DOCSLIB.ORG

Global Analysis of Chaperone Effects Using a Reconstituted Cell-Free Translation System

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Global Analysis of Chaperone Effects Using a Reconstituted Cell-Free Translation System Global analysis of chaperone effects using a reconstituted cell-free translation system Tatsuya Niwaa, Takashi Kanamorib,1, Takuya Uedab,2, and Hideki Taguchia,2 aDepartment of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan; and bDepartment of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved April 19, 2012 (received for review January 25, 2012) Protein folding is often hampered by protein aggregation, which can three chaperone systems are known to act cooperatively: TF and be prevented by a variety of chaperones in the cell. A dataset that DnaK exhibit overlapping cotranslational roles in vivo (13–15). evaluates which chaperones are effective for aggregation-prone Overexpression of DnaK/DnaJ and GroEL/GroES in E. coli proteins would provide an invaluable resource not only for un- rpoH mutant cells, which are deficient in heat-shock proteins, derstanding the roles of chaperones, but also for broader applications prevents aggregation of newly translated proteins (16). GroEL is in protein science and engineering. Therefore, we comprehensively believed to be involved in folding after the polypeptides are re- evaluated the effects of the major Escherichia coli chaperones, trigger leased from the ribosome, although the possible cotranslational factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ∼800 aggregation- involvement of GroEL has also been reported (17–20). prone cytosolic E. coli proteins, using a reconstituted chaperone-free Over the past two decades many efforts have been focused on translation system. Statistical analyses revealed the robustness and elucidating the mechanism of each chaperone as a molecular the intriguing properties of chaperones. The DnaK and GroEL systems machine (4, 9). The next question to be addressed is which drastically increased the solubilities of hundreds of proteins with chaperone is effective for certain proteins. A dataset on the weak biases, whereas trigger factor had only a marginal effect on substrate biases for each chaperone would provide an invaluable solubility. The combined addition of the chaperones was effective for resource, not only for understanding the role of chaperones in a subset of proteins that were not rescued by any single chaperone protein folding but also for applications to protein science and system, supporting the synergistic effect of these chaperones. The engineering. However, the mechanisms by which chaperones resource, which is accessible via a public database, can be used to recognize their substrates are not fully understood. Although BIOCHEMISTRY investigate the properties of proteins of interest in terms of their global analyses of chaperone–protein interactions in cells have solubilities and chaperone effects. been conducted (4, 7, 8, 21–24), no systematic evaluation of chaperone effects under uniform conditions has been performed. chaperonin | Hsp60 | Hsp70 | aggregates | proteome In this context, a reconstituted cell-free translation system that only contains the essential factors for protein synthesis is ideal to ewly synthesized proteins emerging from the ribosome must evaluate the role of chaperones, because the cell-free system is Nfold into their native structures to acquire their functions (1). chaperone-free. Therefore, we chose an E. coli reconstituted cell- Although protein folding is a spontaneous process, in which the free system, the PURE system (25, 26). Using the PURE system, amino acid sequence dictates the native structure (2), nonproduc- we previously analyzed the aggregation propensities of all E. coli tive intermolecular interactions result in aggregate formation (1, 3, proteins under a chaperone-free condition (6). In addition, the 4). Because protein stability is marginal in general, proteins always PURE system has been used to investigate the role of GroE in the have the inherent risk of aggregation (3, 4). Perturbations of cel- folding of some newly synthesized proteins (19, 20, 27). We have lular proteostasis (4), such as cellular stresses or heterologous extended those previous studies to a comprehensive evaluation of recombinant protein expression, often cause protein aggregation in the major E. coli chaperones, TF, DnaK/DnaJ/GrpE (DnaKJE), the cell and the formation of inclusion bodies, which are one of the and GroEL/GroES (GroE), on ∼800 aggregation-prone, cytosolic bottlenecks in various types of biological research, from traditional E. coli proteins. The scheme of the global analysis is shown in Fig. molecular biology to modern synthetic biology (3, 4). 1: the one-by-one synthesis of individual aggregation-prone pro- To counteract the inevitable tendency toward protein aggre- teins in the presence of each chaperone, the quantification of gation, cells have evolved a variety of chaperones (5). Chaperones solubility by a centrifugation-based assay, and the statistical prevent irreversible aggregate formation by binding nonnative analyses of the collected data. This is an “in vitro (reconstituted) proteins and then assisting with productive folding (3, 4). Indeed, ” Escherichia coli proteome approach, in which the properties of thousands of when more than 3,000 proteins were synthesized proteins, including proteins with extremely low abundance in cells, by reconstituted cell-free translation under chaperone-free con- are investigated individually after cell-free translation. This large ditions, a substantial fraction of the proteome, a quarter of the fi dataset is an invaluable resource for investigations of the prop- proteins quanti ed, was aggregation-prone (6), implying that erties of proteins of interest. In addition, the statistical analysis of chaperones are required to rescue the aggregation-prone proteins. the data revealed many intriguing properties of chaperones in The best-characterized chaperones are those in E. coli (4). In terms of substrate recognition. E. coli, three major chaperone systems are known to be involved in the folding of newly synthesized proteins in the cytoplasm (4). The first is trigger factor (TF), which directly associates with the Author contributions: T.N., T.U., and H.T. designed research; T.N. and T.K. performed ribosome and interacts with nascent chains cotranslationally (7). research; T.N., T.K., T.U., and H.T. contributed new reagents/analytic tools; T.N., T.U., The second is DnaK, a member of the Hsp70 family that is and H.T. analyzed data; and T.N., T.U., and H.T. wrote the paper. widely conserved in all kingdoms of life and is considered to act The authors declare no conflict of interest. on a broad spectrum of proteins in cooperation with the This article is a PNAS Direct Submission. cochaperones, DnaJ and GrpE (4, 8, 9). The third is GroEL, 1Present address: GeneFrontier Corporation, Kashiwa, Chiba 277-0882, Japan. – which belongs to a well-conserved chaperonin family (4, 10 12). 2To whom correspondence may be addressed. E-mail: [email protected] or taguchi@ In the presence of ATP, GroEL forms a large cylindrical com- bio.titech.ac.jp. plex with the cochaperonin GroES, which encapsulates substrate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins within its cavity to assist with folding (4, 10–12). These 1073/pnas.1201380109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201380109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 physiological concentration, based on previous assessments of chaperone activities under cell-free conditions (19, 28, 29). The in vitro activities of each chaperone were confirmed (Fig. S1 and Materials and Methods). The [35S]methionine-labeled proteins were electrophoresed on SDS gels and quantified (Fig. 2A). The aggregation propensity was examined by a centrifugation assay (6). Briefly, an aliquot of the translation mixture was centrifuged, and then the supernatant fractions were electrophoresed and quanti- fied (Fig. 1). The solubility was defined as the proportion of the protein in the supernatant fraction to that in the uncentrifuged total protein sample. Typical results are shown in Fig. 2A.Almost all of the proteins (788 of 792) were quantified for their solubilities under each condition. Experimental error (defined as a SD) in the assay has been previously estimated to be 10% (6, 27). Indeed, the analysis in the absence of chaperones was reproducible, because the SD of the solubilities between the current and previous data (6) was less than 10% on average (Fig. S2A), and the solubilities of more than 90% of the translated proteins in the absence of a chaperone (718 of 788) were less than 30% (Fig. S2B). Because all of the translated proteins belong to the aggregation- prone group, which might occlude the exit tunnel in the ribosome, one might ask whether chaperones could facilitate protein syn- thesis by preventing aggregation. However, the presence of the chaperones had little influence on the yields of translated proteins (Fig. S3), suggesting that the overall translation efficiencies were not accelerated by any of the chaperones. Overview of the Dataset. In total, more than 3,000 assays (788 pro- teins × 4 conditions = 3,152) were conducted (Dataset S1). The arranged data, combined with data obtained from our previous ag- gregation analysis, are freely accessible at our online database (eSol database: http://tp-esol.genes.nig.ac.jp). Overall, the
Load more

Recommended publications
  • Coevolutionary Phage Training Leads to Greater Bacterial Suppression and Delays the Evolution of Phage Resistance
    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.02.365361; this version posted November 2, 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. Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance Joshua M. Borin1, Sarit Avrani2, Jeffrey E. Barrick3, Katherine L. Petrie1,4, and Justin R. Meyer1* 1. Division of Biological Sciences, University of California San Diego 92093 2. Department of Evolutionary and Environmental Biology and The Institute of Evolution, University of Haifa 3498838 3. Department of Molecular Biosciences, The University of Texas at Austin 78712 4. Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan 145-0061 *corresponding author Classification BIOLOGICAL SCIENCES, Evolution Keywords Evolution, Coevolution, Resistance, Phage therapy, Phage training Author Contributions JMB, JRM, and SA designed research; JMB, KLP and JRM performed research; JMB and JRM analyzed data; JMB and JRM wrote the paper; SA and JRM provided financial support. All authors edited the manuscript. Significance Statement The evolution of antibiotic resistant bacteria threatens to claim over 10 million lives annually by 2050. This crisis has renewed interest in phage therapy, the use of bacterial viruses to treat infections. A major barrier to successful phage therapy is that bacteria readily evolve phage resistance. One idea proposed to combat resistance is “training” phages by using their natural capacity to evolve to counter resistance.
    [Show full text]
  • XIAO-DISSERTATION-2015.Pdf
    CELLULAR AND PROCESS ENGINEERING TO IMPROVE MAMMALIAN MEMBRANE PROTEIN EXPRESSION By Su Xiao A dissertation is submitted to Johns Hopkins University in conformity with the requirements for degree of Doctor of Philosophy Baltimore, Maryland May 2015 © 2015 Su Xiao All Rights Reserved Abstract Improving the expression level of recombinant mammalian proteins has been pursued for production of commercial biotherapeutics in industry, as well as for biomedical studies in academia, as an adequate supply of correctly folded proteins is a prerequisite for all structure and function studies. Presented in this dissertation are different strategies to improve protein functional expression level, especially for membrane proteins. The model protein is neurotensin receptor 1 (NTSR1), a hard-to- express G protein-coupled receptor (GPCR). GPCRs are integral membrane proteins playing a central role in cell signaling and are targets for most of the medicines sold worldwide. Obtaining adequate functional GPCRs has been a bottleneck in their structure studies because the expression of these proteins from mammalian cells is very low. The first strategy is the adoption of mammalian inducible expression system. A stable and inducible T-REx-293 cell line overexpressing an engineered rat NTSR1 was constructed. 2.5 million Functional copies of NTSR1 per cell were detected on plasma membrane, which is 167 fold improvement comparing to NTSR1 constitutive expression. The second strategy is production process development including suspension culture adaptation and induction parameter optimization. A further 3.5 fold improvement was achieved and approximately 1 milligram of purified functional NTSR1 per liter suspension culture was obtained. This was comparable yield to the transient baculovirus- insect cell system.
    [Show full text]
  • Chloroplast Molecular Chaperone-Assisted Refolding and Reconstitution of an Active Multisubunit Coupling Factor CF1 Core (Atpse/Asembly) GEOFFREY G
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11497-11501, November 1994 Plant Biology Chloroplast molecular chaperone-assisted refolding and reconstitution of an active multisubunit coupling factor CF1 core (ATPse/asembly) GEOFFREY G. CHEN* AND ANDRE T. JAGENDORFt Section of Plant Biology, Cornell University, Ithaca, NY 14853 Contributed by Andre T. Jagendorf, August 8, 1994 ABSTRACT The chloroplast coupling factor 1 (CF1) is logues to E. coli DnaJ and GrpE have not yet been identified composed of five kinds of subuits ith a stoichiometry of in chloroplasts. a4313y6e. Reconstitution of a catalyticay active a3I3Y core Unlike the cpn6O homolog in E. coli designated GroEL, the from urea-denatured subnits at a physiological pH is reported chloroplast cpn6O contains two kinds of subunits, which here. A restoration of approximately 90% of the CF1 ATrase share about 50% sequence homology (12). The stoichiometry activity has been observed. The reconstitution was achieved by of its a and (3 subunits has been suggested to be 1:1 (10). using subunits overexpressed in Eschenicia coli, rfied, and Electron microscopic imaging revealed a structure very sim- combined In the presenoe of MgATP, K+, and a m of ilar to that ofGroEL: a stacked double-ring with a sevenfold several chloroplast m r chaperones at pH 7.5. The rotational symmetry and a central cavity where unfolded combination of chaperonin 60 and chaperonin 24 failed to proteins probably bind (17). recotitute the active CF1 core, as did the GroEL/GroES - The chloroplast cochaperonin (cpn24) is a homologue of (E. coil chaperonin 60/10 homoloues). Characteristics of the cpnl0s, but is different from any cpnl0 described previously, reconstituted ATPase were very cose to those of the native including the E.
    [Show full text]
  • Chaperonin-Assisted Protein Folding: a Chronologue
    Quarterly Reviews of Chaperonin-assisted protein folding: Biophysics a chronologue cambridge.org/qrb Arthur L. Horwich1,2 and Wayne A. Fenton2 1Howard Hughes Medical Institute, Yale School of Medicine, Boyer Center, 295 Congress Avenue, New Haven, CT 06510, USA and 2Department of Genetics, Yale School of Medicine, Boyer Center, 295 Congress Avenue, New Invited Review Haven, CT 06510, USA Cite this article: Horwich AL, Fenton WA (2020). Chaperonin-assisted protein folding: a Abstract chronologue. Quarterly Reviews of Biophysics This chronologue seeks to document the discovery and development of an understanding of – 53, e4, 1 127. https://doi.org/10.1017/ oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. S0033583519000143 It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these Received: 16 August 2019 ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is orga- Revised: 21 November 2019 nized into various topics of physiology and mechanism, for each of which a chronologic order Accepted: 26 November 2019 is generally followed. The text is liberally illustrated to provide firsthand inspection of the key Key words: pieces of experimental data that propelled this field. Because of the length and depth of this Chaperonin; GroEL; GroES; Hsp60; protein piece, the use of the outline as a guide for selected reading is encouraged, but it should also be folding of help in pursuing the text in direct order. Author for correspondence: Arthur L. Horwich, E-mail: [email protected] Table of contents I. Foundational discovery of Anfinsen and coworkers – the amino acid sequence of a polypeptide contains all of the information required for folding to the native state 7 II.
    [Show full text]
  • Mrvr, a Group B Streptococcus Transcription Factor That Controls Multiple Virulence Traits
    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 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 4.0 International license. 1 2 3 4 MrvR, a Group B Streptococcus Transcription Factor that Controls Multiple Virulence Traits 5 6 Allison N. Dammann1, Anna B. Chamby2, Andrew J. Catomeris3, Kyle M. Davidson4, Hervé 7 Tettelin5,6, Jan-Peter van Pijkeren7, Kathyayini P. Gopalakrishna4, Mary F. Keith4, Jordan L. 8 Elder4, Adam J. Ratner1,8, Thomas A. Hooven4,9* 9 10 1 Department of Pediatrics, New York University School of Medicine, New York, NY, USA 11 12 2 University of Vermont Larner College of Medicine, Burlington, VT, USA 13 14 3 Georgetown University School of Medicine, Washington, DC, USA 15 16 4 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 17 18 5 Department of Microbiology and Immunology, University of Maryland School of Medicine, 19 Baltimore, MD, USA 20 21 6 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 22 USA 23 24 7 Department of Food Science, University of Wisconsin, Madison, WI, USA 25 26 8 Department of Microbiology, New York University, New York, NY, USA 27 28 9 Richard King Mellon Institute for Pediatric Research, UPMC Children’s Hospital of Pittsburgh, 29 Pittsburgh, PA, USA 30 31 * Corresponding author 32 E-mail: [email protected] 33 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020.
    [Show full text]
  • Engineering Proteins with GFP: Study of Protein-Protein Interactions in Vivo, Protein Expression and Solubility
    Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mohosin M. Sarkar, M. Sc. Graduate Program in Chemistry The Ohio State University 2009 Dissertation Committee: Thomas J. Magliery, Advisor Dennis Bong Ross E. Dalbey Christopher M. Hadad Copyright by Mohosin M. Sarkar 2009 Abstract Protein–protein interactions (PPIs) play a key role in most biological processes. Many of these interactions are necessary for cell survival. To understand the molecular mechanisms of biological processes, it is essential to study and characterize protein-protein interactions, identify interacting partners and protein interaction networks. There are a number of methods that have been developed to study protein-protein interactions in vitro and in vivo, such as yeast-2-hybrid, fluorescence resonance energy transfer, co-immunoprecipitation, etc. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2- hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly is especially attractive because the GFP chromophore forms spontaneously on protein folding in almost every cell type. However, existing split systems have limitations of evolving cellular fluorescence slowly (3-4 days), failure to evolve at all for some interactions, and also failure to work at a physiological temperature. Among different variants of GFP tested, we found that split folding-reporter GFP (frGFP, a hybrid of EGFP and GFPuv) evolves fluorescence much faster (24 - 30 h) with associating peptides and also evolves fluorescence for the RING domain BRCA1/BARD1 wild type pair.
    [Show full text]
  • Escherichia Coli Dnaj and Grpe Heat Shock Proteins Jointly Stimulate
    Proc. NatI. Acad. Sci. USA Vol. 88, pp. 2874-2878, April 1991 Biochemistry Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK KRZYSZTOF LIBEREK*t, JAROSLAW MARSZALEK*, DEBBIE ANGt, COSTA GEORGOPOULOStt, AND MACIEJ ZYLICZ* *Division of Biophysics, Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland; and tDepartment of Cellular, Viral, and Molecular Biology, University of Utah School of Medicine, Salt Lake City, UT 84132 Communicated by Allan M. Campbell, December 31, 1990 ABSTRACT The products of the Escherichia coli dnaK, when ATP was added to complexes of hsc70 (a constitutive dnaJ, and grpE heat shock genes have been previously shown member of the hsp70 family) and p53 (an anti-oncogenic to be essential for bacteriophage A DNA replication at all protein) (9), immunoglobulin heavy chains and their binding temperatures and for bacterial survival under certain condi- protein BiP (10), and uncoating ATPase complexed with tions. DnaK, the bacterial heat shock protein hsp7O analogue clathrin or membrane vesicles (11, 12). Recently, Beckmann and putative chaperonin, possesses a weak ATPase activity. et al. (13) have shown that the cytosolic hsp70 proteins may Previous work has shown that ATP hydrolysis allows the interact with a large number of newly synthesized proteins. release ofvarious polypeptides complexed with DnaK. Here we These examples suggest that ATP-dependent release ofhsp70 demonstrate that the ATPase activity of DnaK can be greatly from a complex with its substrate is a common feature of the stimulated, up to 50-fold, in the simultaneous presence of the hsp70 family. However, in all the described cases, the DnaJ and GrpE heat shock proteins.
    [Show full text]
  • Horizontal Gene Transfers and Cell Fusions in Microbiology, Immunology and Oncology (Review)
    441-465.qxd 20/7/2009 08:23 Ì ™ÂÏ›‰·441 INTERNATIONAL JOURNAL OF ONCOLOGY 35: 441-465, 2009 441 Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review) JOSEPH G. SINKOVICS St. Joseph's Hospital's Cancer Institute Affiliated with the H. L. Moffitt Comprehensive Cancer Center; Departments of Medical Microbiology/Immunology and Molecular Medicine, The University of South Florida College of Medicine, Tampa, FL 33607-6307, USA Received April 17, 2009; Accepted June 4, 2009 DOI: 10.3892/ijo_00000357 Abstract. Evolving young genomes of archaea, prokaryota or immunogenic genetic materials. Naturally formed hybrids and unicellular eukaryota were wide open for the acceptance of dendritic and tumor cells are often tolerogenic, whereas of alien genomic sequences, which they often preserved laboratory products of these unisons may be immunogenic in and vertically transferred to their descendants throughout the hosts of origin. As human breast cancer stem cells are three billion years of evolution. Established complex large induced by a treacherous class of CD8+ T cells to undergo genomes, although seeded with ancestral retroelements, have epithelial to mesenchymal (ETM) transition and to yield to come to regulate strictly their integrity. However, intruding malignant transformation by the omnipresent proto-ocogenes retroelements, especially the descendents of Ty3/Gypsy, (for example, the ras oncogenes), they become defenseless the chromoviruses, continue to find their ways into even the toward oncolytic viruses. Cell fusions and horizontal exchanges most established genomes. The simian and hominoid-Homo of genes are fundamental attributes and inherent characteristics genomes preserved and accommodated a large number of of the living matter.
    [Show full text]
  • Functional Insights from the Distribution and Role of Homopeptide Repeat-Containing Proteins
    Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Functional insights from the distribution and role of homopeptide repeat-containing proteins Noel G. Faux,1,2,3 Stephen P. Bottomley,1,2 Arthur M. Lesk,1,2,6 James A. Irving,1,2,3 John R. Morrison,5 Maria Garcia de la Banda,2,3,4,7 and James C. Whisstock1,2,3,7 1Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, 2Victorian Bioinformatics Consortium, 3ARC Centre for Structural and Functional Microbial Genomics, and 4School of Computer Science and Software Engineering, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia; 5Monash Institute of Reproduction and Development, Monash University, Clayton, VIC 3168, Australia; 6Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA Expansion of “low complex” repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington’s disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP—involved in important housekeeping processes—retain the repetitive region, suggesting an ancient origin for certain repeats.
    [Show full text]
  • Stress-Induced Expression of the Escherichia Coli Phage Shock Protein Operon Is D,E P Endent on 0 -54 and Modulated by Positive and Negative Feedback Mechanisms
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Stress-induced expression of the Escherichia coli phage shock protein operon is d,e p endent on 0 -54 and modulated by positive and negative feedback mechanisms Lorin Weiner, Janice L. Brissette, 1 and Peter Model z The Rockefeller University, New York, New York 10021 USA The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the or-factor r s4. Negative regulation is mediated by both PspA and the r heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the r 32- and erE-dependent systems, for genes induced by heat and other stresses.
    [Show full text]
  • Proteins to Exosomes Soluble Mycobacterial and Eukaryotic
    Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes This information is current as Victoria L. Smith, Liam Jackson and Jeffrey S. Schorey of September 28, 2021. J Immunol published online 5 August 2015 http://www.jimmunol.org/content/early/2015/08/05/jimmun ol.1403186 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/08/05/jimmunol.140318 Material 6.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 5, 2015, doi:10.4049/jimmunol.1403186 The Journal of Immunology Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes Victoria L. Smith, Liam Jackson, and Jeffrey S. Schorey Exosomes are extracellular vesicles of endocytic origin that function in intercellular communication. Our previous studies indicate that exosomes released from Mycobacterium tuberculosis-infected macrophages contain soluble mycobacterial proteins.
    [Show full text]
  • Characterization of the Dual Functional Effects of Heat Shock Proteins (Hsps
    Zhang et al. Genome Medicine (2020) 12:101 https://doi.org/10.1186/s13073-020-00795-6 RESEARCH Open Access Characterization of the dual functional effects of heat shock proteins (HSPs) in cancer hallmarks to aid development of HSP inhibitors Zhao Zhang1†, Ji Jing2†, Youqiong Ye1, Zhiao Chen1, Ying Jing1, Shengli Li1, Wei Hong1, Hang Ruan1, Yaoming Liu1, Qingsong Hu3, Jun Wang4, Wenbo Li1, Chunru Lin3, Lixia Diao5*, Yubin Zhou2* and Leng Han1* Abstract Background: Heat shock proteins (HSPs), a representative family of chaperone genes, play crucial roles in malignant progression and are pursued as attractive anti-cancer therapeutic targets. Despite tremendous efforts to develop anti-cancer drugs based on HSPs, no HSP inhibitors have thus far reached the milestone of FDA approval. There remains an unmet need to further understand the functional roles of HSPs in cancer. Methods: We constructed the network for HSPs across ~ 10,000 tumor samples from The Cancer Genome Atlas (TCGA) and ~ 10,000 normal samples from Genotype-Tissue Expression (GTEx), and compared the network disruption between tumor and normal samples. We then examined the associations between HSPs and cancer hallmarks and validated these associations from multiple independent high-throughput functional screens, including Project Achilles and DRIVE. Finally, we experimentally characterized the dual function effects of HSPs in tumor proliferation and metastasis. Results: We comprehensively analyzed the HSP expression landscape across multiple human cancers and revealed a global disruption of the co-expression network for HSPs. Through analyzing HSP expression alteration and its association with tumor proliferation and metastasis, we revealed dual functional effects of HSPs, in that they can simultaneously influence proliferation and metastasis in opposite directions.
    [Show full text]