DOCSLIB.ORG

Chaperonin Function: Folding by Forced Unfolding

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Chaperonin Function: Folding by Forced Unfolding R EPORTS (1985); N. Romani et al., ibid. 169, 1169 (1989); C. migrated was measured with the clonotypic antibody 26. J. G. Cyster, J. Exp. Med. 189, 447 (1999). Heufler et al., ibid. 176, 1221 (1992). to TCR KJ1-26 (28). Overnight incubation of day 2 27. G. G. MacPherson, C. D. Jenkins, M. J. Stein, C. Ed- 23. R. Bonecchi et al., ibid. 187, 129 (1998). draining lymph node cells (at 107 cells/ml) in medium wards, J. Immunol. 154, 1317 (1995). 24. Anti-OVA (DO11.10) T cell receptor (TCR) transgenic containing interleukin-2 (IL-2) (4 ng/ml) increased 28. K. Haskins et al., J. Exp. Med. 157, 1149 (1983). 1 lymph node cells (5 3 106 cells) were transferred to the sensitivity of activated KJ1-26 cells to MDC 29. We thank R. Locksley, S. Luther, K. Reif, and A. Weiss for comments on the manuscript; M. Ansel for help BALB/c mice that were immunized 1 day later with (14). Therefore, IL-2–cultured cells were used in ex- with the in vivo transfer experiments; and C. 100-mg OVA in Freund’s complete adjuvant (25). periments to detect chemokine production by puri- McArthur for cell sorting. Supported in part by NIH fied lymph node DCs and stromal cells. Draining (pool of brachial, axillary, and inguinal) and grant AI-40098, the Pew Foundation (J.G.C.), and the nondraining (mesenteric) lymph node cells were iso- 25. E. R. Kearney, K. A. Pape, D. Y. Loh, M. K. Jenkins, American Lung Association (H.L.T.). lated 1 to 5 days later and used in MDC chemotaxis Immunity 1, 327 (1994); K. M. Murphy, A. B. Heim- assays. The frequency of OVA-specific T cells that berger, D. Y. Loh, Science 250, 1720 (1990). 8 January 1999; accepted 23 March 1999 one protein within a complex, which allowed Chaperonin Function: Folding by us to test the entire active chaperonin system and its individual components on the biolog- Forced Unfolding ically relevant time scale of seconds. In nonpermissive conditions RuBisCO Mark Shtilerman,1 George H. Lorimer,2* S. Walter Englander1 folding is blocked. It fails to fold spontane- ously (23) and can reach the native state only The ability of the GroEL chaperonin to unfold a protein trapped in a misfolded with the help of the complete GroEL-GroES- condition was detected and studied by hydrogen exchange. The GroEL-induced ATP system (24). When unfolded RuBisCO unfolding of its substrate protein is only partial, requires the complete chap- is trapped in this way, most of its amide eronin system, and is accomplished within the 13 seconds required for a single hydrogens exchange rapidly with unlabeled system turnover. The binding of nucleoside triphosphate provides the energy water protons, as expected, but a core of 12 for a single unfolding event; multiple turnovers require adenosine triphosphate highly protected hydrogens exhibit exchange hydrolysis. The substrate protein is released on each turnover even if it has not half-lives of 30 min and longer (detected by yet refolded to the native state. These results suggest that GroEL helps partly tritium label) (Fig. 2). The number of slowly folded but blocked proteins to fold by causing them first to partially unfold. The exchanging hydrogens found and their degree structure of GroEL seems well suited to generate the nonspecific mechanical of protection ensures that they represent stretching force required for forceful protein unfolding. amide groups and not side chains (22). The slowly exchanging hydrogens provide multi- The GroEL chaperonin (1, 2) captures non- ever, numerous experiments have shown that ple probe sites that are sensitive to structural native proteins by means of a ring of hydro- the substrate protein is ejected from the cav- stability and change and may or may not phobic residues that line the entrance to the ity with each round of ATP hydrolysis wheth- represent the same sites in different RuBisCO central cavity of its heptameric ring (Fig. 1) er it has reached the native state or not (11). molecules. (3). When GroEL binds adenosine triphos- The iterative annealing model (12) is based The conditions used (pH 8, 22° 6 2°C), phate (ATP) and the GroES cochaperonin, a on the view that the rate-limiting step in slow chosen to promote the rapid exchange of massive structure change doubles the GroEL protein folding is the intramolecular reorga- amide hydrogens that might be transiently cavity volume and occludes its hydrophobic nization of misfolded and trapped protein unmasked by chaperone action [exchange binding surface (4, 5). Spectroscopic evi- segments, dependent on some degree of pro- half-life ;10 ms (22)], require that the dence (6, 7), proteinase protection experi- tein unfolding (13–15). This model proposes trapped hydrogens must be highly protected ments (6, 8), and electron microscopy (4, 9) that ATP hydrolysis is coupled to a forceful in the non-native protein so that their ex- leave no doubt that the substrate protein is unfolding of the misfolded substrate protein change is slow enough to be measurable. transiently encapsulated in the central cavity and its release, either into the protected cen- Some other proteins tested provided similar under the GroES lid. However, despite much tral cavity or to the exterior, so that the numbers of slow hydrogens but the hydro- additional structural and biochemical study misfolding is relieved and forward folding gens were less protected (maltose-binding (1, 2), the manner in which the GroEL struc- can resume. Incompletely folded proteins un- protein, malate dehydrogenase, rhodanese) ture change promotes protein folding remains dergo further iterations, in the biological [see also (19)]. It seems likely that the pro- to be demonstrated. equivalent of optimization through annealing tected RuBisCO hydrogens are sequestered in Two models, not mutually exclusive, are (16), until they achieve the native state. How- a partially folded domain. Nevertheless, un- under consideration. The Anfinsen cage mod- ever, there is no evidence for a GroES- and folded RuBisCO retains sufficient non-native el (10) is based on the view that protein ATP-dependent unfolding reaction on the structure, perhaps in other domains (25), so folding is limited by intermolecular reactions 13-s time scale of the GroEL–adenosine that it is efficiently captured by GroEL. The that produce aggregation. The model propos- triphosphatase cycle. possibility that the slow hydrogens are pro- es that the GroEL cavity provides a seques- We explored GroEL function using un- tected by RuBisCO association or complex tered microenvironment where folding to the folded ribulose-1,5-bisphosphate carboxy- formation with GroEL was ruled out by native state can proceed while the substrate lase-oxygenase (RuBisCO, from Rhodospi- cross-linking experiments that failed to detect protein is protected from aggregation. How- rillum rubrum) labeled by hydrogen-tritium RuBisCO association under these conditions exchange. The role of the individual system and by experiments that compared immediate components and parameters was studied and delayed GroEL addition. 1The Johnson Research Foundation, Department of through their effect on the exchange of the The time course for exchange of the pro- Biochemistry and Biophysics, University of Pennsyl- protected RuBisCO hydrogens. Prior studies tected hydrogens is the same for RuBisCO vania School of Medicine, Philadelphia, PA 19104, USA. 2Department of Chemistry and Biochemistry, of GroEL (17–21) used various hydrogen free in solution and when bound to GroEL University of Maryland, College Park, MD 20742, USA. exchange approaches (22). Tritium exchange (Fig. 2A). A similar result was found for a *To whom correspondence should be addressed. E- provides advantages including sensitivity, ac- unfolded, disulfide-reduced -lactalbumin mail: [email protected] curacy, rapidity, and the ability to focus on (18). To focus on the slowly exchanging hy- 822 30 APRIL 1999 VOL 284 SCIENCE www.sciencemag.org R EPORTS drogens, we incubated labeled RuBisCO in a than its hydrolysis and also that the unfolding hydrogens occurs within the ;45 s necessary small excess of GroEL for 10 min to allow observed does not require repeated system turn- for separation of the protein from the freed replacement of T with H at the rapidly ex- overs. The fact that 2.5 slow hydrogens re- tritium label. To obtain greater time resolu- changing sites. The binary complex was then mained suggests that GroEL does not fully tion, we added ATP to an otherwise complete mixed with GroES and various nucleotides unfold the substrate molecule. It is expected reaction mixture and EDTA was added 5 to (Fig. 2B). The addition of a twofold molar that even a partial unfolding of the protecting 12 s later to quench the reaction. This allows excess of GroES alone had no effect on the structure will tend to labilize protected hydro- a GroEL cycle in progress to continue but exchange rate of the highly protected hydro- gens to exchange (22). precludes further cycling (11). Figure 3 gens, and neither did Mg21-ADP, Mg21- When a stoichiometric mixture of GroEL, shows that the system is committed to the ATP, or Mg21-AMP-PNP in the absence of GroES, and labeled RuBisCO was passed unfolding event that causes the rapid tritium GroES. Similarly, experiments on b-lacta- through a gel filtration column, all the label mase (20) and dihydrofolate reductase (21) emerged at the position of the complex. found no effect when ATP was added to the When ATP was added, the GroEL-bound GroEL complex without GroES. RuBisCO lost all but about two of its protect- In contrast, the addition of GroES and ed hydrogens whereas added ADP had no Mg21-ATP together resulted in the rapid ex- effect, as in the prior experiments. Thus, the change of all but 2.5 of the protected hydro- behavior observed here involves the interac- gens, signaling some unfolding event (Fig.
Load more

Recommended publications
  • "Hsp70 Chaperones"
    Hsp70 Chaperones Advanced article Elizabeth A Craig, University of Wisconsin, Madison, Wisconsin, USA Article Contents . Introduction Jaroslaw Marszalek, University of Gdansk, Gdansk, Poland . Hsp70:Client Protein Interaction Cycle . Proliferation of Hsp70 and J-protein Genes . Function and Evolution of Mitochondrial Hsp70 Systems . Conclusions: Versatility of Hsp70 System Allows for Adaptation to New Functions Online posting date: 15th March 2011 Via their interaction with client proteins, Hsp70 molecu- stress. In some cases they also facilitate transfer of client lar chaperone machines function in a variety of cellular proteins to proteolytic systems, particularly when refolding processes, including protein folding, translocation of into the native state is unachievable. See also: Chaperones, proteins across membranes and assembly/disassembly of Chaperonin and Heat-Shock Proteins The ability of Hsp70 chaperones to be involved in such protein complexes. Such machines are composed of a core diverse cellular functions, whereas relying on a single bio- Hsp70, as well as a J-protein and a nucleotide exchange chemical activity, an adenosine triphosphate (ATP)- factor as co-chaperones. These co-factors regulate the dependent client binding and release cycle, is remarkable. cycle of adenosine triphosphate (ATP) hydrolysis and Here, to illustrate the molecular mechanisms and evo- nucleotide exchange, which is critical for Hsp70’s inter- lutionary history behind the specialisation of Hsp70 sys- action with client proteins. Cellular compartments often tems we focus on mitochondrial Hsp70 systems, as they contain multiple Hsp70s, J-proteins and nucleotide exemplify two major strategies of Hsp70 specialisation: exchange factors. The capabilities of Hsp70s to carry out (i) amplification and diversification of HSP70 genes and diverse cellular functions can result from either special- (ii) multiplication and specialisation of genes encoding isation of an Hsp70 or by interaction of a multifunctional their J-proteins co-chaperones (Figure 1).
    [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]
  • Structure-Function Relationship and Their Role in Protein Folding
    Chapter 8 1 Molecular Chaperones: Structure-Function 2 Relationship and their Role in Protein Folding 3 Bhaskar K. Chatterjee, Sarita Puri, Ashima Sharma, Ashutosh Pastor, 4 and Tapan K. Chaudhuri 5 Abstract During heat shock conditions a plethora of proteins are found to play a 6 role in maintaining cellular homeostasis. They play diverse roles from folding of 7 non-native proteins to the proteasomal degradation of harmful aggregates. A few 8 out of these heat shock proteins (Hsp) help in the folding of non-native substrate 9 proteins and are termed as molecular chaperones. Various structural and functional 10 adaptations make them work efficiently under both normal and stress conditions. 11 These adaptations involve transitions to oligomeric structures, thermal stability, 12 efficient binding affinity for substrates and co-chaperones, elevated synthesis during 13 shock conditions, switching between ‘holding’ and ‘folding’ functions etc. Their 14 ability to function under various kinds of stress conditions like heat shock, cancers, 15 neurodegenerative diseases, and in burdened cells due to recombinant protein pro- 16 duction makes them therapeutically and industrially important biomolecules. 17 Keywords Chaperone assisted folding · Heat shock · Molecular chaperones · 18 Protein folding · Structure-function of chaperones 19 Abbreviations 20 ACD α-crystallin domain 21 ADP Adenosine di-phosphate 22 ATP Adenosine tri-phosphate 23 CCT Chaperonin containing TCP-1 24 CIRCE Controlling inverted repeat of chaperone expression 25 Bhaskar K. Chatterjee, Sarita Puri, Ashima Sharma, and Ashutosh Pastor authors are equally contributed. B. K. Chatterjee · S. Puri · A. Sharma · A. Pastor · T. K. Chaudhuri (*) Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, HauzKhas, New Delhi, India e-mail: [email protected] © Springer International Publishing AG 2018 181 A.
    [Show full text]
  • Role of CCT Chaperonin in the Disassembly of Mitotic Checkpoint Complexes
    Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes Sharon Kaisaria, Danielle Sitry-Shevaha, Shirly Miniowitz-Shemtova, Adar Teichnera, and Avram Hershkoa,1 aUnit of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel Contributed by Avram Hershko, December 14, 2016 (sent for review November 23, 2016; reviewed by Michele Pagano and Alexander Varshavsky) The mitotic checkpoint system prevents premature separation of thebindingoftheATPasetoMCC(13,14).TheenergyofATP sister chromatids in mitosis and thus ensures the fidelity of hydrolysis is apparently used in this process to promote a confor- chromosome segregation. When this checkpoint is active, a mitotic mational change of Mad2 back from C-Mad2 to O-Mad2, leading checkpoint complex (MCC), composed of the checkpoint proteins to loss of its affinity for Cdc20 in MCC and thus to Mad2 release. Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the By the action of TRIP13 and p31comet, MCC is converted to a ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), remnant, called BC-1, in which Cdc20 is still associated with whose action is necessary for anaphase initiation. When the check- BubR1 (15). Cdc20 is released from BubR1 in MCC by a dif- point signal is turned off, MCC is disassembled, a process required ferent process involving the phosphorylation of Cdc20 by Cdk1 for exit from checkpoint-arrested state. Different moieties of MCC (16). We noticed, however, that a considerable part of the release are disassembled by different ATP-requiring processes. Previous of Cdc20 from MCC was due to a phosphorylation-independent, work showed that Mad2 is released from MCC by the joint action comet but ATP-dependent process (16).
    [Show full text]
  • The Chaperonin Folding Machine
    Review TRENDS in Biochemical Sciences Vol.27 No.12 December 2002 627 32 He, B. et al. (1998) Glycogen synthase kinase 3β DNA damage-induced phosphorylation. Science regulates transcriptional activity. Mol. Cell 6, and extracellular signal-regulated kinase 292, 1910–1915 539–550 inactivate heat shock transcription factor 1 by 43 Waterman, M.J. et al. (1998) ATM-dependent 53 Beals, C.R. et al. (1997) Nuclear localization of facilitating the disappearance of transcriptionally activation of p53 involves dephosphorylation and NF-ATc by a calcineurin-dependent, active granules after heat shock. Mol. Cell. Biol. association with 14-3-3 proteins. Nat. Genet. 19, cyclosporin-sensitive intramolecular interaction. 18, 6624–6633 175–178 Genes Dev. 11, 824–834 33 Xia, W. et al. (1998) Transcriptional activation of 44 Stavridi, E.S. et al. (2001) Substitutions that 54 Porter, C.M. et al. (2000) Identification of amino heat shock factor HSF1 probed by phosphopeptide compromise the ionizing radiation-induced acid residues and protein kinases involved in the analysis of factor 32P-labeled in vivo. J. Biol. association of p53 with 14-3-3 proteins also regulation of NFATc subcellular localization. Chem. 273, 8749–8755 compromise the ability of p53 to induce cell cycle J. Biol. Chem. 275, 3543–3551 34 Dai, R. et al. (2000) c-Jun NH2-terminal kinase arrest. Cancer Res. 61, 7030–7033 55 Yang, T.T. et al. (2002) Phosphorylation of NFATc4 targeting and phosphorylation of heat shock 45 Kaeser, M.D. and Iggo, R.D. (2002) Chromatin by p38 mitogen-activated protein kinases. factor-1 suppress its transcriptional activity.
    [Show full text]
  • Chaperonin 10 from Escherichia Coli (C7438)
    Chaperonin 10 from Escherichia coli recombinant, expressed in E. coli overproducing strain Product Number C7438 Storage Temperature 2–8 C Synonym: GroES The folding activity of a 1:1 molar mixture of GroEL and GroES is determined using urea-denatured rhodanese. Product Description At least a 2-fold increase in reactivation of rhodanese is The chaperonin proteins GroES (Chaperonin 10) and observed compared to spontaneous reactivation. GroEL (Chaperonin 60) are heat shock proteins that assist the folding of nascent and heat-destabilized The inhibition of the ATPase activity of GroEL by GroES proteins.1 GroES (Chaperonin 10) is a complex of at a molar ratio of 2:1 (GroES:GroEL) is determined to 6-8 units of a 10 kDa monomer, with a multimer be 40-60%. molecular mass of 60-80 kDa. Aggregation studies on chaperonin 10 monomer from different species have Purity: 95% (SDS-PAGE) been reported.2 GroEL (Chaperonin 60) is a tetradecamer of a 58 kDa monomer, with a multimer Precautions and Disclaimer molecular mass of 800 kDa. For R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for GroEL and GroES can be used to stabilized labile information regarding hazards and safe handling proteins and reactivate denatured proteins. Refolding practices. and reactivation of denatured enzymes such as the photosynthetic enzyme RuBisCO,3 mitochondrial Preparation Instructions rhodanese,4 and glutamine synthase5 have been The product is soluble in water (0.5 mg/mL). reported. Storage/Stability Folding of proteins by these chaperonins requires It is recommended to store the product desiccated at GroEL, Mg-ATP (GroEL has ATPase activity), and in 2–8 C.
    [Show full text]
  • Chaperonins: Nanocarriers with Biotechnological Applications
    nanomaterials Review Chaperonins: Nanocarriers with Biotechnological Applications Sergio Pipaón 1 , Marcos Gragera 1 , M. Teresa Bueno-Carrasco 1, Juan García-Bernalt Diego 1 , Miguel Cantero 1, Jorge Cuéllar 1 , María Rosario Fernández-Fernández 1 and José María Valpuesta 1,2,* 1 Centro Nacional de Biotecnología (CNB-CSIC). Darwin, 3. 28049 Madrid, Spain; [email protected] (S.P.); [email protected] (M.G.); [email protected] (M.T.B.-C.); [email protected] (J.G.-B.D.); [email protected] (M.C.); [email protected] (J.C.); [email protected] (M.R.F.-F.) 2 Unidad Asociada de Nanobiotecnología (IMDEA Nanociencia), 28049 Madrid, Spain * Correspondence: [email protected] Abstract: Chaperonins are molecular chaperones found in all kingdoms of life, and as such they assist in the folding of other proteins. Structurally, chaperonins are cylinders composed of two back-to-back rings, each of which is an oligomer of ~60-kDa proteins. Chaperonins are found in two main conformations, one in which the cavity is open and ready to recognise and trap unfolded client proteins, and a “closed” form in which folding takes place. The conspicuous properties of this structure (a cylinder containing a cavity that allows confinement) and the potential to control its closure and aperture have inspired a number of nanotechnological applications that will be described in this review. Keywords: molecular chaperones; chaperonins; nanocarriers; nanoreactors Citation: Pipaón, S.; Gragera, M.; Bueno-Carrasco, M.T.; García-Bernalt 1. Chaperonins: Structure and Function Diego, J.; Cantero, M.; Cuéllar, J.; In the crowded environment of the cell, most proteins need assistance to acquire their Fernández-Fernández, M.R.; Valpuesta, J.M.
    [Show full text]
  • I ANTIBIOTIC DISCOVERY TARGETING BACTERIAL GROEL
    ANTIBIOTIC DISCOVERY TARGETING BACTERIAL GROEL/GROES CHAPERONIN SYSTEMS Trent A. Kunkle Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Science in the Department of Biochemistry and Molecular Biology Indiana University August 2018 i Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Master of Science. ____________________________ Steven M Johnson, Ph.D., Chair ____________________________ Millie M. Georgiadis, Ph.D. Master’s Thesis Committee ____________________________ Quyen Q. Hoang, Ph.D. ii DEDICATION To my mother, Donna, and my father, Alex, for supporting me through everything. iii ACKNOWLEDGEMENTS Dr. Steven Johnson and Jose Victorino for compound synthesis; Dr. Sanofar Abdeen for research training and performing the GroEL/ES-dRho refolding assay and native Rho enzymatic activity counter-screen, the HSP60/10-dMDH refolding assay, and the S. aureus biofilm and gain-of-resistance assays; Dr. Anne-Marie Ray for performing the human cell cytotoxicity assays and training in cell culturing techniques. iv Trent A. Kunkle ANTIBIOTIC DISCOVERY TARGETING BACTERIAL GROEL/GROES CHAPERONIN SYSTEMS The Centers for Disease Control (CDC) and World Health Organizations (WHO) have highlighted six species of highly drug-resistant bacteria, commonly termed the ESKAPE pathogens, that new antibacterials are urgently needed to treat). The ESKAPE pathogens account for over two-million infections and have healthcare costs upwards of $20 billion dollars annually. Over the past several decades, pharmaceutical companies have drastically reduced their research programs for developing new antibacterial agents. As well, bacteria are predisposed to rapidly generate resistance against these “me too” drugs, making this strategy a temporary stop-gap in our ability to fight these pathogens.
    [Show full text]
  • Chaperonin-Facilitated Protein Folding: Optimization of Rate and Yield by an Iterative Annealing Mechanism MATTHEW J
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4030-4035, April 1996 Biochemistry Chaperonin-facilitated protein folding: Optimization of rate and yield by an iterative annealing mechanism MATTHEW J. TODD*, GEORGE H. LORIMER*, AND D. THIRUMALAIt *Central Research and Development Department, DuPont Company Experimental Station, Wilmington, DE 19880-0402; and tInstitute for Physical Science and Technology, University of Maryland, College Park, MD 20742 Communicated by I. C. Gunsalus, Environmental Research Laboratory, Gulf Breeze, FL, December 15, 1995 (received for review June 1, 1995) ABSTRACT We develop a heuristic model for chapero- mulate; if deep enough, these traps can retard the appearance of nin-facilitated protein folding, the iterative annealing mech- the native state. Especially for large proteins, spontaneousfolding anism, based on theoretical descriptions of "rugged" confor- of all of the molecules to the native state may not occur in vitro mational free energy landscapes for protein folding, and on on a biologicallymeaningful time scale.t (ii) With energy from the experimental evidence that (i) folding proceeds by a nucle- coordinated hydrolysis of ATP, the chaperonins provide a mech- ation mechanism whereby correct and incorrect nucleation anism to traverse the landscape, circumventing the barriers lead to fast and slow folding kinetics, respectively, and (ii) separating the misfolded structures and the native state, as chaperonins optimize the rate and yield of protein folding by suggested on theoretical (6) and on experimental (11, 12) an active ATP-dependent process. The chaperonins GroEL grounds. The interaction of the chaperonin with the substrate and GroES catalyze the folding of ribulose bisphosphate protein is stochastic in nature; the appearance of the native state carboxylase at a rate proportional to the GroEL concentra- is the consequence of multiple rounds of binding and release tion.
    [Show full text]
  • Chaperone Machines for Protein Folding, Unfolding and Disaggregation
    REVIEWS Chaperone machines for protein folding, unfolding and disaggregation Helen Saibil Abstract | Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20–30 kDa domains over distances of 20–50 Å and rotations of up to 100º. Autophagy Protein quality control, also known as proteostasis, diverse proteins? Most of the main chaper­ones use cycles A process in which intracellular constitutes the regulation of protein synthesis, folding, of ATP binding and hydrolysis to act on non-native poly- material is enclosed in a unfolding and turnover. It is mediated by chaperone peptides, facilitating their folding or unfolding6. Others membrane compartment and and protease systems, together with cellular clearance simply have a ‘handover’ role, protecting nascent sub­ delivered to the lysosome autophagy (vacuole in yeast) for mechanisms such as and lysosomal degrada- units during the assembly process. Some ATP-dependent degradation and recycling tion. These quality control systems have an essential chaperones, also known as protein remodelling factors, of the macromolecular role in the life of cells, ensuring that proteins are cor- mediate targeted disassembly, unfolding or even rever- constituents.
    [Show full text]
  • Deletion of Heat Shock Protein 60 in Adult Mouse Cardiomyocytes Perturbs Mitochondrial Protein Homeostasis and Causes Heart Failure
    Cell Death & Differentiation (2020) 27:587–600 https://doi.org/10.1038/s41418-019-0374-x ARTICLE Deletion of heat shock protein 60 in adult mouse cardiomyocytes perturbs mitochondrial protein homeostasis and causes heart failure 1 1 1 2 1 3 1 1 Feifei Fan ● Yaoyun Duan ● Feili Yang ● Christa Trexler ● Hong Wang ● Lei Huang ● Yali Li ● Huayuan Tang ● 4 2 4 5 2 1 Gang Wang ● Xi Fang ● Jie Liu ● Nan Jia ● Ju Chen ● Kunfu Ouyang Received: 12 March 2019 / Revised: 10 May 2019 / Accepted: 5 June 2019 / Published online: 17 June 2019 © The Author(s) 2019. This article is published with open access Abstract To maintain healthy mitochondrial enzyme content and function, mitochondria possess a complex protein quality control system, which is composed of different endogenous sets of chaperones and proteases. Heat shock protein 60 (HSP60) is one of these mitochondrial molecular chaperones and has been proposed to play a pivotal role in the regulation of protein folding and the prevention of protein aggregation. However, the physiological function of HSP60 in mammalian tissues is not fully understood. Here we generated an inducible cardiac-specific HSP60 knockout mouse model, and demonstrated that HSP60 deletion in adult mouse hearts altered mitochondrial complex activity, mitochondrial membrane potential, and ROS production, and eventually led to dilated cardiomyopathy, heart failure, and lethality. Proteomic analysis was performed in purified control and mutant mitochondria before mutant hearts developed obvious cardiac abnormalities, and revealed a list of mitochondrial-localized proteins that rely on HSP60 (HSP60-dependent) for correctly folding in mitochondria. We also utilized an in vitro system to assess the effects of HSP60 deletion on mitochondrial protein import and protein stability after import, and found that both HSP60-dependent and HSP60-independent mitochondrial proteins could be normally imported in mutant mitochondria.
    [Show full text]
  • Involvement of a Eukaryotic-Like Ubiquitin-Related Modifier
    ARTICLE Received 20 Mar 2015 | Accepted 25 Jul 2015 | Published 8 Sep 2015 DOI: 10.1038/ncomms9163 OPEN Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius Rana S. Anjum1,*, Sian M. Bray1,*, John K. Blackwood1, Mairi L. Kilkenny1, Matthew A. Coelho1,w, Benjamin M. Foster1,w, Shurong Li1, Julie A. Howard2, Luca Pellegrini1, Sonja-Verena Albers3,w, Michael J. Deery2 & Nicholas P. Robinson1 In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin- like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved. 1 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. 2 Department of Biochemistry and Cambridge Systems Biology Centre, Cambridge Centre for Proteomics, Cambridge CB2 1QR, UK. 3 Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany.
    [Show full text]