaminyltransferase VI, similar to 2712 854,7145 KOW 2713 8547604 KOW 2714 8547655 KOW 2715 85.47774 PAPIN, similar to 2716 8547995 KOW 271 7 8548319 KOW 2718 8548686 KOW 2719 8548841 KOW 2.720 8549011 KOW 2721 85496.03 KOW 2722 8549721 spe ctrin, alpha, erythrocytic 1 (elliptocytosis 2) 2723 85497.59 KOW 2724 855.0245 KOW 2725 855O248 ferlin 2726 855.0356 KOW 2727 855O688 LWamide neuropeptide precursor protein, similar to 2728 8551342 laminin receptor 1; Laminin receptor-1 (67 kD); 67 kD, ribosomal protein SA, similar to 2729 8551.404 KOW 2730 8551428 KOW 2731 8551530 KOW 2732 855.1750 KOW 2733 8552428 down-regulated by Ctnnb1, a, similar to 2734 8552574 heat shock 70 kD protein 9B (mortalin-2) (H. Sapiens), similar to 2735 8552843 KOW 2736 855.3054 KOW 2737 855.3524. KOW 2738 8553646 KOW 2739 8553709 RIKEN cDNA 1810055D05 gene, similar to US 2004/0101874 A1 May 27, 2004 66

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2740 8553922 succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (H. Sapiens) similar to 2741 855.4092 Unknown 2742 855.4792 Unknown 2743 8554892 protein phosphatase 4 regulatory subunit 2 (H. Sapiens), similar to 2744 85.55498 Unknown 2745 8555697 SALL1 (sal (Drosophila)-like, similar to 2746 85.55923 Unknown 2747 8556527 protein tyrosine phosphatase, receptor type, G 2748 8557013 Unknown 2749 855.7341 Unknown 2750 8557515 ring finger protein 23; RING-B box-coiled coil-B30.2, similar to 2751 8557535 Unknown 2752 8557606 Unknown 2753 8557689 Unknown 2754 8558040 Unknown 2755 8558112 C-terminal binding protein 1 (H. Sapiens), similar to 2756 8558130 cyclin G associated kinase (H. Sapiens), similar to 2757 8558177 Unknown 2758 8558348 Unknown 2759 8558362 Unknown 2760 8558762 Unknown 2761 855.9050 Unknown 2762 855.9054 Unknown 2763 8.5591.69 GrpE-like protein cochaperone 2764 855.9889 Unknown 2765 855.9896 Unknown 2766 855.9969 Unknown 2767 855.9997 Unknown 2768 856OO88 Unknown 2769 856O396 Unknown 2770 85.60536 Unknown 2771 85.60871 Unknown 2772 85.60910 SGC32445 protein 2773 8561153 Unknown 2774 8561225 Unknown 2775 8561342 Unknown 2776 8561850 Unknown 2777 85621.64 Unknown 2778 8562264 Unknown 2779 8562403 gag, similar to 2780 8562447 Unknown 2781 8562613 Unknown 2782 8562676 Unknown 2783 8562743 Unknown 2784 8562778 Unknown 2785 8562814 Unknown 2786 8562826 Unknown 2787 85.63O24 Unknown 2788 8563079 Unknown 2789 856,3446 Unknown 2790 85.64249 Unknown 2791 85.652OO Unknown 2792 85.65553 Unknown 2793 85.65735 Unknown 2794 85.65792 Unknown 2.795 85.65965 Unknown 2796 85.66008 Unknown 2797 85.66051 Unknown 2798 856,6469 CDC14 cell division cycle 14 homolog B (S. cerevisiae) (H. Sapiens), similar to 2799 85.66582 Unknown 28OO 85.67546 Unknown 28O1 8568O15 Unknown 28O2 8568092 Unknown 28O3 85681OO Unknown 2804 85687.32 Unknown 2805 8568834 Unknown 2806 8568892 T-COMPLEX PROTEIN 1, GAMMA SUBUNIT (TCP-1-GAMMA) (CCT GAMMA), similar to 2807 8568988 Unknown US 2004/0101874 A1 May 27, 2004 67

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 28O8 8569016 Unknown 2809 8569389 Unknown 2810 8569391. Unknown 2811 8569544 Unknown 2812 8569728 Unknown 2813 8569926 Unknown 2814 8570016 Unknown 2815 85.70037 Unknown 2816 8571373 Unknown 2817 8571864 Unknown 281.8 8572080 tubulin, beta polypeptide 4, member Q (H. Sapiens), similar to 2819 8572219 Unknown 282O 8572532 Unknown 2821 8572576 DKFZP434J193 protein (H. sapiens), similar to 2822 8572752 Unknown 2823 8573432 Unknown 2824 8573604 Unknown 2825 8573884. Sec24-related protein C 2826 8574091 (H. Sapiens), similar to 2827 8574564 Unknown 2828 85.74897 cathepsin L, similar to 2829 8575O14 Unknown 283O 8575020 Unknown 2831 8575034 Unknown 2832 8575353 Unknown 2833 8575792 Unknown 2834 8575881 solute carrier family 9 (sodium/hydrogen exchanger), isoform 3, similar to 2835 8575937. Unknown 2836 8576372 Unknown 2837 8576435 glycoprotein beta-Gal 3'-sulfotransferase (H. Sapiens), similar to 2838 8576618 Unknown 2839 8576708 Unknown 2840 8576758 Unknown 2841 8576861. Unknown 2842 8577160 Unknown 284.3 8577199 suppression of tumorigenicity 5 2844 8577427 Unknown 2845 8577553 Unknown 2846 8577877 glutamate receptor, metabotropic 5 (H. Sapiens), similar to 2847 8578024 Unknown 2848 8578981 voltage gated potassium channel Kv3.2b, similar to 2849 8579037 glyceraldehyde-3-phosphate dehydrogenase, similar to 2850 8579791. Unknown 2851 858OO15 Unknown 2852 85.80073 Unknown 2853 858O116 solute carrier family 4, sodium bicarbonate cotransporter, member 8 (H. Sapiens), similar to 2854 858O149 Unknown 2855 8580193 Unknown 2856 8580223 Unknown 2857 8580396 Unknown 2858 8580585 Unknown 2859 8580633 phosphoinositide-3-kinase, class 2, gamma polypeptide 286O 8581005 Unknown 2861 8581215 Unknown 2862 8581598 Unknown 2863 8581873 Unknown 2864 8582200 Unknown 2865 8582274 Unknown 2866 8582343 Unknown 2867 8582592 Unknown 2868 8582682 CG9109 gene product, similar to 2869 8582865. Unknown 2870 8583213 Unknown 2871 8583325 Unknown 2872 8583345 Unknown 2873 8583383 Unknown 287.4 8583657 Unknown 2875 8583725 multidomain presynaptic cytomatrix protein Piccolo, similar to 2876 8583727 Unknown 2877 8584.065 Unknown US 2004/0101874 A1 May 27, 2004 68

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS 2878 8584949 Unknown 2879 8585335 Unknown 288O 8585686 Unknown 2881 8586054 Unknown 2882 8586298 Unknown 2883 8586333 splicing factor 3b, subunit 3, 130 kD 2884 858.6459 putative, similar to 2885 8586610 Unknown 2886 8587004 Unknown 2887 8587044 Unknown 2888 8587O67 Unknown 2889 8587111 Unknown 2890 8587387 Unknown 2891 8587810 arachidonate 12-lipoxygenase, 12R type (H. Sapiens), similar to 2892 8588235 Unknown 2893 858.8450 Unknown 2894 858.8517 Unknown 2.895 8589035 Unknown 2896 85890.65 WW domain binding protein-2, similar to 2897 8589260 Unknown 2898 8589408 Unknown 2899 85.89876 Unknown 29OO 8590023 Unknown 29O1 8590390 RNI-like protein, similar to 29O2 8590417 Unknown 2903 8590816 Unknown 2904 8591174 Unknown 2905 8591441 ND B14.5a NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7 (14.5 kD, B14.5a) 2906 8591813 Unknown 2907 8592023. Unknown 2908 8592.069 Unknown 2909 8592852 Unknown 2910 8593545 Unknown 2911 8593908 Unknown 2912 8593939 secretory protein 45 kDa, similar to 291.3 8594.017 Unknown 291.4 8594189 Unknown 2915 8594359 Unknown 2916 8594592 Unknown 2917 8594594 Unknown 2918 8594767 Unknown 2919 859.4954 Unknown 292O 8594992 Unknown 2921 8595.043 Unknown 2922 8595057 Unknown 2923 8595318 Unknown 2924 8595340 Unknown 2925 8595665 Unknown 2926 8596319 glycerol kinase (H. Sapiens), similar to 2927 8596413 Unknown 2928 8596484 Unknown 2929 8596861 RAS-RELATED PROTEIN RAB-15, similar to 293O 8597225 Unknown 2931 8597549 ZINC FINGER PROTEIN 268 (ZINC FINGER PROTEIN HZF3), similar to 2932 8597551 Unknown 2933 8597742 Unknown 2934 8598132 Unknown 2935 85982.91 kinesin family member C3 2936 85.98462 Unknown 2937 8598482 Unknown 2938 8598674. Unknown 2939 8598989 Unknown 2940 85991.37 zinc finger protein 2 (A1-5) 2941 8599227 Unknown 2.942 8599297 EphB1 2943 8599533 polyhomeotic 2 protein, similar to 2.944 8599587 Unknown 2945 86OO174 Unknown 2.946 8600186 Unknown 2947 8600274 Unknown US 2004/0101874 A1 May 27, 2004 69

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS

2948 86OO32O Unknown 2949 8600459 axonal transport of synaptic vesicles 2950 86OO477 Unknown 2.951 8600510 Unknown 2952 86OO673 replication initiation region protein (60 kD) (H. Sapiens), similar to 2953 86OO792 Unknown 2954 86OO878 Unknown 2955 86OO890 Unknown 2956 86012SO Unknown 2957 86O1419 Unknown 2958 86O1439 Unknown 2959 86O1460 Unknown 2960 86O1629 huntingtin interacting protein-1-related (H. Sapiens), similar to 2961 86O1927 Unknown 2962 86O2O66 Unknown 2963 86O2347 Unknown 2964 86O2.382 chromosome condensation-related SMC-associated protein 1 2965 86O2858 PUTATIVE NUCLEOSIDE DIPHOSPHATE KINASE (NDK) (NDP KINASE), similar to 2966 Unknown 2967 Unknown 2968 Unknown 2969 solute carrier family 1 (glial high affinity glutamate transporter), member 2 2970 Unknown 2971 Unknown 2972 Unknown 2973 PHOSPHATIDYLINOSITOL 3-KINASE REGULATORY SUBUNIT (IB PI3 KINASE P101 SUBUNIT) (PTDINS-3-KINASE P101) (PI3K) (P101-PI3K), similar to 2974 86O4379 Unknown 2975 86O452O Unknown 2976 86O4537 rab-related GTP-binding protein 2977 86O4876 exostoses (multiple) 2 (H. Sapiens), similar to 2978 86OSO74 Unknown 2979 86OS322 Unknown 298O 8605.359 Unknown 2981 8606573 Unknown 2982 8645167 annexin A2 2983 86,76544 Unknown 2984 8676570 Unknown 2985 86,76847 Unknown 2986 8.860829 optic atrophy 1, isoform 1 2987 8.860843 optic atrophy 1, isoform 7 2988 891.6767 Unknown 2989 8916841 Unknown 2990 89592O2 leucine-rich PPR-motif containing; leucine-rich protein mRNA 2991 9115954 dynein, axonemal, heavy polypeptide 5 2992 9263915 Unknown 2993 9353103 Unknown 2994 9526647 Oxidored-nitro domain-containing protein 2995 9584,385 Unknown 2996 9684O29 Unknown 2.997 974.3821 integrin beta 1 isoform 1C-2 precursor; integrin VLA-4 beta subunit; fibronectin receptor beta subunit 2998 9850567 breast carcinoma amplified sequence 3 2999 992.31 O2 holocarboxylase synthetase (biotin-proprionyl-Coenzyme A-carboxylase (ATP-hydrolysing) ligase); HolocarbyOxylase synthetase; holocarboxylase synthetase 9923233 sterol carrier protein 2 992,3611 Unknown 99.23717 rhysin 2 992.3721 pre-T-cell receptor alpha precursor 99.23757 golgi autoantigen, golgin subfamily a, 2; golgin-95 20070212 voltage-dependent anion channel 3 20070798 androgen-regulated short-chain dehydrogenase/reductase 1 2O1274.08 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A hiolasefenoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit; Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolasef 3008 2O127473 glucose regulated protein, 58 kD 3009 20127510 peroxisomal long-chain acyl-coA thioesterase; peroxisomal long-chain US 2004/0101874 A1 May 27, 2004 70

TABLE 1-continued

HUMAN HEART MITOCHONDRIAL PROTEINS

SEO ID GENBANK NO: ACC. NO. DESCRIPTION OF MITOCHONDRIAL PROTEINS acyl-coA thioesterase; putative protein 3O10 20140018 mitochondrial ribosomal protein S9, precursor (MRP-S9) 3O11 2014O250 Sideroflexin 1 3O12 20141424 Short chain 3-hydroxyacyl-CoA dehydrogenase, mitochondrial precursor (HCDH) 3O13 20141538 Homeobox protein Hox-C12 (Hox-3F) 3O14 20141568 Isocitrate dehydrogenase INADP), mitochondrial precursor (Oxalosuccinate decarboxylase) (IDH) (NADP+-specific ICDH) (IDP) (ICD-M) 3O15 20141580 Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP) 3O16 20141765 Succinyl-CoA ligase GDP-forming alpha-chain, mitochondrial precursor (Succinyl-CoA synthetase, alpha chain) (SCS-alpha) 3017 20141946 DNA topoisomerase II, beta isozyme 301.8 2014.7036 transient receptor potential cation channel protein 3019 20150348 Deoxy Hbalphayq, A Mutant Of Hba 3O2O 20151189 Glutamate Dehydrogenase-Apo Form 3O21 20178093 Suppressor of cytokine signaling 7 (SOCS-7) (Nck, Ash and phospholipase C gamma-binding protein) (Nck-associated protein 4) (NAP-4) 3O22 20268814 CD36 antigen (collagen type I receptor, thrombospondin receptor) 3O23 20270305 synaptotagmin-like 5 3024 20270399 polycystic kidney and hepatic disease 1 3O25 226207 dihydrolipoamide S-acetyltransferase

0157 Table 2 presents a selected subset of the 3025 human heart mitochondrial proteins that are disclosed in TABLE 2 Table 1 and in the Sequence Listing. The mitochondrial MITOCHONDRIAL FUNCTIONS OF SELECTED proteins of Table 2 are organized according to particular COMPONENTS OF THE HUMAN HEART mitochondrial function classifications as indicated,- based on MTOCHONDRIAL PROTEOME analysis of amino acid sequences and GENBANK annota- MITOCHONDRIAL FUNCTION GENEBANK SEO ID tions, a number of the entries in Table 2 may use earlier CLASSIFICATION ACCNO. NO: GENBANK Accession numbers which differ from those Amino acid metabolism 2. s shown in Table 1, but the sequences of Such GENBANK 4504O67 75 Accession numbers can each be matched to a sequence in the 3.A. s: Sequence Listing of the instant application using Sequence 1545863 52O database Searching Software tools as exemplified above and 2653507 76 as known to the art (e.g., Basic Local Alignment Search Tool 35182283027640 519491 (“BLAST), http://www.ncbi.nim.nih.gov/BLAST, Alts- 4764412 240 chul, J. Mol. Biol. 219:555-565, 1991, Henikoff et al., Proc. 5. Natl. Acad. Sci. USA 89:10915-10919, 1992; PSI-BLAST, Amino acid metabolism Total 12 ALIGN, MEGALIGN; WISETOOLS. CLUSTAL W. Apoptosis 22861.45 59 Thompson et al., 1994 Nucl. Ac. Res. 22:4673; CAP, www- t .no.embnet. org/clustalw.html; FASTA/FASTP, Pearson, 2382773 58 1990 Proc. Nat. Acad. Sci. USA 85:2444, available from D. 47294.75 O1 Hudson, Univ. of Virginia, Charlottesville, Va.). As 4761496 717 described above, each amino acid Sequence provides a Apoptosis Total ois, OO polypeptide Structure from which a Sample can be analyzed C-compound metabolism 1354.222 40 to determine, on the basis of Structure, whether a modified 47.58498 405 polypeptide as provided herein may be present in the E. s Sample. AS also described above, each functional classifi- 1436,533 36 cation refers to a defined biological activity measureable 2230075 359 according to methods provided herein and known to the art, 3. Such that the invention contemplates determination in a 40431.87 38 sample of whether a polypeptide that exhibits altered bio- 4724751 695 logical activity is present. US 2004/0101874 A1 May 27, 2004 71

TABLE 2-continued TABLE 2-continued

MTOCHONDRIAL FUNCTIONS OF SELECTED MITOCHONDRIAL FUNCTIONS OF SELECTED COMPONENTS OF THE HUMAN HEART COMPONENTS OF THE HUMAN HEART MITOCHONDRIAL PROTEOME MTOCHONDRIAL PROTEOME

MITOCHONDRIAL FUNCTION GENEBANK SEO ID MITOCHONDRIAL FUNCTION GENEBANK SEO ID CLASSIFICATION ACCNO. NO: CLASSIFICATION ACCNO. NO: C-compound metabolism Total 1O Complex 3 Total 1O Carrier 113463 153 Complex 4 117103 211 4505,775 157 226209 221 4557.403 155 1262.581 2O7 7657347 532 45O2985 213 11141.885 851 4502987 218 12232421 92O 45O2989 217 12653827 531 45O2991 219 13632616 152 45O2993 22O 13647558 151 4758O38 210 14747216 154 4758040 215 14752O24 850 13629150 209 14774525 156 13637833 216 Carrier Total 12 13648426 237 Complex 1 13013 599 16196598 212 262579 583 Complex 4 Total 14 26,258O 592 Complex 5 114549 84 4505.355 62O 1262582 8O 4505.357 609 4502297 87 4505.359 613 45O23O3 93 4505.361 611 5901896 89 4505.365 617 6005717 88 4505.367 605 115261.49 85 46891.04 610 13272855 81 4758768 6OO 13543618 83 475.8772 621 14774139 91 4758776 6O7 Complex 5 Total 1O 4758784 614 DNA synthesis 118749 497 4758786 6O1 1709123 281 4758790 588 4153874 840 4758792 586 11225260 283 4826848 612 DNA synthesis Total 4 4826852 608 31645 355 4894.370 619 107554 752 6O41669 616 129070 750 7657369 591 136066 921 OO92657 585 387O11 751. O1795.99 622 4557032 467 O764847 618 1143O299 4O1 O835O25 596 12653371 684 O835087 584 13436413 350 2005918 369 14043654 831 14761208 356 30971.56 598 3272567 6O2 15553127 403 1599.1827 402 3272.568 604 Glycolysis Total 13 3528960 590 Guanine-related 106.185 372 3637608 606 121009 379 4336775 623 386,745 38O 4769051 615 13352SO 784 4777313 587 45O4049 378 53.07634 595 4506517 764 Complex 1 Total 37 6005772 747 Complex 2 47.5908O 865 10047118 344 3639114 792 10945.428 516 4727.486 867 1105.5998 376 6157047 791 14745808 377 Complex 2 Total 4 15779126 375 Complex 3 117759 944 16181084 343 117863 947 Guanine-related Total 13 190804 946 Inositol-related 10848O 688 1351360 934 124505 433 9297078 933 1399.105 682 11128O19 233 45058O1 686 13631678 945 10835O23 431 13649658 948 11436778 435 14736223 942 14724557 683 14775827 943 14728229 687 US 2004/0101874 A1 May 27, 2004 72

TABLE 2-continued TABLE 2-continued

MTOCHONDRIAL FUNCTIONS OF SELECTED MITOCHONDRIAL FUNCTIONS OF SELECTED COMPONENTS OF THE HUMAN HEART COMPONENTS OF THE HUMAN HEART MITOCHONDRIAL PROTEOME MTOCHONDRIAL PROTEOME

MITOCHONDRIAL FUNCTION GENEBANK SEO ID MITOCHONDRIAL FUNCTION GENEBANK SEO ID CLASSIFICATION ACCNO. NO: CLASSIFICATION ACCNO. NO: 14760649 432 Total 34 14783738 434 Lipoprotein 2294.79 48O Inositol-related Total 1O 1082692 693 Kinase/phosphatase 130749 45 4826914 691 1103677 573 9438229 692 1709242 6SO 1347OO94 70 45O3269 246 14721241 485 4505153 510 Lipoprotein Total 6 4506091 551 Nucleotide metabolism 45O2O13 28 45.57769 522 45.02457 78 7439346 737 4503375 258 OO4712O 437 86,71846 2O)4 1526789 430 13654685 79 2643716 738 14776778 77 26544O7 574. Nucleotide metabolism Total 6 2659007 733 Protease 45O22O1 3O 283O367 803 45O2S63 137 3606056 28O 7656.959 139 3631907 553 10047106 144 36.46385 222 12408656 136 364,861 8O2 12643637 24 3938619 224 12654627 517 419446 11 14772672 138 4721507 8O1 1478OO55 727 473,3904 799 16741033 726 4736227 774 Protease Total 1O 47.4037 12 Protein targeting 123571 385 4749765 10 1091688 390 478292 732 1346317 387 4784.064 552. 40O8131 184 47854.05 7O6 SO321.81 915 53O1488 418 58O2970 33 603359 808 691271.4 916 Kinase/phosphatase Total 3O 7657257 917 Lipid metabolism O82723 722 7662673 918 169204 286 99.10382 533 762533 148 2655195 391 3273228 18 3645.492 389 45O1869 22 4603.309 386 45O2327 97 Protein targeting Total 13 45036O7 295 ras/GTPase 1657266 789 4503609 296 58O3135 755 4503651 322 1359874 371 4504975 484 1436135 761 4557.817 869 2652715 648 4557.833 724 2751117 704 4758312 297 3569962 845 O835059 319 3651229 772 276,083 323 3652324 760 433OO7 678 3786129 417 640566 421 3794.267 757 2669909 483 4211570 2O2 2707570 304 424.9144 754. 280SO21 19 474O792 1390 3435350 327 ras/GTPase Total 14 36,396.28 13 Receptor 184477 771 3647276 465 1OO194 257 3653O49 2O 116878 316 4041699 310 4504733 436 4043451 373 4.87729 763 4725848 21 11968152 852 4729.783 252 13632266 894 4730775 42O 13650874 748 47.46487 815 1473.2886 895 14744234 646 4764159 14 16161569 788 47642O2 419 Receptor Total 1. 4769776 674 Redox 802.150 662 4781.245 324 45O260 143 4557.845 775 US 2004/0101874 A1 May 27, 2004 73

TABLE 2-continued TABLE 2-continued

MTOCHONDRIAL FUNCTIONS OF SELECTED MITOCHONDRIAL FUNCTIONS OF SELECTED COMPONENTS OF THE HUMAN HEART COMPONENTS OF THE HUMAN HEART MITOCHONDRIAL PROTEOME MTOCHONDRIAL PROTEOME

MITOCHONDRIAL FUNCTION GENEBANK SEO ID MITOCHONDRIAL FUNCTION GENEBANK SEO ID CLASSIFICATION ACCNO. NO: CLASSIFICATION ACCNO. NO: 69.12536 633 Transcription Total 27 11399.466 239 Translation 1706611 3OO 114-16669 632 450.3507 311 12804319 142 4758118 243 13112O23 199 5032051 6 13236495 753 7661872 474 13529257 4 7705626 543 13627233 42 77O6349 546 13994325 744 11771.48 535 14735899 235 1416393 538 Redox Total 13 1424.404 544 Stress 45O3731 33 1559927 542 4758192 8OO 1596859 537 5453902 634 3027604 547 7643782 383 3123976 73 1363.1440 675 35594O4 534 1425OO63 676 363152 549 147554.36 874 3648964 35 Stress Total 7 3.89923 541 Structural 13194,197. 459 4O28389 539 1364.3253 460 40284.05 545 14124976 46 4165270 536 14730782 462 4285174 299 15305472 924 515081 548 Structural Total 5 5295574 469 TCA cycle 417178 450 5298022 540 O71834 256 Translation Total 25 170477 45 Transport 2871.4 52 7185O2 16 11.4374 579 5031777 448 1172554 1394 5174539 500 1359715 578 11321581 872 1588292 130 11321.583 868 45O3057 225 11374664 452 572993.7 518 12804901 449 573OO33 848 13627252 658 7799988 470 13639817 505 892387O 408 14740547 342 10716563 135 14782O63 5O1 1083522O 94 15318843 17 11612670 690 161926.38 446 128O3281 1395 TCA cycle Total 16 13376991 1396 Transcription 105294 48 1354O606 875 107912 905 13649217 1393 1033182 1400 141496O7 186 1582692 888 14739472 710 2565032 904 14767738 134 45064.45 78O 14778381 294 4507389 3O1 1697.4753 849 6678455 908 Transport Total 22 6912440 287 Tumor-related 12O749 498 98.84738 67 132164 768 1096171 783 11774.38 123 1761696 119 45O7643 930 1890755 782 1056.7164 348 2653775 394 108351.55 928 2734816 741 1086,3907 397 3242069 647 1224-6901 929 3787197 242 12643796 770 39.38539 232 13529047 912 4730158 889 13650639 515 474.2266 781 14725,399 898 4748858 910 14755336 931 4766373 765 15076827 665 4790190 847 15296,762 1388 5296351 859 16160929 769 53OO149 558 Tumor-related Total 16 5451854 530 Zinc finger 1177230 1401 6163124 926 2117022 1402 US 2004/0101874 A1 May 27, 2004 74

Prior to complex I isolation and electrophoresis, mitochon TABLE 2-continued dria were prepared identically from all hearts which were freshly collected, frozen and thawed immediately prior to MTOCHONDRIAL FUNCTIONS OF SELECTED COMPONENTS OF THE HUMAN HEART analysis. FIG. 4 shows the MALDI spectra of peptides from MITOCHONDRIAL PROTEOME the human complex I subunit, NDUFS4 (see Table 3), and its bovine homologue from five different preparations cor MITOCHONDRIAL FUNCTION GENEBANK SEO ID responding to Seven different hearts (five human, including CLASSIFICATION ACCNO. NO: one pooled Sample of mitochondria from three individual 2317769 71.4 hearts, and two bovine hearts). The relative intensities of m/z. 3O21386 1403 1329.6 and 1361.6 (corresponding to peptides without and 4507979 1404 4827O65 1405 with dioxidized tryptophan, FIG. 4A) and 1112.5 and 545418O 14O7 1128.5 (corresponding to peptides without and with oxidized 7671629 464 methionine, FIG. 4B) were used as a rough measure of 14211907 1410 protein oxidation. No correlation was found between the 14286.186 1406 1467.0360 1409 extent of tryptophan oxidation and that of methionine oxi 14755316 3025 dation, Suggesting that they occurred via different mecha 14755456 1408 nisms. Zinc finger Total 13 0.161 The dioxidation of tryptophan was clearly discern able in FIG. 4A (i) and (ii) in which complex I was purified by different methods, Sucrose density gradient centrifugation Example 4 or immunoprecipitation, respectively, but corresponded to mitochondria from the same human heart. This finding OXIDATIVE POST TRANSLATIONAL Suggested that the method of preparation was not a factor in MODIFICATION OF TRYPTOPHAN RESIDUES determining the extent of oxidation, but rather that Such IN CARDIAC MITOCHONDRIAL PROTEINS oxidation was a characteristic of the donor from which the 0158. This example shows the distribution of Sample was obtained (in this case, a 41-year-old male N-formylkynurenine, a product of the dioxidation of tryp Caucasian who died of brain cancer). The other human tophan residues in proteins, throughout the human heart donor, displaying far less extensive oxidation of tryptophan mitochondrial proteome. This oxidized amino acid was as seen in FIG. 4A (iii), was a 62-year-old female Caucasian associated with a distinct Subset of proteins, including an who died of intracranial bleeding. In contrast, NDUFS4 over-representation of complex I Subunits as well as com from a pool of mitochondria from three human hearts plex V subunits and enzymes involved in redox metabolism. displayed an extensively oxidized tryptophan-containing No relationship was observed between the tryptophan modi peptide FIG. 4A (iv). Again the degree of oxidation in the fication and methionine oxidation, a known artifact of pooled Sample was not commensurate with the degree of Sample handling. AS the mitochondria were isolated from oxidation for the methionine-containing fragment FIG. 4B normal human heart tissue and not Subject to any artificially (iv). induced oxidative StreSS, the Susceptible tryptophan residues in this group of proteins appeared, according to non-limiting 0162 Distribution of the oxidatively modified tryptophan theory, to be “hot spots” for oxidation in close proximity to in the MS/MS spectra dataset described in the preceding a Source of reactive oxygen species (ROS) in respiring Examples was assessed by reanalyzing the data with mitochondria. N-formylkynurenine selected as a differential modification 0159) LC/MS/MS data generated from the human heart of tryptophan (+32) using the SonarMSMS algorithm mitochondrial proteome project as described in the preced according to the Supplier's instructions (Genomic Solutions, ing Examples, as well as data for human and bovine proteins Ann Arbor, Mich.). Table 3 lists N-formylkynurenine-con prepared by Sucrose density gradient centrifugation as taining peptides found with peptide expect Scores (Epep) described above, or by immunoprecipitation using antibod values s1x10° (99% confidence); also listed in Table 3 are ies against complex V (ATP synthase) and/or complex I the identifiers for the mitochondrial polypeptide Sequences (NADH dehydrogenase) proteins (see, Table 2), were que from which these peptides derived. Of this list of 51 peptide ried against the human or bovine Subsets of GenBank using sequences from 39 proteins, 9 subunits of complex I had the Sonar MSMS searching algorithm (Genomic Solutions, N-formylkyenurine-containing tryptic peptides and included Ann Arbor, Mich.) with oxidation of methionine (+16 u) and two newly discovered subunits (Table 1, NCBI/Genbank tryptophan (+32 u) specified as differential modifications. Acc. Nos. 13938.442 and 17455445, now 21754001). This Corresponding MALDI spectra were manually inspected. Subset of proteins was used to compare tryptophan oxidation FIG. 3 shows oxidation products of tryptophan from pro Versus methionine oxidation as a function of the ability to teins, including N-formylkynurenine (Structure 2). observe a peptide in any given LC/MS/MS experiment. As 0160 Modifications to complex I subunits in bovine shown in FIG. 5, the numbers of distinct peptides containing heart mitochondria in response to the oxidative StreSS caused methionine (A) and tryptophan (B) were plotted for a given by peroxynitrite treatment were Studied in Vitro, and yielded complex I subunit which had a Sonar MSMS Epep score of evidence of oxidized tryptophan in Several Subunits, both by s1x10°, and on each plot FIG. 5 indicates whether the MALDI TOF and by LC/MS/MS. Surprisingly, the relative corresponding oxidized residue was observed. Methionine intensities of the peaks in the MALDI spectra corresponding oxidation appeared to be directly related to the number of to peptides containing N-formylkynurenine were also high observable peptides that would be expected if oxidation in untreated mitochondria from Some bovine and human were a random Sample-handling artifact. In contrast, tryp heart preparations, although there was Substantial variation. tophan oxidation appeared to be much more Specific to US 2004/0101874 A1 May 27, 2004 75

Selected Subunits, with the greatest modification being noted kDa reductase/isomerase Subunit). In addition, five Subunits for NDUFV1 (51 kDa flavoprotein 1) and NDUFA9 (a 39 of the iron-protein component were oxidized.

TABLE 3

PEPTIDES CONTAINING DOUBLY OXIDIZED TRYPTOPHAN FROM THE CARDIAC MITOCHONDRIAL PROTEOME. Peptide Derived from NCBIf Genbank PROTEIN PEPTIDE Epep Acc. No. DESCRIPTION VFEISPFEPwTR 1.4OE-05 6681764 NDUFA9 FGPIPLGSLGwK 2.3OE-04 6681764 NDUFA9 wLSAEEDVKPAK 18OE-03 6681764 NDUFA9 HAGGVTGGw NLLAVIPGGS 2.1OE-04 2014.9568 NDUFV1 STPLIPK GDARPAEIDSLw'SK 9.4OE-04 2014.9568 NDUFV1 GPDwLGEIK 2.4OE-03 2014.9568 NDUFV1 LAALPENPPAIDwAYYK 3.2OE-05 5453559 ATPase d FO TIDwVAFAEIIPONOK 2.1OE-03 5453559 ATPase d FO YPYwPHOPIENL 7.2OE-03 5453559 ATPase d FO wVVGDENYGEGSSR 8.4OE-08 3600098 aconitase precursor WAEKEGwBLDIR 4.OOE-04 3600098 aconitase precursor LWSNGGLADFTVFAK 2.90E-06 18044943 acyl-Coenzyme A dehydrogenase, very long chain IFGSEAAwk 3.90E-03 18044943 acyl-Coenzyme A dehydrogenase, very long chain ALGVLAOLIwSR 1.1OE-05 4758076 citrate synthase precursor DYwNTLNSGR 7.1OE-04 4758076 citrate synthase precursor KLETAVNLAwTAGNSNTR 1.6OE-05 45O7879 VDAC-1 wNTDNTLGTEITVEDOLAR 5.3OE-03 45O7879 VDAC-1 VVDGAVGAOwLAEFR 4.7OE-05 17458911 dihydrolipoamide S-acetyltransferase VPEANSSwMDTVIR 6.6OE-04 17458911 dihydrolipoamide S-acetyltransferase SAVTALwok 3.7OE-03 4504349 beta globin LLVVYPWTOR 4.3OE-03 4504349 beta-globin RPPEPTTPwOEDPEPEDENL 6.8OE-08 13938442 neuronal protein YEK (ND17.3) NLTOYSwLLDGFPR 1.00E-06 1992.3437 adenylate kinase 3 alpha like FDLNSPWEAFPVYR 2.1OE-OS 1136O2O6 NDUFS3 ASGLGLAwVGR 2.6OE-05 4758714 microsomal glutathione S-transferase 3 GYVEDLWK 2.90E-05 12001992 brain myO25 ASSTSPVEISEwLDOK 4.OOE-05 4503607 electron transfer flavoprotein alpha polypeptide GRPTSTNPIASIFAwTR 6.4OE-05 4504575 isocitrate dehydrogenase 2 (NADP+), mitochondrial GLLTYTSWEDALSR 1.4OE-04 21.411235 NDUFS1 IPwFOYPIIYDIR 1.90E-04 6005854 D-prohibitin GLSDGEwCLVLNVwOK 2.5OE-04 229361 Myoglobin ASwSSLSMDEK 3.OOE-04 592.1895 Cytochrome c oxidase subunit IV isoform 1 LDDLVNWAR 5.3OE-04 21750696 NDUFS7 TLLWTELFR 7.8OE-04 45OS371 NDUFS8 SYGANFSwNK 8.7OE-04 1352896O NDUFS4 ASLEHALVGSPwCGGEPR 9.90E-04 13676336 long-chain acyl-coA thioesterase peroxisomal wEVADLOPOLK 1.2OE-03 21903482 Ubiquinol-cytochrome C reductase complex core protein 2 YEGFFSLWK 1.3OE-03 21361114 mitochondrial carrier; Oxoglutarate carrier US 2004/0101874 A1 May 27, 2004 76

TABLE 3-continued

PEPTIDES CONTAINING DOUBLY OXIDIZED TRYPTOPHAN FROM THE CARDIAC MITOCHONDRIAL PROTEOME. Peptide Derived from NCBIf Genbank PROTEIN PEPTIDE Epep Acc. No. DESCRIPTION LITTOOwLIK 1.4OE-03 13272660 ATP synthase 6 LWEPLVEEPPADOwK 1.5OE-03 4826848 NDUFAS IDEALITWTK 2.OOE-03 15991833 hexokinase 1 wDGOETTLVR 3.3OE-03 458862 fatty acid binding protein, heart; hFABP HwLDSPwPGFFTLDGOPR 3.4OE-03 20541592 2-oxoglutarate dehydroqenase E1 component, mitochondrial precursor AwNGSAEGPGKVER 4.3OE-03 21754001 Unnamed protein product (NDUFB11) ELWFSDDPNVTK 4.7OE-03 4757732 programmed cell death 8 (apoptosis-inducing factor AIF) EOwDTIEELIR 5.3OE-03 4503301 2,4-dienoyl CoA reductase 1 precursor GAwSNVLR 5.3OE-03 86754 carrier ANT wYYNAAGFNK 5.3OE-03 5454152 UCR ubiquinone binding protein (VI) ELDSITPEVLPGwK 5.5OE-03 8131894 Mitofilin APLAEEw)NMTMK 8.1OE-03 4505093 monoamine oxidase B LATFwYYAK 9.1OE-03 22096328 ATP synthase G chain, mitochondrial

0163 From the foregoing it will be appreciated that, fications may be made without deviating from the Spirit and although Specific embodiments of the invention have been Scope of the invention. Accordingly, the invention is not described herein for purposes of illustration, various modi limited except as by the appended claims.

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing Section. A copy of the "Sequence Listing” is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/sequence.html?DocID=20040101874). An electronic copy of the “Sequence Listing” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 119(b)(3).

What is claimed is: therefrom identifying a mitochondrial target for thera 1. A method for identifying a mitochondrial target for peutic intervention. therapeutic intervention in treatment of a disease associated 2. The method of claim 1 wherein the modified polypep with altered mitochondrial function, comprising: tide exhibits altered biological activity. (a) determining a presence, in a biological Sample from a 3. The method of claim 1 wherein the biological sample Subject known to have or Suspected of having a disease is Selected from the group consisting of blood, skin, Skeletal asSociated with altered mitochondrial function, of at muscle, liver and cartilage. least one modified polypeptide, Said modified polypep 4. The method of claim 1 wherein the disease associated tide comprising at least one modification to a polypep with altered mitochondrial function is selected from the tide having an amino acid Sequence as Set forth in any group consisting of Alzheimer's disease, diabetes mellitus, one of SEQ ID NOS 1-3025; and Parkinson's disease, Huntington's disease, osteoarthritis, (b) correlating the modification with at least one disease dystonia, Leber's hereditary optic neuropathy (LHON), asSociated with altered mitochondrial function, and mitochondrial encephalopathy, lactic acidosis, and Stroke US 2004/0101874 A1 May 27, 2004 77

(MELAS), myoclonic epilepsy ragged red fiber Syndrome polypeptide having an amino acid Sequence as Set forth in (MERRF) and cancer. any one of SEQ ID NOS 1-3025. 5. The method of claim 1 wherein the modification is 12. A method for identifying a risk for having or a Selected from the group consisting of an amino acid Substi presence of a disease associated with altered mitochondrial tution, an amino acid insertion, an amino acid deletion, a function, comprising: posttranslational modification and an altered expression (a) determining a presence, in a biological Sample from a level. Subject Suspected of having a disease associated with 6. The method of claim 4 wherein the posttranslational altered mitochondrial function, of at least one modified modification is Selected from the group consisting of gly polypeptide, Said modified polypeptide comprising at cosylation, phosphorylation, nitration, nitrosylation, amida least one modification to a polypeptide having an tion, fatty acylation and oxidative modification. amino acid Sequence as Set forth in any one of SEQ ID NOS 1-3025, wherein the modification correlates with 7. A method of identifying an agent for treating a disease at least one disease associated with altered mitochon asSociated with altered mitochondrial function, comprising: drial function, and therefrom identifying a risk for or (a) contacting a candidate agent with a biological Sample presence of disease. from a Subject having a disease associated with altered 13. A method of identifying an agent for treating a disease mitochondrial function, wherein Said Sample comprises asSociated with altered mitochondrial function, comprising: at least one polypeptide that exhibits altered biological (a) contacting a candidate agent with an isolated polypep activity which accompanies Said disease and wherein tide that exhibits altered biological activity which the polypeptide is Selected from the group consisting of accompanies a disease associated with altered mito (i) a polypeptide having an amino acid Sequence as Set chondrial function, wherein the polypeptide is Selected forth in any one of SEQ ID NOS 1-3025 and (ii) a from the group consisting of (i) a polypeptide having an modified polypeptide that comprises at least one modi amino acid Sequence as Set forth in any one of SEQ ID fication to a polypeptide having an amino acid NOS 1-3025 and (ii) a modified polypeptide that com sequence as set forth in any one of SEQ ID NOS prises at least one modification to a polypeptide having 1-3025; and an amino acid Sequence as Set forth in any one of SEQ ID NOS 1-3025; and (b) determining an increase or decrease in the altered biological activity of the polypeptide in the presence of (b) determining an increase or decrease in the altered the candidate agent relative to the level of the altered biological activity of the polypeptide in the presence of biological activity in the absence of the candidate the candidate agent relative to the level of the altered agent, and therefrom identifying an agent for treating a biological activity in the absence of the candidate disease associated with altered mitochondrial function. agent, and therefrom identifying an agent for treating a disease associated with altered mitochondrial function. 8. The method of claim 7 wherein the altered biological 14. The method of claim 13 wherein the disease associ activity is an indicator of altered mitochondrial function that ated with altered mitochondrial function is selected from the is Selected from the group consisting of ATP biosynthesis, group consisting of Alzheimer's disease, diabetes mellitus, oxidative phosphorylation, calcium uptake, calcium release, Parkinson's disease, Huntington's disease, osteoarthritis, maintenance of inner mitochondrial membrane potential, dystonia, Leber's hereditary optic neuropathy (LHON), mitochondrial permeability transition, ETC-mediated elec mitochondrial encephalopathy, lactic acidosis, and Stroke tron transport and intermembrane Space protein release. (MELAS), myoclonic epilepsy ragged red fiber Syndrome 9. The method of claim 7 wherein the sample is selected (MERRF), and cancer. from the group consisting of a cell, a mitochondria enriched 15. The method of claim 13 wherein the isolated polypep Sample, an isolated mitochondrion and a Submitochondrial tide is present in a preparation that is Selected from the group particle. consisting of a Submitochondrial particle, a proteoliposome 10. The method of claim 7 wherein the disease associated and a mitochondrial protein fraction. with altered mitochondrial function is selected from the 16. A method of identifying an agent for treating a disease group consisting of Alzheimer's disease, diabetes mellitus, asSociated with altered mitochondrial function, comprising: Parkinson's disease, Huntington's disease, Osteoarthritis, (a) administering a candidate agent to a Subject having a dystonia, Leber's hereditary optic neuropathy (LHON), disease associated with altered mitochondrial function; mitochondrial encephalopathy, lactic acidosis, and Stroke and (MELAS), myoclonic epilepsy ragged red fiber Syndrome (b) determining, in a first biological Sample obtained from (MERRF), and cancer. the Subject prior to the Step of administering the can 11. A method of treating a disease associated with altered didate agent and in a Second biological Sample obtained mitochondrial function comprising administering to a Sub from the Subject Subsequent to the Step of administering ject in need thereof an agent that compensates for at least the candidate agent, wherein each of Said first and one biological activity of a polypeptide that exhibits altered Second Samples comprises at least one polypeptide that biological activity which accompanies said disease, wherein exhibits altered biological activity which accompanies the polypeptide is Selected from the group consisting of (i) Said disease and wherein the polypeptide is Selected a polypeptide having an amino acid Sequence as Set forth in from the group consisting of (i) a polypeptide having an any one of SEQ ID NOS 1-3025 and (ii) a modified amino acid Sequence as Set forth in any one of SEQ ID polypeptide that comprises at least one modification to a NOS 1-3025 and (ii) a modified polypeptide that com US 2004/0101874 A1 May 27, 2004 78

prises at least one modification to a polypeptide having 18. The method of claim 16 wherein the sample is selected an amino acid Sequence as Set forth in any one of SEQ from the group consisting of a cell, a mitochondria enriched ID NOS 1-3025, Sample, an isolated mitochondrion and a Submitochondrial an increase or decrease in the altered biological activity of particle. the polypeptide in the Second Sample relative to the level of the altered biological activity in the first 19. The method of claim 16 wherein the disease associ Sample, and therefrom identifying an agent for treating ated with altered mitochondrial function is selected from the a disease associated with altered mitochondrial func group consisting of Alzheimer's disease, diabetes mellitus, tion. Parkinson's disease, Huntington's disease, osteoarthritis, 17. The method of claim 16 wherein the altered biological dystonia, Leber's hereditary optic neuropathy (LHON), activity is an indicator of altered mitochondrial function that is Selected from the group consisting of ATP biosynthesis, mitochondrial encephalopathy, lactic acidosis, and Stroke oxidative phosphorylation, calcium uptake, calcium release, (MELAS), myoclonic epilepsy ragged red fiberSyndrome maintenance of inner mitochondrial membrane potential, (MERRF), and cancer. mitochondrial permeability transition, ETC-mediated elec tron transport and intermembrane Space protein release.