DOCSLIB.ORG

Tryptic Dissection and Reconstitution of Translocation Activity for Nascent Presecretory Proteins Across Microsomal Membranes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Tryptic Dissection and Reconstitution of Translocation Activity for Nascent Presecretory Proteins Across Microsomal Membranes Proc. Natd. Acad. Sci. USA Vol. 76, No. 4, pp. 1795-1799, April 1979 Cell Biology Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes (prolactin mRNA/translation in reticulocyte lysate/signal and ribosome receptor domain/transport domain/signal peptidase) PETER WALTER, ROBERT C. JACKSON*, MADELEINE M. MARCUS, VISHWANATH R. LINGAPPA, AND GUNTER BLOBEL Laboratory of Cell Biology, The Rockefeller University, New York, New York 10021 Communicated by Fritz Lipmann, December 26,1978 ABSTRACT The ability of microsomal membranes to tionally competent components which were separated by translocate nascent presecretory proteins across their lipid bi- centrifugation. Translocation activity of the membrane can be layer into the intravesicular space was investigated by using regenerated by recombination of the fractions. trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity METHODS of microsomal membranes into components that can be sepa- rated into two fractions, one soluble and the other membrane Preparation of Stripped Microsomal Membranes. Rough bound. The trypsinized membrane fraction has lost its translo- microsomal membranes were prepared from freshly excised cation activity. Addition of the trypsin-generated soluble frac- dog pancreas (11) as described (12), with the following excep- tion, however, results in reconstitution of translocation activity. tions: all buffers contained 1 mM dithiothreitol and the rough These results are compatible with the notion proposed in the microsomes were collected by centrifugation for 2.5 hr at signal hypothesis that the translocation activity of the micro- somal membrane resides in transmembrane protein(s). We 140,000 X gay. All procedures were done at 2-4°C. The re- propose that trypsin effects solubilization from the membrane sulting pellet, consisting of rough microsomes, was resuspended of cytosol-exposed domain(s) involved in recognition of the by manual homogenization in a Potter-Elvehjem homogenizer signal sequence or ribosome or both, leaving behind mem- in solution A (250 mM sucrose/50 mM triethanolamine-HCI, brane-integrated domain(s) that provide the environment for pH 7.5/1 mM dithiothreitol) to a concentration of 50 A2g the passage of the nascent chain across the membrane. Signal units/ml. [All absorbance determinations of membrane solu- peptidase activity was unaffected by trypsinization of micro- somal vesicles consistent with a localization of the active site tions were done in 2% sodium dodecyl sulfate (NaDodSO4).] of this enzyme on the cisternal side of the vesicles. Membrane-bound polysomes and peripheral membrane proteins were removed by extracting rough microsomes first Most of the secretory proteins (1) and several membrane pro- with EDTA and then with high salt. A solution of rough mi- teins (2-4) so far investigated are synthesized as preproteins crosomes was incubated for 2 min at 2-40C with 1 vol of solu- containing an amino-terminal extension (signal peptide) when tion B (like solution A, except that it was 46 mM in EDTA); their mRNAs are translated in a cell-free system in the absence 20-ml aliquots were transferred into 45-ml centrifuge tubes of microsomal membranes. The signal peptide portion of the underlayered with 15 ml of solution C (like solution A, except nascent chain has been proposed (5) to contain the necessary that it contained 0.5 M sucrose) and centrifuged for 1 hr at information to direct the translating ribosome to form a func- 140,000 X gav- The resulting pellet of EDTA-stripped micro- tional complex with receptor(s) of the rough endoplasmic re- somal membranes was resuspended in solution A to a concen- ticulum membrane and thereby to provide the conditions for tration of 50 A2g units/ml (yield after EDTA stripping was a unidirectional translocation of the nascent peptide chain across t--30-40% of the A280 of the original rough microsomes). Ice- the membrane into the intracisternal space. Before the nascent cold 2.0 M KCl was slowly added to a final concentration of 0.5 chain is completed, the signal peptide is removed by signal M. The membranes were centrifuged for 1 hr at 140,000 X gv, peptidase, an endoprotease located on the cisternal side of the Excess KCI was removed from the sedimented membranes by membrane (6), except for chicken ovalbumin (7), which is resuspending them in solution A and centrifuging again through proposed to contain an uncleavable signal sequence (8). a cushion of 15 ml of solution C for 1 hr at 140,000 X gav The Little is known about the recognition-transport machinery resulting pellet was resuspended in solution A to a concentration in the membrane or the ribosomal components interacting with of 40 A20 units/ml (yield after KC1 stripping was -60-70% of it. Two membrane proteins, restricted in their location to rough the A20 of the EDTA-stripped microsomes). Aliquots were endoplasmic reticulum, have been proposed (9) to function as frozen in liquid N2 and stored at -80°C. This preparation is ribosome receptors, primarily on the basis of their physical as- called stripped microsomal membranes. sociation with membrane-bound ribosomes. Furthermore, it Limited Trypsin Digestion of Stripped Microsomal has been reported (10) that microsomal membranes can be Membranes. All operations were performed at 1-40C. Volumes largely depleted of their translocation activity when extracted for digestion were 15-500,l. For a typical trypsin treatment, with a solution of relatively high ionic strength (500 mM KCl) 78 ,ul of stripped microsomal membranes was incubated for 30 and that activity can be regenerated when the salt extract is min on ice with 2 ,lI of a trypsin solution of the appropriate added back to the depleted membranes. concentration so that the final concentration of trypsin was We present a way to dissect the translocation activity in the 5-600 ,ug/ml. The trypsin solution was freshly prepared by microsomal membrane by limited trypsinization into func- dilution of a stock solution (10 mg of trypsin per ml in 10 mM triethanolamine-HCl, pH 7.5) kept in small aliquots at -800C. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviation: NaDodSO4, sodium dodecyl sulfate. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Department of Biochemistry, Dartmouth Medical this fact. School, Hanover, NH 03755. 1795 Downloaded by guest on September 28, 2021 1796 Cell Biology: Walter et at. Proc. Natl. Acad. Sci. USA 76 (1979) Trypsin digestion was terminated by addition of 10,ul of RESULTS Trasylol. Aliquots of this mixture were assayed directly in the To investigate the nature of the translocation activity that allows cell-free protein-synthesizing system (see Fig. 1) or fractionated the nascent chains of secretory proteins to be transferred into from aliquots by high-speed centrifugation into a membrane the cisternae of the rough endoplasmic reticulum, we sought and a supernatant fraction. limited proteolysis of microsomal vesicles as a means to dissect To prepare the membrane fraction, we layered a 40-,ul ali- this activity into functional components. The rationale was that quot of the digestion mixture over a discontinuous sucrose the translocation activity might be represented by transmem- gradient consisting of 50 t1A of 0.5 M sucrose over 25 ,l of 2.0 brane protein(s) (5) that should contain cytosol-exposed do- M sucrose in 175-Aul nitrocellulose tubes; both sucrose solutions main(s) that are readily accessible to proteolytic enzymes. On also contained 10 mM triethanolamine1HCl, pH 7.5/1 mM the premise that these domains are globular in nature, condi- dithiothreitol/1% Trasylol. Centrifugation for 5 min at 30 tions were sought that would permit their proteolytic severance lb./inch2 (1 lb./inch2 = 6.9 kPa), generating -100,000 X g., in a form sufficiently intact so that their readdition to the pro- was in a Beckman Airfuge kept in a cold room (4-8°C). The teolytically depleted and inactivated membranes would restore membranes banded neatly at the 2.0 M/0.5 M sucrose interface translocation activity. and, by a Hamilton syringe, could be quantitatively recovered Translocation activity of microsomal membranes can be (after removal of the 2.0 M sucrose layer). The original volume assayed in cell-free protein-synthesizing systems (12, 15, 16). of 40 A.l was restored by addition of 20 MAl of 10mM triethano- The activity is tightly coupled to translation-i.e., it is expressed lamine.HCl, pH 7.5/1 mM dithiothreitol. Membranes prepared only during but not after synthesis of the polypeptide chain; this way will abbreviated moreover, it is accompanied by cleavage ("processing") of the be Mb(x), in which x indicates the signal peptide (13, 15, 17). We used a nuclease-treated rabbit concentration of trypsin in ,ug/ml of digestion mixture. reticulocyte lysate system programmed with bovine pituitary To prepare the supernatant fraction, we centrifuged the RNA (12, 13). In the absence of microsomal membranes there remaining 45-Al aliquot for 10 min at 30 lb./inch2 in the Air- is synthesis of a larger molecule, preprolactin, whereas in the fuge. The supernatant was withdrawn with a Hamilton syringe presence of adequate amounts of membranes there is synthesis and recentrifuged for 10 min at 30 lb./inch2. Supernatants primarily of "processed" prolactin which is similar, if not prepared this way will be abbreviated Sup(x), in which x in- identical, to mature prolactin (13). Among the two forms of dicates the concentration of trypsin in ,ug/ml of digestion prolactin molecules that are synthesized in the presence of mixture. One equivalent of Mb(x) or Sup(x) is arbitrarily de- microsomal membranes, only the "processed" prolactin, but fined as the amount of material that is derived from the not the unprocessed preprolactin molecules, were segregated equivalent (0.035 A28o unit) of stripped microsomal mem- within the microsomal vesicles (12, 13).
Load more

Recommended publications
  • The Secretory Proprotein Convertase Neural Apoptosis-Regulated Convertase 1 (NARC-1): Liver Regeneration and Neuronal Differentiation
    The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation Nabil G. Seidah*†, Suzanne Benjannet*, Louise Wickham*, Jadwiga Marcinkiewicz*, Ste´phanie Be´langer Jasmin‡, Stefano Stifani‡, Ajoy Basak§, Annik Prat*, and Michel Chre´ tien§ *Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC, H2W 1R7 Canada; ‡Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4 Canada; and §Regional Protein Chemistry Center and Diseases of Aging Unit, Ottawa Health Research Institute, Ottawa Hospital, Civic Campus, 725 Parkdale Avenue, Ottawa, ON, K1Y 4E9 Canada Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved December 5, 2002 (received for review September 10, 2002) Seven secretory mammalian kexin-like subtilases have been iden- LP251 (Eli Lilly, patent no. WO 02͞14358 A2) recently cloned tified that cleave a variety of precursor proteins at monobasic and by two pharmaceutical companies. NARC-1 was identified via dibasic residues. The recently characterized pyrolysin-like subtilase the cloning of cDNAs up-regulated after apoptosis induced by SKI-1 cleaves proproteins at nonbasic residues. In this work we serum deprivation in primary cerebellar neurons, whereas LP251 describe the properties of a proteinase K-like subtilase, neural was discovered via global cloning of secretory proteins. Aside apoptosis-regulated convertase 1 (NARC-1), representing the ninth from the fact that NARC-1 mRNA is expressed in liver ϾϾ member of the secretory subtilase family. Biosynthetic and micro- testis Ͼ kidney and that the gene localizes to human chromo- sequencing analyses of WT and mutant enzyme revealed that some 1p33-p34.3, no information is available on NARC-1 ac- human and mouse pro-NARC-1 are autocatalytically and intramo- tivity, cleavage specificity, cellular and tissue expression, and lecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)2 motif, a biological function.
    [Show full text]
  • Chymotrypsin-Like Peptidases in Insects
    Chymotrypsin-like peptidases in insects Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Fachbereich Biologie/Chemie der Universität Osnabrück vorgelegt von Gunnar Bröhan Osnabrück, Mai 2010 TABLE OF CONTENTS I Table of contents 1. Introduction 1 1.1. Serine endopeptidases 1 1.2. The structure of S1A chymotrypsin-like peptidases 2 1.3. Catalytic mechanism of chymotrypsin-like peptidases 6 1.4. Insect chymotrypsin-like peptidases 9 1.4.1. Chymotrypsin-like peptidases in insect immunity 9 1.4.2. Role of chymotrypsin-like peptidases in digestion 14 1.4.3. Involvement of chymotrypsin-like peptidases in molt 16 1.5. Objective of the work 18 2. Material and Methods 20 2.1. Material 20 2.1.1. Culture Media 20 2.1.2. Insects 20 2.2. Molecular biological methods 20 2.2.1. Tissue preparations for total RNA isolation 20 2.2.2. Total RNA isolation 21 2.2.3. Reverse transcription 21 2.2.4. Quantification of nucleic acids 21 2.2.5. Chemical competent Escherichia coli 21 2.2.6. Ligation and transformation in E. coli 21 2.2.7. Preparation of plasmid DNA 22 2.2.8. Restriction enzyme digestion of DNA 22 2.2.9. DNA gel-electrophoresis and DNA isolation 22 2.2.10. Polymerase-chain-reaction based methods 23 2.2.10.1. RACE-PCR 23 2.2.10.2. Quantitative Realtime PCR 23 2.2.10.3. Megaprimer PCR 24 2.2.11. Cloning of insect CTLPs 25 2.2.12. Syntheses of Digoxigenin-labeled DNA and RNA probes 26 2.2.13.
    [Show full text]
  • Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage
    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.11.378711; this version posted November 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage A. Manuel Liaci1, Barbara Steigenberger2,3, Sem Tamara2,3, Paulo Cesar Telles de Souza4, Mariska Gröllers-Mulderij1, Patrick Ogrissek1,5, Siewert J. Marrink4, Richard A. Scheltema2,3, Friedrich Förster1* Abstract The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo- microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments. Keywords Signal Peptidase Complex, Signal Peptide, Protein Maturation, Membrane Thinning, cryo-EM, Crosslinking Mass Spectrometry, Molecular Dynamics Simulations, Protein Secretion, ER Translocon 1Cryo-Electron Microscopy, Bijvoet Centre for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
    [Show full text]
  • Trypsinogen Isoforms in the Ferret Pancreas Eszter Hegyi & Miklós Sahin-Tóth
    www.nature.com/scientificreports OPEN Trypsinogen isoforms in the ferret pancreas Eszter Hegyi & Miklós Sahin-Tóth The domestic ferret (Mustela putorius furo) recently emerged as a novel model for human pancreatic Received: 29 June 2018 diseases. To investigate whether the ferret would be appropriate to study hereditary pancreatitis Accepted: 25 September 2018 associated with increased trypsinogen autoactivation, we purifed and cloned the trypsinogen isoforms Published: xx xx xxxx from the ferret pancreas and studied their functional properties. We found two highly expressed isoforms, anionic and cationic trypsinogen. When compared to human cationic trypsinogen (PRSS1), ferret anionic trypsinogen autoactivated only in the presence of high calcium concentrations but not in millimolar calcium, which prevails in the secretory pathway. Ferret cationic trypsinogen was completely defective in autoactivation under all conditions tested. However, both isoforms were readily activated by enteropeptidase and cathepsin B. We conclude that ferret trypsinogens do not autoactivate as their human paralogs and cannot be used to model the efects of trypsinogen mutations associated with human hereditary pancreatitis. Intra-pancreatic trypsinogen activation by cathepsin B can occur in ferrets, which might trigger pancreatitis even in the absence of trypsinogen autoactivation. Te digestive protease precursor trypsinogen is synthesized and secreted by the pancreas to the duodenum where it becomes activated to trypsin1. Te activation process involves limited proteolysis of the trypsinogen activation peptide by enteropeptidase, a brush-border serine protease specialized for this sole purpose. Te activation peptide is typically an eight amino-acid long N-terminal sequence, which contains a characteristic tetra-aspartate motif preceding the activation site peptide bond, which corresponds to Lys23-Ile24 in human trypsinogens.
    [Show full text]
  • Role of the Amino Terminus in Intracellular Protein Targeting to Secretory Granules Teresa L
    In Vitro Mutagenesis of Trypsinogen: Role of the Amino Terminus in Intracellular Protein Targeting to Secretory Granules Teresa L. Burgess,* Charles S. Craik,** Linda Matsuuchi,* and Regis B. Kelly* * Department of Biochemistry and Biophysics, and *Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 Abstract. The mouse anterior pituitary tumor cell expressed in AtT-20 cells to determine whether intra- line, AtT-20, targets secretory proteins into two distinct cellular targeting could be altered. Replacing the tryp- intracellular pathways. When the DNA that encodes sinogen signal peptide with that of the kappa-immu- trypsinogen is introduced into AtT-20 cells, the protein noglobulin light chain, a constitutively secreted is sorted into the regulated secretory pathway as protein, does not alter targeting to the regulated secre- efficiently as the endogenous peptide hormone ACq'H. tory pathway. In addition, deletion of the NH2-terminal In this study we have used double-label immunoelec- "pro" sequence of trypsinogen has virtually no effect tron microscopy to demonstrate that trypsinogen on protein targeting. However, this deletion does affect colocalizes in the same secretory granules as ACTH. the signal peptidase cleavage site, and as a result the In vitro mutagenesis was used to test whether the in- enzymatic activity of the truncated trypsin protein is formation for targeting trypsinogen m the secretory abolished. We conclude that neither the signal peptide granules resides at the amino (NH2) terminus of the nor the 12 NH2-terminal amino acids of trypsinogen protein. Mutations were made in the DNA that en- are essential for sorting to the regulated secretory codes trypsinogen, and the mutant proteins were pathway of AtT-20 cells.
    [Show full text]
  • Chem331 Tansey Chpt4
    3/10/20 Adhesion Covalent Cats - Proteases Roles Secretion P. gingivalis protease signal peptidases Immune Development Response T-cell protease matriptase 4 classes of proteases: Serine, Thiol (Cys), Acid Blood pressure Digestion regulation (Aspartyl), & Metal (Zn) trypsin renin Function Protease ex. Serine Cysteine trypsin, subtilisin, a-lytic Complement protease protease Nutrition Cell fusion protease Fixation hemaglutinase Invasion matrixmetalloproteases CI protease Evasion IgA protease Reproduction Tumor ADAM (a disintegrain and Invasion Adhesion collagenase and metalloproteinase) Fertilization signal peptidase, viral Processing acronase proteases, proteasome Signaling caspases, granzymes Fibrinolysis Pain Sensing tissue plasminogen kallikrein Acid Metallo- actvator protease protease Animal Virus Hormone Replication Processing HIV protease Kex 2 Substrate Specificity Serine Proteases Binding pocket is responsible for affinity Ser, His and Asp in active site Ser Proteases Multiple Mechanism Chymotrypsin Mechanism •Serine not generally an active amino acid for acid/base catalysis Nucleophilic attack •Catalytic triad of serine, histadine and aspartate responsible for the reactivity of serine in this active site •Covalent catalysis, Acid/base Catalysis, transition state bindingand Proximity mechanisms are used •Two phase reaction when an ester is used – burst phase - E +S initial reactions – steady state phase - EP -> E + P (deacylation) – The first step is the covalent catalysis - where the substrate actually is bound to the enzyme itself
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Intrinsic Evolutionary Constraints on Protease Structure, Enzyme
    Intrinsic evolutionary constraints on protease PNAS PLUS structure, enzyme acylation, and the identity of the catalytic triad Andrew R. Buller and Craig A. Townsend1 Departments of Biophysics and Chemistry, The Johns Hopkins University, Baltimore MD 21218 Edited by David Baker, University of Washington, Seattle, WA, and approved January 11, 2013 (received for review December 6, 2012) The study of proteolysis lies at the heart of our understanding of enzyme evolution remain unanswered. Because evolution oper- biocatalysis, enzyme evolution, and drug development. To un- ates through random forces, rationalizing why a particular out- derstand the degree of natural variation in protease active sites, come occurs is a difficult challenge. For example, the hydroxyl we systematically evaluated simple active site features from all nucleophile of a Ser protease was swapped for the thiol of Cys at serine, cysteine and threonine proteases of independent lineage. least twice in evolutionary history (9). However, there is not This convergent evolutionary analysis revealed several interre- a single example of Thr naturally substituting for Ser in the lated and previously unrecognized relationships. The reactive protease catalytic triad, despite its greater chemical similarity rotamer of the nucleophile determines which neighboring amide (9). Instead, the Thr proteases generate their N-terminal nu- can be used in the local oxyanion hole. Each rotamer–oxyanion cleophile through a posttranslational modification: cis-autopro- hole combination limits the location of the moiety facilitating pro- teolysis (10, 11). These facts constitute clear evidence that there ton transfer and, combined together, fixes the stereochemistry of is a strong selective pressure against Thr in the catalytic triad that catalysis.
    [Show full text]
  • Specific, Gene Expression Signatures in HIV-1 Infection1
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Qingsheng Li Publications Papers in the Biological Sciences 2009 Microarray Analysis of Lymphatic Tissue Reveals Stage- Specific, Gene Expression Signatures in HIV-1 Infection1 Qingsheng Li University of Minnesota, [email protected] Anthony J. Smith University of Minnesota Timothy W. Schacker University of Minnesota John V. Carlis University of Minnesota Lijie Duan University of Minnesota See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/biosciqingshengli Li, Qingsheng; Smith, Anthony J.; Schacker, Timothy W.; Carlis, John V.; Duan, Lijie; Reilly, Cavan S.; and Haase, Ashley T., "Microarray Analysis of Lymphatic Tissue Reveals Stage- Specific, Gene Expression Signatures in HIV-1 Infection1" (2009). Qingsheng Li Publications. 8. https://digitalcommons.unl.edu/biosciqingshengli/8 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Qingsheng Li Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Qingsheng Li, Anthony J. Smith, Timothy W. Schacker, John V. Carlis, Lijie Duan, Cavan S. Reilly, and Ashley T. Haase This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ biosciqingshengli/8 NIH Public Access Author Manuscript J Immunol. Author manuscript; available in PMC 2013 January 23. Published in final edited form as: J Immunol. 2009 August 1; 183(3): 1975–1982. doi:10.4049/jimmunol.0803222. Microarray Analysis of Lymphatic Tissue Reveals Stage- Specific, Gene Expression Signatures in HIV-1 Infection1 $watermark-text $watermark-text $watermark-text Qingsheng Li2,*, Anthony J.
    [Show full text]
  • The Role of Elastases in Pancreatic Diseases
    The role of elastases in pancreatic diseases Ph.D. Thesis Anna Zsófia Tóth M.D. Supervisors: Prof. Péter Hegyi, M.D., Ph D., D.Sc. Prof. Miklós Sahin-Tóth, M.D., Ph.D., D.Sc Doctoral School of Theoretical Medicine Szeged 2018. Content Content ....................................................................................................................................... 1 1. List of abbreviations ........................................................................................................... 3 2. Introduction ......................................................................................................................... 5 2.1. Exocrin pancreatic insufficiency ................................................................................. 5 2.2. Pancreatic elastases ...................................................................................................... 5 2.3. Pancreatic function tests based on detection of elastases ............................................ 7 2.4. Possible role of elastases in chronic pancreatitis ......................................................... 8 3. Aims .................................................................................................................................. 10 3.1. ScheBo Pancreatic Elastase 1 Test Study .................................................................. 10 3.2. Genetic analysis study ............................................................................................... 10 4. Materials and methods .....................................................................................................
    [Show full text]
  • Proline Residues in the Maturation and Degradation of Peptide Hormones and Neuropeptides
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 234, number 2, 251-256 FEB 06071 July 1988 Review Letter Proline residues in the maturation and degradation of peptide hormones and neuropeptides Rolf Mentlein Anatomisches Institut der Christian-Albrechts Universitiit zu Kiel, OlshausenstraJe 40.2300 Kiel I, FRG Received 4 May 1988; revised version received 23 May 1988 The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro- sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosyn- thesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which arc then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localiza- tion of the protease is not clarified. Neuropeptide; Peptide hormone; Proteolytic processing; Degradation; Dipeptidyl (amino)peptidase; Prolyl endopeptidase 1. INTRODUCTION 2. DIRECTION OF THE PROTEOLYTIC MATURATION OF REGULATORY The unique cyclic and imino structure of the PEPTIDES BY PROLINE RESIDUES amino acid proline influences not only the confor- mation of peptide chains, but also restricts the at- Regulatory peptides are synthesized as part of tack of proteases.
    [Show full text]
  • Structural and Kinetic Analysis of Escherichia Coli Signal Peptide Peptidase A
    Structural and Kinetic Analysis of Escherichia coli Signal Peptide Peptidase A by Apollos C. Kim B.A. (Psychology), Simon Fraser University, 2001 B.Sc. (Biology), Seoul National University, Korea, 1992 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biochemistry Faculty of Science Apollos C. Kim 2013 SIMON FRASER UNIVERSITY Summer 2013 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for “Fair Dealing.” Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately. Approval Name: Apollos C. Kim Degree: Doctor of Philosophy (Molecular Biology and Biochemistry) Title of Thesis: Structural and kinetic analysis of Escherichia coli signal peptide peptidase A Examining Committee: Chair: Frederic Pio, Associate Professor Mark Paetzel Senior Supervisor Associate Professor Nicholas Harden Supervisor Professor Edgar C. Young Supervisor Associate Professor Dipankar Sen Internal Examiner Professor Ross MacGillivray External Examiner Professor Department of Biochemistry and Molecular Biology University of British Columbia Date Defended/Approved: June 19, 2013 ii Partial Copyright Licence iii Abstract Secretory proteins contain a signal peptide at their N-terminus. The signal peptide functions to guide proteins to the membrane and is cleaved off by signal peptidase. The remnant signal peptides must be removed from the membrane to prevent their accumulation which can lead to membrane destabilization. Escherichia coli signal peptide peptidase A (SppAEC) has been identified as a major enzyme that processes the remnant signal peptide to smaller fragments.
    [Show full text]