DOCSLIB.ORG

Mitochondrial Ribonuclease P Structure Provides Insight Into the Evolution of Catalytic Strategies for Precursor-Trna 5′ Processing

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mitochondrial Ribonuclease P Structure Provides Insight Into the Evolution of Catalytic Strategies for Precursor-Trna 5′ Processing Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing Michael J. Howarda, Wan Hsin Limb, Carol A. Fierkea,c,1, and Markos Koutmosd,1 aDepartment of Biological Chemistry, bChemical Biology Program, and cDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109; and dDepartment of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814 Edited* by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved August 27, 2012 (received for review May 31, 2012) Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of This structure clearly demonstrates that there is no structural tRNA precursors. Typically these enzymes are ribonucleoproteins homology between PRORP1 and any of the proteins associated with a conserved RNA component responsible for catalysis. How- with either the bacterial or the nuclear human RNase P (4, 5), ever, protein-only RNase P (PRORP) enzymes process precursor revealing that this protein evolved independently from the ri- tRNAs in human mitochondria and in all tRNA-using compartments bonucleoprotein RNase P. Furthermore, the combination of of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and structural and biochemical data suggests that the RNA and conserved among many eukaryotes, having evolved recently as protein-based catalysts use distinct strategies to bind and cleave yeast mitochondrial genomes encode an RNase P RNA. Here we pre-tRNA. In PRORP1, the pentatricopeptide repeat (PPR) report the crystal structure of PRORP1 from A. thaliana at 1.75 Å domain enhances pre-tRNA binding affinity, in contrast to the resolution, revealing a prototypical metallonuclease domain teth- combination of RNA–tRNA and protein–pre-tRNA leader ered to a pentatricopeptide repeat (PPR) domain by a structural contacts in the ribonucleoprotein RNase P (5). Whereas both zinc-binding domain. The metallonuclease domain is a unique PRORP1 and bacterial RNase P are magnesium-dependent high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) enzymes, the metal ions are coordinated by carboxylate side domain that is a member of the PIN domain-like fold superfamily, chains and nonbridging phosphate oxygens/nucleotide carbonyl BIOCHEMISTRY including the FLAP nuclease family. The structural similarity be- groups (5), respectively. These studies allow us to begin to tween PRORP1 and the FLAP nuclease family suggests that they compare, at a molecular level, the catalytic strategies and mo- evolved from a common ancestor. Biochemical data reveal that lecular recognition used by the protein-only and RNA-based conserved aspartate residues in PRORP1 are important for catalytic RNase P enzymes. activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results Results provide a foundation for understanding tRNA maturation in Global Architecture of PRORP1. The crystal structure of a functional organelles. Furthermore, these studies allow for a molecular-level recombinant protein-only RNase P from A. thaliana (residues comparison of the catalytic strategies used by the only known 76–572 with the mitochondrial signal sequence deleted) was naturally evolved protein and RNA-based catalysts that perform solved by single-wavelength anomalous dispersion (SAD) and the same biological function, pre-tRNA maturation, thereby pro- was refined to a resolution of 1.98 Å (Rwork = 18.7%, Rfree = viding insight into the differences between the prebiotic RNA 22.0% with good stereochemistry; Table S1). One molecule is world and the present protein-dominated world. present per asymmetric unit and the final refined model includes residues 95–570 (Fig. 1). PRORP1 adopts a conformation catalytic mechanism | magnesium | molecular recognition that resembles an upside-down “V”, where two arms, each ∼70 Å in length, fold at an angle of ∼56°. Arm 1 is composed of 11 α β ccording to the RNA world hypothesis, RNA played dual -helices, whereas arm 2 is composed of a parallel -sheet fl α roles as carrier of genetic information and catalyst in anked by -helices (Fig. 1). The two arms are attached to a core A β a prebiotic world. However, over eons of evolution, proteins with domain consisting of an antiparallel -sheet. These discrete their expanded 20-aa alphabet and greater structural and func- structural elements represent three domains: the PPR domain (residues 95–292; red in Fig. 1C), the central domain (residues tional complexity took over many RNA-based functions. Struc- 328–357 and 534–570; yellow in Fig. 1C), and the metallonuclease tural insights into this evolutionary transition are limited because domain (residues 358–533; blue in Fig. 1C). of the lack of examples of RNA and protein macromolecules that perform the same biological function in nature. One notable PPR Domain Is Important for Activity and Substrate Affinity. PPR exception is ribonuclease P (RNase P) that catalyzes maturation motifs are often found in tandem and are composed of a helix- of the 5′ end of tRNA across all domains of life. Until recently, turn-helix fold of ∼35 aa (6). These motifs have been implicated all known RNase P enzymes included a catalytic RNA compo- in RNA binding and editing activities and are prevalent among nent. The discovery of a protein-only RNase P [proteinacous RNase P (PRORP)] from human mitochondria and Arabidopsis thaliana has dramatically shifted this paradigm (1–3). These Author contributions: M.J.H., W.H.L., C.A.F., and M.K. designed research; M.J.H., W.H.L., enzymes represent a unique class of metallonucleases and are and M.K. performed research; M.J.H., C.A.F., and M.K. analyzed data; and M.J.H., C.A.F., conserved among many eukaroytes (1, 2). A. thaliana encodes and M.K. wrote the paper. fl three PRORP enzymes (PRORP1, -2, and -3) that catalyze pre- The authors declare no con ict of interest. tRNA processing (2). PRORP1 localizes to the mitochondria *This Direct Submission article had a prearranged editor. and chloroplast whereas PRORP2 and PRORP3 localize to the Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4G23–4G26). nucleus (2), suggesting that protein-based enzymes catalyze pre- 1To whom correspondence may be addressed. E-mail: fi[email protected] or Markos. tRNA maturation in these cellular locations (2, 3). To gain [email protected]. mechanistic and evolutionary insights into the PRORP enzyme This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. family, we crystallized PRORP1 from A. thaliana. 1073/pnas.1209062109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1209062109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Fig. 1. Crystal structure of PRORP1. (A) Structure of PRORP1 (helices shown in blue, strands shown in yellow). (B) Topology map of secondary structure elements. (C) Cartoon depiction of the PRORP1 domain arrangements: PPR domain (residues 95–292; red), central domain (residues 328–357 and 534–570; yellow), and metallonuclease domain (residues 358–533; blue). (D) Electrostatic surface potential. proteins targeted to mitochondria and chloroplasts (7–9). The (11). Similarly, we posit that the PPR domain of PRORP1 PPR domain in PRORP1 is composed of 11 α-helices forming facilitates interactions with pre-tRNA. 5.5 consecutive PPR repeats, each consisting of a helix-turn-helix An N-terminal truncation of PRORP1 (Δ245) that removes 8 hairpin (Figs. 1 and 2A). These tandem helical repeats associate of the 11 helices in the PPR domain decreases pre-tRNA binding to form a right-handed superhelical structure that resembles affinity by 34-fold and catalytic activity by ≥2,000-fold (Fig. 3C) the arrangement of domains in tetratricopeptide repeat (TPR) with little effect on zinc binding (Table S2). Because the PPR motifs. As a result, the PPR domain forms an inner concave domain is located far away from the metal-binding active site, we surface facing the putative active site of the metallonuclease propose that this domain is important for properly orientating domain. A comparative search based on the PPR domain using the leader of pre-tRNA in the active site rather than playing the Dali server (10) reveals structural similarity with a plethora a direct role in catalysis. The electrostatic surface potential for of proteins (or protein domains) that notably include mitochon- PRORP1 (calculated using Apbs) (15) (Fig. 1D) reveals that the drial RNA polymerase (mtRNAP) (11), Get4 (12), proteosomal concave surface facing the putative active site has an overall subunit Rpn6 (13), and small glutamine-rich tetratricopeptide neutral charge, suggesting that the PPR–nucleic acid interaction repeat protein (hSGT) (14) (Fig. 2B). Except for mtRNAP, which is not mainly electrostatic. The only positive patch is found in the contains the first and only structural data on PPR motifs, all of base of the PPR domain closest to the active site, including side the homologous domains contain TPR motifs that are involved in chains from the interior-facing helices and connecting loops. The protein–protein interactions (6). The PPR domain of mtRNAP PPR domain could interact directly with pre-tRNA through con- is proposed to interact with upstream DNA promoter sequences tacts between this positive patch and the negative phosphodiester 2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209062109 Howard et al. Downloaded by guest on October 2, 2021 Fig. 2. PPR domain architecture and structural Zn-binding site. (A) Tandem repeats of the PPR domain. Domain and profile analysis of PRORP1 using PROSITE predicts three PPR motifs (PPR1, residues 96–130; PPR3, residues 175–209; and PPR4, residues 210–244) on the basis of sequence consensus. The structure reveals two additional helix-turn-helix hairpins that adopt the PPR motif (PPR2, residues 131–174; and PPR5, residues 245–278) despite their divergence from the canonical PPR sequence.
Load more

Recommended publications
  • Supporting Information
    Supporting Information Figure S1. The functionality of the tagged Arp6 and Swr1 was confirmed by monitoring cell growth and sensitivity to hydeoxyurea (HU). Five-fold serial dilutions of each strain were plated on YPD with or without 50 mM HU and incubated at 30°C or 37°C for 3 days. Figure S2. Localization of Arp6 and Swr1 on chromosome 3. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 3L, Tel 3R, CEN3, and the RP gene are shown under the panels. Figure S3. Localization of Arp6 and Swr1 on chromosome 4. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) in the whole chromosome region are compared. The position of Tel 4L, Tel 4R, CEN4, SWR1, and RP genes are shown under the panels. Figure S4. Localization of Arp6 and Swr1 on the region including the SWR1 gene of chromosome 4. The binding of Arp6- FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position and orientation of the SWR1 gene is shown. Figure S5. Localization of Arp6 and Swr1 on chromosome 5. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 5L, Tel 5R, CEN5, and the RP genes are shown under the panels. Figure S6. Preferential localization of Arp6 and Swr1 in the 5′ end of genes. Vertical bars represent the binding ratio of proteins in each locus.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Ribonuclease P
    14 Ribonuclease P Sidney Altman Department of Molecular, Cellular and Developmental Biology Yale University New Haven, Connecticut 06520 Leif Kirsebom Department of Microbiology Biomedical Center Uppsala University Uppsala S-751 23, Sweden RNase P was first characterized in Escherichia coli as an enzyme that is required for the processing of the 5؅ termini of tRNA in the pathway of biosynthesis of tRNA from precursor tRNAs (ptRNAs; Altman and Smith 1971; for review, see Altman 1989). Subsequently, it was discov- ered that this enzyme consisted of one protein and one RNA subunit, the latter being the catalytic subunit (Guerrier-Takada et al. 1983). In retro- spect, it should not be surprising that an enzyme with the chemical compo- sition of RNase P exists. Ribosomes are also ribonucleoproteins (RNPs), but they are much more complicated than RNase P. It seems unreasonable that ribosomes, with three RNAs and about 50 proteins, would exist with- out less complex RNPs having come into existence first and having per- sisted throughout evolution. Indeed, relatively simple RNPs with and without catalytic activity have been discovered with regularity during the past 20 years. What important questions about RNase P have not yet been an- swered? Certainly, for the biochemist, problems not yet solved for E. coli RNase P include a full understanding of enzyme–substrate interac- tions, RNA–protein subunit interactions, and the chemical details of the hydrolysis reaction. In every case, much progress has been made, but much has yet to be learned. A second issue of interest to biochemists and “evolutionists” is the puzzle presented by the different compositions of RNase P from eubac- teria (one catalytic RNA and one protein subunit) and from eukaryotes (one RNA subunit and several protein subunits: no understanding yet of which subunit is responsible for catalysis).
    [Show full text]
  • Catalybc Rnas
    Felix Loosli ITG/KIT (Karlsruhe) Developmental neurobiology of vertebrates Introduc*on to Gene*cs Cataly@c RNAs Lewin‘s GeneX Chapter 23 Albert‘s Molecular Biology of the Cell 5th ed. Chapter 6 Ribozyme : a RNA that has catalytic activity Group I and II self splicing introns Ribonuclease P (RNase P: t-RNA maturaon) Viroids Ribosome Evolu@onary consideraons Tetrahymena thermophilus Thomas R. Cech rRNA self-splicing Splicing occurred without added cell extract Sidney Altman Cataly@c RNA of RNase P which contains a RNA molecule essen@al for catalysis rRNA genes Mitochondrial/Chloroplast genes Bound G nucleo@de (cofactor) aacks phosphodiester bond Two consecuve transesterificaons; no ATP required 2nd structure of RNA is essen@al promoter β-gal β-gal tetrahymena intron transfect E. coli transcrip@on and splicing blue colonies on agar plate GTP conc. >rRNA conc. change of 2nd structure of products (products are ‘removed’ from the reac@on) -> reac@on proceeds to comple@on In vitro self splicing is very slow, in vivo proteins have accessory funcons Group I introns share a common 2nd structure: core structure is the ac@ve site Group I introns can also catalyse: Sequence specific RNA cleavage (ribo endonuclease) RNA ligase Phospatase (not related to self-splicing ac@vity) Group II Introns May Code for Multifunction Proteins • Group II introns can autosplice in vitro, but are usually assisted by protein activities encoded by the intron • A single open reading frame codes for a protein with reverse transcriptase activity, maturase activity, a DNA-binding
    [Show full text]
  • Detection of a Novel Avian Influenza a (H7N9)
    Fan et al. BMC Infectious Diseases 2014, 14:541 http://www.biomedcentral.com/1471-2334/14/541 RESEARCH ARTICLE Open Access Detection of a novel avian influenza A (H7N9) virus in humans by multiplex one-step real-time RT-PCR assay Jian Fan1†, David Cui1†, Siuying Lau3, Guoliang Xie1, Xichao Guo1, Shufa Zheng1, Xiaofeng Huang3, Shigui Yang2, Xianzhi Yang1, Zhaoxia Huo1, Fei Yu1, Jianzhou Lou1, Li Tian1, Xuefen Li1, Yuejiao Dong1, Qiaoyun Zhu1 and Yu Chen1,2* Abstract Background: A novel avian influenza A (H7N9) virus emerged in eastern China in February 2013. 413 confirmed human cases, including 157 deaths, have been recorded as of July 31, 2014. Methods: Clinical specimens, including throat swabs, sputum or tracheal aspirates, etc., were obtained from patients exhibiting influenza-like illness (ILIs), especially from those having pneumonia and a history of occupational exposure to poultry and wild birds. RNA was extracted from these samples and a multiplex one-step real-time RT-PCR assay was developed to specifically detect the influenza A virus (FluA). PCR primers targeted the conserved M and Rnase P (RP) genes, as well as the hemagglutinin and neuraminidase genes of the H7N9 virus. Results: The multiplex assay specifically detected the avian H7N9 virus, and no cross-reaction with other common respiratory pathogens was observed. The detection limit of the assay was approximately 0.05 50% tissue culture infective doses (TCID50), or 100 copies per reaction. Positive detection of the H7N9 virus in sputum/tracheal aspirates was higher than in throat swabs during the surveillance of patients with ILIs. Additionally, detection of the matrix (M) and Rnase P genes aided in the determination of the novel avian H7N9 virus and ensured the quality of the clinical samples.
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • A Role of Human Rnase P Subunits, Rpp29 and Rpp21, in Homology
    www.nature.com/scientificreports OPEN A role of human RNase P subunits, Rpp29 and Rpp21, in homology directed-repair of double-strand Received: 22 December 2016 Accepted: 22 March 2017 breaks Published: xx xx xxxx Enas R. Abu-Zhayia1, Hanan Khoury-Haddad1, Noga Guttmann-Raviv1, Raphael Serruya2, Nayef Jarrous2 & Nabieh Ayoub1 DNA damage response (DDR) is needed to repair damaged DNA for genomic integrity preservation. Defective DDR causes accumulation of deleterious mutations and DNA lesions that can lead to genomic instabilities and carcinogenesis. Identifying new players in the DDR, therefore, is essential to advance the understanding of the molecular mechanisms by which cells keep their genetic material intact. Here, we show that the core protein subunits Rpp29 and Rpp21 of human RNase P complex are implicated in DDR. We demonstrate that Rpp29 and Rpp21 depletion impairs double-strand break (DSB) repair by homology-directed repair (HDR), but has no deleterious effect on the integrity of non-homologous end joining. We also demonstrate that Rpp29 and Rpp21, but not Rpp14, Rpp25 and Rpp38, are rapidly and transiently recruited to laser-microirradiated sites. Rpp29 and Rpp21 bind poly ADP-ribose moieties and are recruited to DNA damage sites in a PARP1-dependent manner. Remarkably, depletion of the catalytic H1 RNA subunit diminishes their recruitment to laser-microirradiated regions. Moreover, RNase P activity is augmented after DNA damage in a PARP1-dependent manner. Altogether, our results describe a previously unrecognized function of the RNase P subunits, Rpp29 and Rpp21, in fine- tuning HDR of DSBs. The human genome is susceptible to endogenous and exogenous DNA damaging agents1, 2.
    [Show full text]
  • 4. Chemical Stability of Nucleic Acids
    4. Chemical stability of nucleic acids NH2 N 8 N H O 2 Heterocycle: stable - O N P O N H except for reactions with alkylating O HO agents, nitrite and radicals Cleavage of the N-glycosidic bond Purinyl-N-glycosides are cleaved by acid HO OH or hydrazine treatment Cleavage of the phosphodiester linkage: RNA: hydrolyzed by 0.3 M KOH DNA: stable Chemical hydrolysis of phosphoesters Comparison: Ester hydrolysis of carboxylic acid O - O HO- O - R O-R´ + O-R´ R O-R´ R OH OH O tetrahedral transition state Acyl cleavage R O- + HO-R´ Proof: Reaction in 18O labeled water O + H2O, H O + R O R OH HO Alkyl cleavage in specific cases only Chemical hydrolysis of phosphoesters 1.Phosphotriester Apical ligand 1 M NaOH OMe O O 24 h, 20 °C Equatorial ligand - P O P MeO OMe MeO P MeO OMe OMe OMe O- + OH MeOH Trimethyl phosphate Dimethyl phosphate Transition state: dsp3-hybrid! Phosphoryl cleavage Chemical hydrolysis of phosphoesters 2.Phosphodiester 1 M NaOH O 100 °C O P P MeO O-Me MeO O- + MeOH O- O- HO- Dimethyl phosphate Methyl phosphate Alkyl cleavage Half life time: 16 days! Chemical hydrolysis of phosphoesters 2.Phosphodiester HO- H Cyclic phosphodiester (ring strain!) H O - O HO O O O P P O- O O-Me O P O - O- O O O- 2-Hydroxyethyl-methyl-phosphate 2-Hydroxyethyl phosphate Δ H = -26.8 kJ/mol Cf: Dimethyl phosphate: -7.5 kJ/ mol Half life time at 25 °C 25 min! Neighboring group participation Anchimeric assistance OH O H+ O OH OH O-Me + P P P O O H2O O O-Me O O O 2-Hydroxyethyl-methyl Ethylene-phosphate phosphate O 70% 30% P O O „Pseudorotation“: Interchange
    [Show full text]
  • Global Proteome Changes in Liver Tissue 6 Weeks After FOLFOX Treatment of Colorectal Cancer Liver Metastases
    Proteomes 2016, 4, 30; doi:10.3390/proteomes4040030 S1 of S5 Supplementary Materials: Global Proteome Changes in Liver Tissue 6 Weeks After FOLFOX Treatment of Colorectal Cancer Liver Metastases Jozef Urdzik, Anna Vildhede, Jacek R. Wiśniewski, Frans Duraj, Ulf Haglund, Per Artursson and Agneta Norén Table S1. Clinical data. FOLFOX All Patients No Chemotherapy Clinical Characteristics Expressed By Treatment p-Value (n = 15) (n = 7) (n = 8) Gender (male) n (%) 11 (73%) 5 (63%) 6 (86%) 0.57 Age (years) median, IQR 59 (58–69) 63 (58–70) 58 (53–70) 0.40 BMI median, IQR 25.6 (24.1–30.0) 25.0 (23.7–27.6) 29.8 (24.2–31.0) 0.19 Number of FOLFOX cures median, IQR - 5 (4–7) - Delay after FOLFOX cessation median, IQR - 6 (5–8) - (weeks) Major liver resection n (%) 12 (80%) 7 (88%) 5 (71%) 0.57 Bleeding preoperatively (mL) median, IQR 600 (300–1700) 450 (300–600) 1100 (400–2200) 0.12 Transfusion units median, IQR 0 (0–0) 0 (0–0) 0 (0–0) 1.00 peroperatively Transfusion units median, IQR 0 (0–0) 0 (0–0) 0 (0–0) 0.69 postoperatively Hospital stay (days) median, IQR 10 (8–15) 10 (8–14) 10 (7–16) 0.78 Clavien complication grade 3 n (%) 5 (33%) 2 (25%) 3 (43%) 0.61 or more Table S2. Classifying proteins according Recursive Feature Elimination-Support Vector Machine model resulting in list of 184 proteins with classifying error rate 20%. Gene Names Fold Unique Protein Names Ranks Peptides Alt. Protein ID Change Peptides MCM2 DNA replication licensing factor MCM2 0 3.40 12 12 CAMK2G; Calcium/calmodulin-dependent protein kinase type CAMK2A; II subunit gamma;
    [Show full text]
  • Supplementary Materials
    Supplementary materials Sagittal Craniosynostosis with Uncommon Anatomical Pathologies in a 56-Year-Old Male Cadaver Andrey Frolov, Craig Lawson, Joshua Olatunde, James T. Goodrich, and John R. Martin, III Methods CT Imaging The embalmed cadaveric head specimen underwent a CT scanning at SLU Hospital using Siemens SOMATOM Definition Flash system (140 kV; 119 mAs; slice thickness: 3mm by 3mm interval; detector size: 0.6 mm, 128 rows of detectors, pitch of 0.6). The images were obtained with standard resolution and analyzed using Syngo Fast-View software. Measurement of Skull Bone Thickness The thickness of mesocephalic skull bones was determined exactly as described in [1] using a digital caliper. The respective male skulls were purchased from Osta International (White Rock, Canada). The scaphocephalic bone thickness was derived from the respective calibrated CT images of the embalmed scaphocephalic head at the anatomical points matching those of mesocephalic skulls. The direct measurement of bone thickness in the scaphocephalic head which would require its maceration was not performed due to the specimen preservation for additional anatomical studies. Anatomical Dissection Craniectomy A craniectomy was conducted to examine the extent of the scaphocephaly in the intracranial space. The dissection of the specimen was performed at Saint Louis University School of Medicine’s Practical Anatomy and Surgical Education Learning Center at the 27th Annual Craniofacial Surgery and Transfacial Approaches to the Skull Base workshop by James T. Goodrich, M.D., Ph.D., D.Sc. (Honoris Causis) (Albert Einstein College of Medicine, New York, NY). The specimen was thawed at room temperature for 48 hours prior to dissection.
    [Show full text]
  • Ribonuclease P: Anenzyme with an Essential Rnacomponent
    Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3717-3721, August 1978 Biochemistry Ribonuclease P: An enzyme with an essential RNA component (endoribonuclease/precursor tRNA substrates/RNA subunit) BENJAMIN C. STARK*, RYSZARD KOLEt, EMMA J. BOWMAN*, AND SIDNEY ALTMAN§ Department of Biology, Yale University, New Haven, Connecticut 06520 Communicated by Joseph G. Gall, June 8,1978 ABSTRACT The activity of ribonuclease P on precursor same buffer. The RNase P eluted after the rRNA peak and tRNA substrates from Escherichia coli can be abolished by before the 4S RNA peak. The active fractions were pooled and pretreatment of this enzyme with micrococcal nuclease or concentrated as described above and then applied to a Sephadex pancreatic ribonuclease A, as well as by proteases and by ther- mal denaturation. Highly purified RNase P exhibits one prom- G-200 column (1 X 90 cm) equilibrated and eluted in the same inent RNA and one prominent polypeptide com nent when manner as the Sepharose 4B column. The RNase P eluted just examined in polyacrylamide gels containing sodum dodecyl after the void volume. This material was again pooled, con- sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is centrated, redissolved in 2 M (NH4)2SO4 in buffer B, applied characteristic of a protein-RNA complex. The activity of RNase to an n-octyl-Sepharose (a gift of H. Taira, Yale University, P is inhibited by various RNA molecules. The presence of a New Haven, CT) column (0.5 X 2 cm) that had been equili- discrete RNA component in RNase P appears to be essential for brated with 2 M in buffer and then eluted with enzymatic function.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]