DOCSLIB.ORG

Unveiling the Regulatory Network for Di/Tri-Peptide Utilization in Saccharomyces Cerevisiae

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Unveiling the Regulatory Network for Di/Tri-Peptide Utilization in Saccharomyces Cerevisiae University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae Houjian Cai University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Microbiology Commons Recommended Citation Cai, Houjian, "Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1649 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Houjian Cai entitled "Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Jeffrey M. Becker, Major Professor We have read this dissertation and recommend its acceptance: David A. Brian, Pamela L. C. Small, Todd Reynolds, Bruce McKee Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Houjian Cai entitled “Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Jeffrey M. Becker Major Professor We have read this dissertation and recommend its acceptance: David A. Brian . Pamela L. C. Small . Todd Reynolds . Bruce McKee . Accepted for the Council: Anne Mayhew . Vice Chancellor and Dean of Graduate Studies (Original signatures are on file with official student records.) Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Houjian Cai May 2006 DEDICATION My faith was never shaken. It took me 15 years from the time when I planned to earn a Ph.D degree. This work is dedicated to my family: To my passed eldest brother, who mistakenly sacrificed himself to the distorted era of his country when he still was not able to think clearly; whose spirit is my everlasting driving force for life. To my passed grandmother, who raised me up on her back, and her mother-image is in my memory forever. It was greatly unfortunate that I could not finish up my study before she passed away. To my wife, who has supported me and always makes me smarter. To my brothers, Houyong and Houren, and sister, Sujuan for caring about me and taking care of the family when I was away from home in the past years. To my mother, who always worries about my living outside the country and never gave up her faith for my success. Finally, to my daughter, Lena, who brings the joy to me. Her sweet smile makes me energetic whenever I am tired. ii ACKNOWLEDGEMENTS Life is really un-predictable. Fifteen years ago I had never known that I would finish up my Ph.D study in Dr. Becker’s lab. It is my fortune that I have met so many great people in the journey of reaching this degree. The encouragement and support of these people has made possible the realization of my dream, getting a Ph.D degree. It is more than difficult to adequately express my gratitude for their help. I would like to acknowledge Dr. Dittmar Hahn for providing me an opportunity to study in his lab, which brought me from Europe to the US. He showed me how to work efficiently in being a scientist. I would also like to thank Dr. Gary Stacy for recruiting me to the University of Tennessee, Knoxville. The dissertation committee members made it possible for me to go smoothly in my projects. I would like to thank Dr. Pamela Small, Dr. David Brian, Dr. Todd Reynolds, and Dr. Bruce McKee, for being interested in my ideas and making my time as a graduate student the most challenging adventure I have yet undertaken. I would also like to thank Dr. Dong Xu provided me a lot of fresh ideas when I started my project. My appreciation goes out to Dr. Fred Naider for providing me the toxic peptides continually, and giving me his faith for whatever I am working on. If I could attribute any one aspect as having contributed most to my success, it would be the talented and supportive people in Becker’s lab. I can not express more of my gratitude to the “lab mom” Dr. Melinda Hauser. I started my lab rotation with her. It was her charming knowledge that became one of reasons to work in the lab. She has been a dictionary for me throughout the time in the lab on everything. My special thanks also iii go to Dr. Tom Masi, a king of molecular biology in the lab. I could always find answers from him about the experimental difficulties, and he usually is the person to crack the first version of my terrible writing. I owed him for spending a lot of free time on understanding my writing at home. Special thanks to Sarah Kauffman, a super-technician, who conquered the boring and helped me to transfer a plasmid more than two hundred times. Thanks to Dr. Amy Wiles, who taught me a lot of English, showed me American culture, and discussed American political issues. She shared the Transporter niche with me in the lab and provided a lot of valuable ideas. Appreciation to Dr. Yong-Hun Lee, who always challenged me in science with the final conclusion “it is not enough” and “so what”. Thanks to my friend, Dr. Cagdas Son, a king in the protein chemistry and computer knowledge in the lab, who showed me a lot of tricks in working with the protein and solving all kinds of computer problems. I enjoyed his humor and shared a lot of life experience. Thanks to Steve Minkin, who came through many times when I needed a laugh. His enthusiasm to political issues and optimum to the life made me understand more about this country. Thanks to Dr. Byung-Kwon Lee, who gave me a lot of suggestion and advice in the scientific field. Special gratitude to Dr. Agnieszka Janiak for a lot of constructive discussions about my project. Thanks to Heejung Kim, George Umanah, Li-Yin Huang, who showed great passion in the lab. I appreciate the enjoyable moment they have brought to me. To my mentor, teacher, Dr. Jeffrey Becker, more than anyone else, I am very grateful for the opportunity he gave to me to work in his lab. In Chinese, we said “one day being your mentor, forever being your father”. In past five years, Jeff’s influence and lessons have permeated my life and instilled me how to face the challenge and conquer iv difficulties. He provided me with strength, support and direction, and gave enormous freedom, patience, and trust in my research. His critical thinking, self-confidence, and open-mind influence my research in the future. I am proud to declare my thanks. v ABSTRACT Dipeptides and tripeptides serve as important sources of amino acids, nitrogen and carbon for the growth of all organisms. To identify genes involved in the regulation of small peptide utilization, I performed a systematic, functional examination of this process in a haploid, non-essential, single-gene deletion mutant library of Saccharomyces cerevisiae. In addition, we used high-throughput phenotyping in which we grew yeast cells on 284 different dipeptides or 11 tripeptides as the sole nitrogen source to dissect different mechanisms of di/tri-peptide utilization in seven genetically diverse strains. I have identified 103 candidate genes involved in regulating peptide utilization: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. Since in S. cerevisiae the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tri-peptides and is a key player in the di/tri- peptide utilization process, I focused our studies on the regulation of PTR2 expression, Ptr2p-GFP localization, and dipeptide uptake assays. Forty-two genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes apparently did not affect expression or localization. Together with the high- throughput phenotyping study, we have identified gene components involved in regulation of dipeptide utilization in many cellular processes: 1) proteins involved in up- or down-regulation of PTR2 transcription including the chromatin remodeling INO80 protein complex (Arp5p, Arp8p, Ies6p and Taf14p), transcription factors (Cup9p, Rpn4p, Stp2p, and Dal81p), polymerase mediators (Ssn3p/Srb10p, Ssn8p/Srb11p, Ssn2p/Srb9p, and Srb8p), and mRNA maturation (Kem1p and Pat1p); 2) proteins involved in the Ptr2p vi trafficking system including ESCRT I, II, III protein complex; 3) proteins participating in metabolic processes including the pyruvate dehydrogenase complex (Pdx1p, Lpd1, Pdb1p, and Lat1p), the fatty acid synthase complex (Oar1p, Mct1p, and Etr1p), and the glycine decarboxylase multienzyme complex (Gcv2p, Gcv3p, and Lpd1p); 4) proteins involved in other cellular processes, such as Ybt1p, a protein located at the vacuole membrane likely related to the storage of dipeptides in the cytosol and Dal5p previously identified as an allantoate/ureidosuccinate permease, which facilitates di/tri-peptide transport.
Load more

Recommended publications
  • Entrez Symbols Name Termid Termdesc 117553 Uba3,Ube1c
    Entrez Symbols Name TermID TermDesc 117553 Uba3,Ube1c ubiquitin-like modifier activating enzyme 3 GO:0016881 acid-amino acid ligase activity 299002 G2e3,RGD1310263 G2/M-phase specific E3 ubiquitin ligase GO:0016881 acid-amino acid ligase activity 303614 RGD1310067,Smurf2 SMAD specific E3 ubiquitin protein ligase 2 GO:0016881 acid-amino acid ligase activity 308669 Herc2 hect domain and RLD 2 GO:0016881 acid-amino acid ligase activity 309331 Uhrf2 ubiquitin-like with PHD and ring finger domains 2 GO:0016881 acid-amino acid ligase activity 316395 Hecw2 HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 GO:0016881 acid-amino acid ligase activity 361866 Hace1 HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 GO:0016881 acid-amino acid ligase activity 117029 Ccr5,Ckr5,Cmkbr5 chemokine (C-C motif) receptor 5 GO:0003779 actin binding 117538 Waspip,Wip,Wipf1 WAS/WASL interacting protein family, member 1 GO:0003779 actin binding 117557 TM30nm,Tpm3,Tpm5 tropomyosin 3, gamma GO:0003779 actin binding 24779 MGC93554,Slc4a1 solute carrier family 4 (anion exchanger), member 1 GO:0003779 actin binding 24851 Alpha-tm,Tma2,Tmsa,Tpm1 tropomyosin 1, alpha GO:0003779 actin binding 25132 Myo5b,Myr6 myosin Vb GO:0003779 actin binding 25152 Map1a,Mtap1a microtubule-associated protein 1A GO:0003779 actin binding 25230 Add3 adducin 3 (gamma) GO:0003779 actin binding 25386 AQP-2,Aqp2,MGC156502,aquaporin-2aquaporin 2 (collecting duct) GO:0003779 actin binding 25484 MYR5,Myo1e,Myr3 myosin IE GO:0003779 actin binding 25576 14-3-3e1,MGC93547,Ywhah
    [Show full text]
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [Show full text]
  • Processing of Antigenic Peptides by Aminopeptidases
    June 2004 Biol. Pharm. Bull. 27(6) 777—780 (2004) 777 Current Topics Aminopeptidases in Health and Disease Processing of Antigenic Peptides by Aminopeptidases Akira HATTORI* and Masafumi TSUJIMOTO Laboratory of Cellular Biochemistry, RIKEN; 2–1 Hirosawa, Wako, Saitama 351–0198, Japan. Received January 7, 2004 Antigenic peptides presented to major histocompatibility complex (MHC) class I molecules are generated in the cytosol during degradation of cellular proteins by the ubiquitin-proteasome proteolytic pathway. Proteasome can generate N-extended precursors as well as final epitopes, and then the precursors are processed to mature epitopes by aminopeptidases. Both cytosolic peptidases (i.e. puromycin-sensitive aminopeptidase, bleomycin hy- drolase and interferon-g-inducible leucine aminopeptidase) and recently identified metallo-aminopeptidase lo- cated in the endoplasmic reticulum (i.e. adipocyte-derived leucine aminopeptidase/endoplasmic reticulum aminopeptidase 1 and leukocyte-derived arginine aminopeptidase) can generate final epitopes from precursor peptides. Some of these aminopeptidases are also considered to destroy certain antigenic peptides to limit the antigen presentation. Taken together, it is getting evident that aminopeptidases located in the cytosol and the lumen of endoplasmic reticulum play important roles in the generation of antigenic peptides presented to MHC class I molecules. Key words aminopeptidase; antigen processing; major histocompatibility complex (MHC) class I; antigen presentation; protea- some; protein degradation
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • Enzymatic Hydrolysis and Peptide Mapping of Potato Pulp Protein
    Enzymatic Hydrolysis and Peptide Mapping of Potato Pulp Protein Von der Naturwissenschaftlichen Fakultät der Universität Hannover zur Erlangung des Grades Doktorin der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von Chulaporn Kamnerdpetch, M.Sc. geboren in Bangkok, Thailand Hannover 2006 Hauptreferent Prof. Dr. Thomas Scheper Institut für Technische Chemie Universität Hannover Koreferent Prof. Dr. Bernd Hitzmann Institut für Technische Chemie Universität Hannover Tag der Promotion 29. Mai 2006 Erklärung Ich versichere, dass ich diese Dissertation selbstständig und nur unter Verwendung der angegebenen Hilfsmittel und Quellen durchgeführt habe. Diese Arbeit wurde nicht als Diplomarbeit oder ähnliche Prüfungsarbeit verwendet. Chulaporn Kamnerdpetch Hannover, den 29. Mai 2006 ACKNOWLEDGEMENTS This thesis is the result of my four years research work whereby I have been accompanied and supported by many people. It is a pleasant aspect that I have now the opportunity to express my sincere gratitude for all of them who made this thesis possible. The first person I would like to thank is my supervisor Prof. Dr. Thomas Scheper for giving me the opportunity to take part on the doctoral program at the Institut für Technische Chemie der Universität Hannover. I appreciate very much for his enthusiastic and enthusing support. He gave me an encourage independent thinking and the freedom to try out my ways. I would like to thank to Prof. Dr. Bernd Hitzmann for his kindness acceptance as my co-referee. I wish to express my thank to Dr. Cornelia Kasper for preparing my publication and proof reading. It is a great pleasure for me to thank Dr. Pichai Namparkai for proof reading as well.
    [Show full text]
  • JASON MARC GOLDSTEIN the Isolation, Characterization
    JASON MARC GOLDSTEIN The Isolation, Characterization and Cloning of Three Novel Peptidases From Streptoccocus gordonii: Their Potential Roles in Subacute Bacterial Endocarditis (Under the Direction of JAMES TRAVIS) Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora yet is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Colonization and adherence mechanisms have been identified, yet factors necessary to sustain growth remain unidentified. Strain FSS2 produced three extracellular aminopeptidase activities during growth in neutral pH- controlled batch cultures. The first included a serine-class dipeptidyl-aminopeptidase, an x-Pro DPP (Sg-xPDPP) found as an 85 kDa monomer by SDS-PAGE while appearing as a homodimer under native conditions. Kinetic studies indicated a unique and stringent x- Pro specificity comparable to the DPPIV/CD26 and lactococcal x-Pro DPP families. Isolation of the full-length gene uncovered a 759-amino acid polypeptide with a mass of 87,115 Da and theoretical pI of 5.6. Significant homology was found with PepX gene family members from Lactobacillus ssp. and Lactococcus ssp., and putative streptococcal x-Pro DPPs. The second activity was a putative serine-class arginine aminopeptidase (Sg- RAP) with some cysteine-class characteristics. It was found as a protein monomer of 70 kDa under denaturing conditions. Nested PCR cloning enabled the isolation of a 324 bp- long DNA fragment encoding the protein’s 108 amino acid N-terminus. Culture activity profiles and N-terminal sequence analysis indicated the release of this protein from the cell surface.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier Et Al
    US 200601 10747A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier et al. (43) Pub. Date: May 25, 2006 (54) PROCESS FOR IMPROVED PROTEIN (60) Provisional application No. 60/591489, filed on Jul. EXPRESSION BY STRAIN ENGINEERING 26, 2004. (75) Inventors: Thomas M. Ramseier, Poway, CA Publication Classification (US); Hongfan Jin, San Diego, CA (51) Int. Cl. (US); Charles H. Squires, Poway, CA CI2O I/68 (2006.01) (US) GOIN 33/53 (2006.01) CI2N 15/74 (2006.01) Correspondence Address: (52) U.S. Cl. ................................ 435/6: 435/7.1; 435/471 KING & SPALDING LLP 118O PEACHTREE STREET (57) ABSTRACT ATLANTA, GA 30309 (US) This invention is a process for improving the production levels of recombinant proteins or peptides or improving the (73) Assignee: Dow Global Technologies Inc., Midland, level of active recombinant proteins or peptides expressed in MI (US) host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant (21) Appl. No.: 11/189,375 protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recom (22) Filed: Jul. 26, 2005 binant protein expression. The process can improve protein production or can improve protein quality, for example, by Related U.S. Application Data increasing solubility of a recombinant protein. Patent Application Publication May 25, 2006 Sheet 1 of 15 US 2006/0110747 A1 Figure 1 09 010909070£020\,0 10°0 Patent Application Publication May 25, 2006 Sheet 2 of 15 US 2006/0110747 A1 Figure 2 Ester sers Custer || || || || || HH-I-H 1 H4 s a cisiers TT closers | | | | | | Ya S T RXFO 1961.
    [Show full text]
  • Proteasome Inhibitors: from Research Tools to Drug Candidates
    Chemistry & Biology 8 (2001) 739^758 www.elsevier.com/locate/chembiol Review Proteasome inhibitors: from research tools to drug candidates Alexei F. Kisselev*, Alfred L. Goldberg Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115, USA Received 13 December 2000; revisions requested 29 March 2001; revisions received 12 June 2001; accepted 19 June 2001 First published online 12 July 2001 Abstract The 26S proteasome is a 2.4 MDa multifunctional ATP- inhibitors are now in clinical trials for treatment of multiple dependent proteolytic complex, which degrades the majority of cancers and stroke. ß 2001 Elsevier Science Ltd. All rights re- cellular polypeptides by an unusual enzyme mechanism. Several served. groups of proteasome inhibitors have been developed and are now widely used as research tools to study the role of the ubiquitin^ Keywords: Proteasome inhibitor; proteasome pathway in various cellular processes, and two Ubiquitin^proteasome pathway; Drug candidate 1. Introduction of cyclins and inhibitors of cyclin-dependent kinases [7], while degradation of transcriptional regulators, such as The ubiquitin^proteasome pathway is the major proteo- c-Jun, E2F-1 and L-catenin (see [8] for review) is essential lytic system in the cytosol and nucleus of all eukaryotic for the regulation of cell growth and gene expression. cells. This ATP-dependent pathway was discovered more Similarly, degradation by the proteasome of activated pro- than 20 years ago [1,2], but the involvement of the pro- tein kinases, e.g. src and protein kinase C [9,10], is critical teasome particle was demonstrated only in the late 1980s for the termination of certain signal transduction cascades.
    [Show full text]
  • Studies of Structure and Function of Tripeptidyl-Peptidase II
    Till familj och vänner List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I. Eriksson, S.; Gutiérrez, O.A.; Bjerling, P.; Tomkinson, B. (2009) De- velopment, evaluation and application of tripeptidyl-peptidase II se- quence signatures. Archives of Biochemistry and Biophysics, 484(1):39-45 II. Lindås, A-C.; Eriksson, S.; Josza, E.; Tomkinson, B. (2008) Investiga- tion of a role for Glu-331 and Glu-305 in substrate binding of tripepti- dyl-peptidase II. Biochimica et Biophysica Acta, 1784(12):1899-1907 III. Eklund, S.; Lindås, A-C.; Hamnevik, E.; Widersten, M.; Tomkinson, B. Inter-species variation in the pH dependence of tripeptidyl- peptidase II. Manuscript IV. Eklund, S.; Kalbacher, H.; Tomkinson, B. Characterization of the endopeptidase activity of tripeptidyl-peptidase II. Manuscript Paper I and II were published under maiden name (Eriksson). Reprints were made with permission from the respective publishers. Contents Introduction ..................................................................................................... 9 Enzymes ..................................................................................................... 9 Enzymes and pH dependence .............................................................. 11 Peptidases ................................................................................................. 12 Serine peptidases ................................................................................. 14 Intracellular protein
    [Show full text]
  • The Role of Tricorn Protease and Its Aminopeptidase-Interacting Factors in Cellular Protein Degradation
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 95, 637±648, November 25, 1998, Copyright 1998 by Cell Press The Role of Tricorn Protease and Its Aminopeptidase-Interacting Factors in Cellular Protein Degradation Noriko Tamura, Friedrich Lottspeich, complexes that invariably contain ATPase subunits ren- Wolfgang Baumeister,* and Tomohiro Tamura dering protein degradation energy dependent. The oc- Max-Planck-Institut fuÈ r Biochemie currence of self-compartmentalizing proteases in all D-82152 Martinsried three domains of life bears testimony of an old evolution- Germany ary principle. In prokaryotic cells lacking membrane- bounded compartments, ATP-dependent self-compart- mentalizing proteases such as the proteasome, HslV, ClpP, or Lon are responsible for the bulk of the protein Summary turnover. Beyond facilitating the control of proteolysis, the con- Tricorn protease was previously described as the core finement of the proteolytic activity to a nanocompart- enzyme of a modular proteolytic system displaying ment inside these assemblies also provides the struc- multicatalytic activity. Here we elucidate the mode of tural basis for the processive mode of action that is cooperation between Tricorn and its interacting fac- characteristic for these proteases: they do not release tors, and we identify two additional factors, F2 and F3, fragments after a single cleavage, but proceed to make closely related aminopeptidases of 89 kDa. In conjunc- multiple cleavages before finally discharging the degra- tion with these three factors, Tricorn degrades oligo- dation products (Thompson et al., 1994; Akopian et al., peptides in a sequential manner, yielding free amino 1997).
    [Show full text]
  • The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-To-ER Retrograde Transport
    Supplementary Materials The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport Bustamante HA., et al. Figure S1. siRNA sequences directed against human PSMD14 used for Validation Stage. Figure S2. Primer pairs sequences used for RT-qPCR. Figure S3. The PSMD14 DUB inhibitor CZM increases the Golgi apparatus area. Immunofluorescence microscopy analysis of the Golgi area in parental H4 cells treated for 4 h either with the vehicle (DMSO; Control) or CZM. The Golgi marker GM130 was used to determine the region of interest in each condition. Statistical significance was determined by Student's t-test. Bars represent the mean ± SEM (n =43 cells). ***P <0.001. Figure S4. CZM causes the accumulation of KDELR1-GFP at the Golgi apparatus. HeLa cells expressing KDELR1-GFP were either left untreated or treated with CZM for 30, 60 or 90 min. Cells were fixed and representative confocal images were acquired. Figure S5. Effect of CZM on proteasome activity. Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM or MG132, for 90 min. Protein extracts were used to measure in vitro the Chymotrypsin-like peptidase activity of the proteasome. The enzymatic activity was quantified according to the cleavage of the fluorogenic substrate Suc-LLVY-AMC to AMC, and normalized to that of control cells. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). **P <0.01; n.s., not significant. Figure S6. Effect of CZM and MG132 on basal macroautophagy. (A) Immunofluorescence microscopy analysis of the subcellular localization of LC3 in parental H4 cells treated with either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h.
    [Show full text]
  • Contribution of Endo- and Exopeptidases to Formation of Non-Protein N During Ensiling of Alfalfa
    Contribution of endo- and exopeptidases to formation of non-protein N during ensiling of alfalfa Dr. Xusheng Guo,1 W. Cheng,1 L. Tao,2 Yu Zhu,2 F.Y. Yang 2 & H. Zhou2 1.School of Life Science, Lanzhou University 1. International Centre for Tibetan Plateau Ecosystem Management (ICTPEM), Lanzhou University 2. Institute of Grassland Science, China Agricultural University Hämeenlinna, Finland 2-4 July 2012 Introduction Alfalfa (Medicago Sativa L.) is well known for its high nutritive value However, after ensiling: N use efficiency Extensive ppyroteolysis Reduce True protein NPN(Peptide, FAA, NH3-N etc.) Silage DM intake (44-87% of Total N; Muck, 1987) Silage Fermentation Proteolysis in ensiled forage mainly results from plant proteases (Ohshima and McDonald, 1978; McKersie, 1981; Heron et al., 1988). Proteases (peptidases) are divided into 2 classes (NC-IUBMB, 1992): Exopeptidase Endopeptidase Objectives Proteases (peptidases, E.C.3.4） Endopeptidases Exopeptidases ？！ Ser ine pep tidase (E .C .3 .4 .21) Aminopeptidase (EC 3.4.11) Carboxypeptidase (EC 3.4.16) Cysteine peptidase (E.C.3.4.22) Dipeptidase (EC 3.4.13) Aspartic Peptidase (E.C.3.4.23) Dipeptidyl-peptidase (EC 3.4.14) Metallopeptidase (E.C.3.4.24) Tripeptidyl-peptidase (EC 3.4.14) Peptidyl-dipeptidase (EC 3.4.15) Aims of our research were: 1. To clarify the classes of exo- and endopeptidases that are involved in proteolysis within ensiled alfalfa. 2. To determine the contribution of these peptidases to the formation of different NPN compounds (peptide-N, FAA-N, and NH3-N) during
    [Show full text]