Portal de Periódicos da CAPES

Molecular Chaperones in Cellular Protein Folding: The Birth of a Field

2014; Cell Press; Volume: 157; Issue: 2 Linguagem: Inglês

10.1016/j.cell.2014.03.029

1097-4172

Arthur L. Horwich,

Enzyme Structure and Function

The early decades of Cell witnessed key discoveries that coalesced into the field of chaperones, protein folding, and protein quality control. The early decades of Cell witnessed key discoveries that coalesced into the field of chaperones, protein folding, and protein quality control. In January 1974, at the front of the first issue of Cell, Benjamin Lewin provided an opening announcement entitled "A Journal of Exciting Biology," establishing the goal of publishing the elucidation of systems responsible for cellular function and phenotype. For those reading across all or part of the 40 year span currently being celebrated, there can be no question that the goal has been met beyond all expectation. I can think of so many astonishing revelations that were first brought to light in Cell. The Cell paper from Chow et al., 1977Chow L.T. Gelinas R.E. Broker T.R. Roberts R.J. Cell. 1977; 12: 1-8Abstract Full Text PDF PubMed Scopus (693) Google Scholar describing splicing—"An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA"—stands out to me as the most dazzling early paper (coinciding with the equally stunning paper of Berget et al., 1977Berget S.M. Moore C. Sharp P.A. Proc. Natl. Acad. Sci. USA. 1977; 74: 3171-3175Crossref PubMed Scopus (848) Google Scholar). In looking back and taking stock of an area close to my own heart, I would say that, as a collective, the papers investigating the molecular machines that govern the folded state of proteins inside of the cell—the chaperones—are equally distinguished in describing biology that was unexpected and exciting. Here, I'll discuss how a number of diverse lines of inquiry, published during the first two decades of Cell's history, coalesced into the field of chaperones, protein folding, and protein quality control as we now know it. The term "molecular chaperone" hadn't been coined at the time Cell was launched. That had to wait until 1978, when Ron Laskey used the term to describe nucleoplasmin, a protein that binds and conveys histones into the nuclear compartment, shielding positive charge of the histones via its own acidic character (Laskey et al., 1978Laskey R.A. Honda B.M. Mills A.D. Finch J.T. Nature. 1978; 275: 416-420Crossref PubMed Scopus (537) Google Scholar). Obviously the term got repurposed to machines that bind nonnative proteins—I think with Ron's blessing, as indicated by his willingness to attend early meetings of the field, speak about nuclear biology, and strum a few songs in the beer frame. As concerns protein folding at the launch of Cell, Christian Anfinsen had recently received the Nobel Prize in Chemistry (1972) for work showing that the primary structure of a protein contains all of the information necessary for folding to the native state, which lies at an energetic minimum (Anfinsen, 1973Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5137) Google Scholar). Who could have thought at that point that thermodynamics would not be enough to produce the native active form of proteins inside of the cell? Who would have imagined that kinetic assistance by a dedicated group of protein machines, in most cases utilizing ATP, would be essential for the proper folding of a large cohort of proteins? That realization emerged from two contemporaneous but initially disconnected sets of observations. On one hand, it became clear that many proteins could not spontaneously refold in a test tube in the same way as ribonuclease in Anfinsen's early experiments, lodging instead in insoluble aggregates that could be sedimented to the bottom of the tube. In addition, in the cellular context, as expression of mammalian proteins in E.coli was undertaken in the late 1970s and early 1980s, it became clear that many expressed proteins were subject to misfolding, aggregation, and localization into terminal inclusion bodies (Williams et al., 1982Williams D.C. Van Frank R.M. Muth W.L. Burnett J.P. Science. 1982; 215: 687-689Crossref PubMed Scopus (244) Google Scholar, Marston, 1986Marston F.A.O. Biochem. J. 1986; 240: 1-12Crossref PubMed Scopus (752) Google Scholar, Haase-Pettingell and King, 1988Haase-Pettingell C.A. King J. J. Biol. Chem. 1988; 263: 4977-4983Abstract Full Text PDF PubMed Google Scholar; Figure 1). Thus, in these situations, there seemed to be kinetic difficulties during protein folding. On the other hand, a class of proteins known as heat shock proteins was becoming the subject of considerable scrutiny. In 1962, regions of Drosophila salivary gland chromosomes were observed to become "puffed" during heat shock (Figure 2). RNAs induced under these conditions were shown by in situ hybridization to be produced from these regions (Spradling et al., 1975Spradling A. Penman S. Pardue M.L. Cell. 1975; 4: 395-404Abstract Full Text PDF PubMed Scopus (170) Google Scholar, McKenzie et al., 1975McKenzie S.L. Henikoff S. Meselson M. Proc. Natl. Acad. Sci. USA. 1975; 72: 1117-1121Crossref PubMed Scopus (240) Google Scholar). It became clear with molecular cloning of the abundant heat-induced RNA that one of these regions encoded a 70 kDa heat-shock-induced protein (Schedl et al., 1978Schedl P. Artavanis-Tsakonas S. Steward R. Gehring W.J. Mirault M.-E. Goldschmidt-Clermont M. Moran L. Tissières A. Cell. 1978; 14: 921-929Abstract Full Text PDF PubMed Scopus (101) Google Scholar). At about the same time, it was observed that a characteristic set of heat-inducible proteins, including a 70 kDa protein, was manifest in both E.coli (Lemaux et al., 1978Lemaux P.G. Herendeen S.L. Bloch P.L. Neidhardt F.C. Cell. 1978; 13: 427-434Abstract Full Text PDF PubMed Scopus (155) Google Scholar, Yamamori et al., 1978Yamamori T. Ito K. Nakamura Y. Yura T. J. Bacteriol. 1978; 134: 1133-1140Crossref PubMed Google Scholar, Bardwell and Craig, 1984Bardwell J.C.A. Craig E.A. Proc. Natl. Acad. Sci. USA. 1984; 81: 848-852Crossref PubMed Scopus (346) Google Scholar) and metazoan fibroblasts (Kelley and Schlesinger, 1978Kelley P.M. Schlesinger M.J. Cell. 1978; 15: 1277-1286Abstract Full Text PDF PubMed Scopus (327) Google Scholar). It seemed likely that these inducible proteins would be protective to the cell under stress. Was there a link between heat-shock-induced proteins and the kinetic challenges of in vivo protein folding? The work of Pelham was particularly telling with respect to heat shock. He observed that Drosophila Hsp70 expressed in mouse L cells or monkey COS cells enabled rapid recovery of nucleolar damage following heat shock (Pelham, 1984Pelham H.R.B. EMBO J. 1984; 3: 3095-3100Crossref PubMed Scopus (366) Google Scholar). He subsequently analyzed release of Hsp70 from the nuclei isolated from heat-shocked cells, observing tight binding of Hsp70 to the nuclei relative to the nonshocked cells and rapid and complete release upon the addition of ATP (Lewis and Pelham, 1985Lewis M.J. Pelham H.R.B. EMBO J. 1985; 4: 3137-3143Crossref PubMed Scopus (247) Google Scholar). A model based on these findings was presented in a Cell Minireview (Pelham, 1986Pelham H.R.B. Cell. 1986; 46: 959-961Abstract Full Text PDF PubMed Scopus (1145) Google Scholar), proposing a cycle of action wherein Hsp70 binds to incipiently aggregating proteins (as produced by heat shock) and pries them apart through recurrent cycles of binding and release associated with ATP binding and hydrolysis. Because Hsp70 was known to strongly bind to hydrophobic column matrices, it was proposed that it recognizes hydrophobic surfaces of the misfolding proteins and prevents them from driving aggregation. It was a prescient model. Indeed, recognition by molecular chaperones generally involves the binding of hydrophobic surfaces specifically exposed in nonnative proteins by hydrophobic surfaces proffered by the chaperones themselves, each chaperone family offering a different geometry of binding surface (Bukau and Horwich, 1998Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2426) Google Scholar). Subsequent binding of ATP then produces allosterically mediated movement of the binding surface that releases the protein substrate (Zhuravleva et al., 2012Zhuravleva A. Clerico E.M. Gierasch L.M. Cell. 2012; 151: 1296-1307Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, Kityk et al., 2012Kityk R. Kopp J. Sinning I. Mayer M.P. Mol. Cell. 2012; 48: 863-874Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Clare et al., 2012Clare D.K. Vasishtan D. Stagg S. Quispe J. Farr G.W. Topf M. Horwich A.L. Saibil H.R. Cell. 2012; 149: 113-123Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Pelham's observations concerning the action of Hsp70 fit well with concurrent data that emerged from studies of two other 70 kDa proteins, the immunoglobulin-binding protein (BiP) inside of the endoplasmic reticulum (ER) and the clathrin-uncoating ATPase in the cytosol. In the former case, a 70 kDa protein was found to bind selectively to immunoglobulin heavy chains prior to their association with light chains, indicating once again a protein-protein interaction, here potentially facilitating oligomeric assembly (Haas and Wabl, 1983Haas I.G. Wabl M. Nature. 1983; 306: 387-389Crossref PubMed Scopus (591) Google Scholar). In the latter case, studies of Rothman and coworkers (Schlossman et al., 1984Schlossman D.M. Schmid S.L. Braell W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (288) Google Scholar, Chappell et al., 1986Chappell T.G. Welch W.J. Schlossman D.M. Palter K.B. Schlesinger M.J. Rothman J.E. Cell. 1986; 45: 3-13Abstract Full Text PDF PubMed Scopus (422) Google Scholar) and of Ungewickell, 1985Ungewickell E. EMBO J. 1985; 4: 3385-3391PubMed Google Scholar indicated that a 70 kDa protein was an ATP-dependent mediator of uncoating clathrin cages from vesicles during endocytosis, releasing clathrin triskelions. This amounted to an action more like that described by Pelham, in which binding of the 70 kDa protein mediates disassembly of a protein complex—in the case of clathrin, an action carried out under normal physiologic conditions (by what we now know to be the constitutively expressed heat shock 70 "cognate" protein, Hsc70 [Xing et al., 2010Xing Y. Böcking T. Wolf M. Grigorieff N. Kirchhausen T. Harrison S.C. EMBO J. 2010; 29: 655-665Crossref PubMed Scopus (92) Google Scholar]). Shortly thereafter, cytosolic Hsp70 proteins became implicated in transport of protein precursors into ER and mitochondria. The chaperone binds the protein to be transported in the cytosol, apparently preventing its hydrophobic surfaces from producing aggregation and holding it in an unfolded state that could engage with and pass through translocation machinery (Chirico et al., 1988Chirico W.J. Waters M.G. Blobel G. Nature. 1988; 332: 805-810Crossref PubMed Scopus (837) Google Scholar, Deshaies et al., 1988Deshaies R.J. Koch B.D. Werner-Washburne M. Craig E.A. Schekman R. Nature. 1988; 332: 800-805Crossref PubMed Scopus (1002) Google Scholar, Eilers and Schatz, 1986Eilers M. Schatz G. Nature. 1986; 322: 228-232Crossref PubMed Scopus (466) Google Scholar). These events were not stress related, indicating a constitutive need for the action of 70 kDa class chaperone proteins. The apparently disparate worlds of protein folding and molecular chaperones converged in the 1980s, with the characterization of a separate class of heat-inducible ATP-hydrolyzing proteins: oligomeric double-ring protein complexes composed of ∼60 kDa subunits, the Hsp60s. These complexes were first implicated as playing a role in oligomeric assembly. Genetic defects in the bacterial GroE operon (Georgopoulos et al., 1972Georgopoulos C.P. Hendrix R.W. Kaiser A.D. Wood W.B. Nat. New Biol. 1972; 239: 38-41Crossref PubMed Scopus (132) Google Scholar, Takano and Kakefuda, 1972Takano T. Kakefuda T. Nat. New Biol. 1972; 239: 34-37Crossref PubMed Scopus (80) Google Scholar) were observed to affect the ability of propagating phages to assemble, but they also affected bacterial cell growth. Likewise, a role in oligomeric assembly was ascribed to the similarly sized Rubisco subunit-binding protein, which could associate with newly translated large subunits of Rubisco inside of the chloroplast stroma, but not with mature Rubisco, formed by assembly of the large subunits with small subunits imported from the cytosol (Barraclough and Ellis, 1980Barraclough R. Ellis R.J. Biochim. Biophys. Acta. 1980; 608: 19-31Crossref PubMed Scopus (189) Google Scholar). John Ellis dubbed these ring complexes chaperonins. The homology of GroEL with Rubisco-binding protein was then appreciated upon sequencing of the respective coding regions (Hemmingsen et al., 1988Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (933) Google Scholar). A role for chaperonins in polypeptide chain folding, as distinct from oligomeric assembly, soon emerged from studies of a yeast mutant affecting a GroEL homolog in the mitochondrial matrix, mitochondrial Hsp60 (Cheng et al., 1989Cheng M.-Y. Hartl F.-U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Nature. 1989; 337: 620-625Crossref PubMed Scopus (670) Google Scholar). In this mutant, proteins entering mitochondria failed to reach native form. Among the first proteins found to be affected in the mutant was a monomeric protein, the Rieske iron-sulfur protein. This suggested that proper polypeptide folding, as opposed to oligomeric protein assembly of already-folded monomers, might be the step facilitated by the chaperonin ring assemblies. This role was further established by the observation that monomeric DHFR imported into mitochondria (by attachment of an N-terminal mitochondrial targeting signal) associated in a nonnative form with the Hsp60 complex and was subsequently released in a native form upon addition of ATP (Ostermann et al., 1989Ostermann J. Horwich A.L. Neupert W. Hartl F.-U. Nature. 1989; 341: 125-130Crossref PubMed Scopus (476) Google Scholar). Hsp60 proved to be an essential gene in yeast, indicating a requirement for its action under all conditions (Cheng et al., 1989Cheng M.-Y. Hartl F.-U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Nature. 1989; 337: 620-625Crossref PubMed Scopus (670) Google Scholar, Reading et al., 1989Reading D.S. Hallberg R.L. Myers A.M. Nature. 1989; 337: 655-659Crossref PubMed Scopus (222) Google Scholar). Mechanistic insights were enabled by in vitro reconstitution experiments. The first reconstitution experiment was carried out with the dimeric Rubisco from R. rubrum. Denatured subunit diluted from denaturant became bound to GroEL, and subsequent addition of ATP and cochaperonin GroES (a single ring composed of 10 kDa "small" subunits) led to production of native, active Rubisco (Goloubinoff et al., 1989Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (546) Google Scholar). Further in vitro refolding experiments with monomeric DHFR and rhodanese confirmed that GroEL/GroES could mediate refolding to the native state of these proteins following their dilution from chaotrope, whereas quantitative aggregation occurred upon dilution into buffer solution (Martin et al., 1991Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (725) Google Scholar). Apparently the chaperonin system could prevent or remove proteins from kinetically trapped states and allow them to reach native form. The chaperonin reaction could be experimentally broken into steps: binding to GroEL prevented wholesale aggregation of the protein substrate, and subsequent addition of GroES/ATP produced the native state over a period of minutes, with GroES appearing to couple the folding reaction to the GroEL ring assembly (Goloubinoff et al., 1989Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (546) Google Scholar, Martin et al., 1991Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (725) Google Scholar). We now know that substrate proteins bind to a surrounding hydrophobic surface in the cavity of an open GroEL ring and are, upon ATP/GroES binding, ejected into a now hydrophilic GroES-encapsulated chamber where they proceed to fold in isolation, without the chance of aggregation (Horwich and Saibil, 2011Horwich A.L. Saibil H.R. The GroEL/GroES chaperonin machine.in: Frank Joachim Molecular Machines in Biology. Cambridge University Press, New York2011: 191-207Crossref Scopus (1) Google Scholar). Thus, the early experiments summarized here established that there are molecular machines that prevent protein aggregation and use ATP to help adjust the conformation of other proteins. Their abundance increases under stress conditions via transcriptional regulation to provide increased capacity to prevent aggregation. Under nonstress conditions, they provide kinetic assistance to folding, apparently necessary due to ongoing mis-steps of protein folding even at physiologic temperature in a milieu that has a large concentration of solute. Not only could the chaperones prevent aggregation from occurring, but they could also help proteins to maintain unfolded conformations when necessary, e.g., when emerging from ribosomes or when passage through membranes required an unfolded state. Finally, in the case of the chaperonins, they directly promote the native state of proteins via folding inside of an encapsulated chamber. Thus, the existence of a diverse and dedicated machinery for protein "management" in the cell had been uncovered. A host of other chaperone machines have been identified in a variety of compartments—too many to list here. In addition, the field has expanded to include the characterization of stress-sensing pathways that provide exquisite regulation of the chaperone systems. It is now also apparent that the chaperone systems link to the ubiquitin-proteasome and autophagy systems as part of a global quality control network. Unexpected discoveries are surely still to come. I'd like to particularly thank Walter Neupert and Ulrich Hartl for a well-timed phone call that triggered a wonderful collaboration to understand the mitochondrial-folding-defective yeast mutant affecting Hsp60. I thank all of the talented people who have worked in my group over the years, as well as my collaborators, with whom I've enjoyed watching our field grow and develop. I thank the NIH and Howard Hughes Medical Institute for generous support of the chaperonin work. And I thank Ben Lewin and his successors for their ongoing interest in our field, which I think has lived up to providing "exciting biology."