Fink, Anthony L. Chaperone-Mediated Protein Folding. Physiol. Rev. 79: 425-449, 1999.
The basic paradigm of molecular chaperones is that they recognize and selectively bind nonnative, but not native, proteins to form relatively stable complexes (48). In most cases, the complexes are dissociated by the binding and hydrolysis of ATP. In addition, there are "specific" molecular chaperones that typically are involved in the assembly of particular multiprotein complexes. Molecular chaperones comprise several highly conserved families of unrelated proteins; many chaperones are also heat shock (stress) proteins. The ubiquitous role of molecular chaperones continues to unfold with more discoveries each year. In the context of in vivo protein folding, chaperones prevent irreversible aggregation of nonnative conformations and keep proteins on the productive folding pathway. In addition, they may maintain newly synthesized proteins in an unfolded conformation suitable for translocation across membranes and bind to nonnative proteins during cellular stress, among other functions. It is likely that most, if not all, cellular proteins will interact with a chaperone at some stage of their lifetime.
The focus of this review is on the functional contribution of chaperones to in vivo protein folding and assembly, especially those chaperones that are promiscuous, in that they show broad specificity for binding nonnative proteins. In addition to several specialized review articles on chaperones, e.g., References 11, 49, 50, 53, 76, 77, 90, 144, 164, 190, 191, 226, 228, 252, there have been two recent monographs published on the subject (60, 150). This review is of necessity selective; the main goal is to furnish an up-to-date overview of the role of the major molecular chaperones involved in protein folding. In view of the vast array of literature on molecular chaperones, and the ease of access to literature citations using the Internet, this article should not be viewed as exhaustive.
Why do we need chaperones? After all, a basic tenet of in vitro protein folding has been the seminal work of Anfinsen (2), which demonstrated that formation of the native protein from the unfolded state is a spontaneous process determined by the global free energy minimum. The results indicated that the native state of small globular proteins is determined by their amino acid sequence. However, the experimental conditions necessary to successfully fold many proteins, especially larger ones, in vitro, are very constrictive, usually requiring very low protein concentration and long incubation times and are usually unphysiological (e.g., relatively low temperatures). In contrast, most cells operate at ambient or homeothermically set temperatures (e.g., 37°C) where the hydrophobic effect will be stronger and thus protein denaturation and aggregation will be bigger problems, and the time-frame available for successful folding is short. Thus there is the need for additional factors for the successful folding of many proteins in vivo. When one considers the crowded cellular environment within a cell, it becomes clear that in vitro folding experiments at low protein concentrations are poor models for what happens in the cell, where a newly synthesized protein is in an environment with little or no "free" water, very high concentrations of other proteins and metabolites, and typically membranes, cytoskeletal elements, and other cellular components. Thus the need for chaperones 1) to prevent aggregation and misfolding during the folding of newly synthesized chains, 2) to prevent nonproductive interactions with other cell components, 3) to direct the assembly of larger proteins and multiprotein complexes, and 4) during exposure to stresses that cause previously folded proteins to unfold, becomes evident. In the few cases where folding has been studied both in vivo and in vitro, it appears that the folding pathways are similar (148, 190).
Cells have solved the problem of misfolding and aggregation, to a considerable extent at least, through the participation of molecular chaperones in the in vivo folding process. Many investigations in the past few years have confirmed the critical role of molecular chaperones in protein folding in the cell. Although much has been learned about the function of chaperones in protein folding, and the general outline of the process is thought to be understood, there are still many important unresolved issues, and new chaperones and cochaperones are still being discovered.
The molecular chaperones involved in the folding of newly synthesized proteins recognize nonnative substrate proteins predominantly via their exposed hydrophobic residues. The major chaperone classes are 40-kDa heat shock protein (HSP40; the DnaJ family), 60-kDa heat shock protein [HSP60; including GroEL and the T-complex polypeptide 1 (TCP-1) ring complexes], 70-kDa heat shock protein (HSP70), and 90-kDa heat shock protein (HSP90). All these chaperones can prevent the aggregation of at least some unfolded proteins. For HSP60 and HSP70, their activity is modulated by the binding and hydrolysis of ATP. The HSP70 (DnaK in Escherichia coli) bind to nascent polypeptide chains on ribosomes, preventing their premature folding, misfolding, or aggregation, as well as to newly synthesized proteins in the process of translocation from the cytosol into the mitochondria and the endoplasmic reticulum (ER). The HSP70 are regulated by HSP40 (DnaJ or its homologs). The HSP60 are large oligomeric ring-shaped proteins known as chaperonins that bind partially folded intermediates, preventing their aggregation, and facilitating their folding and assembly. This family is composed of GroEL-like proteins in eubacteria, mitochondria, and chloroplasts and the TCP-1 (CCT or TRiC) family in the eukaryotic cytosol and the archaea. The HSP60 (GroEL in E. coli) are large, usually tetradecameric proteins with a central cavity in which nonnative protein structures bind. The HSP60 are found in all biological compartments except the ER. The HSP60 are regulated by a cochaperone, chaperonin 10 (cpn10) (GroES in E. coli). In addition to preventing aggregation, it has been suggested that HSP60 may permit misfolded structures to unfold and refold. The HSP90 are associated with a number of proteins and play important roles in modulating their activity, most notably the steroid receptors. A number of other proteins involved in the folding of many newly synthesized proteins are often considered to be molecular chaperones; these include protein disulfide isomerase and peptidyl prolyl isomerase, which catalyze the rearrangement of disulfide bonds and isomerization of peptide bonds around Pro residues, respectively, and are perhaps better considered to be folding catalysts rather than chaperones. As mentioned previously, there are also a number of more specific chaperones that are involved in the folding/assembly of only one, or a very limited number, of particular substrate proteins.
Chaperones are catalysts in the sense that they transiently interact with their substrate proteins but are not present in the final folded product, and also in that they increase the yield of folded protein. However, there is no good evidence that they actually enhance the spontaneous rate of folding itself, although they may appear to do this by minimizing off-pathway reactions.A brief perusal of the literature demonstrates that our knowledge of molecular chaperones is growing at an enormous pace. To put these new discoveries in context, a few more general points are worthy of note. Although in many respects the field of molecular chaperones can now be considered a mature one, in that it has passed its first decade of life, and the broad outlines, at least, are reasonably well established, there are still many outstanding questions. Furthermore, there are many areas of considerable controversy, and many of these relate to fundamental questions. For example, we do not yet know with certainty whether all newly synthesized proteins interact with chaperones, although it is likely that they do. We certainly do not know much about all the interactions between the various chaperones themselves, as well as with newly synthesized proteins or other chaperone target proteins. As discussed in this review, there are significant controversies concerning which chaperones interact first with nascent polypeptides, and even whether all nascent polypeptides interact with chaperones. The GroEL family of chaperones has been intensively studied, especially in the context of in vitro protein folding, yet it is not clear just how important a role this family (the cpn60 chaperonins and their TCP-1 eukaryotic homologs) play in the folding of most proteins in the cell. We are only now beginning to get a picture of the apparently ubiquitous role of the HSP90 family in many critical processes in the cell, especially those involving protein-protein interactions. Recently, several new "accessory" proteins have been discovered, which apparently act as "cochaperones." Again, their significance to protein folding and denaturation in the cell in general is unclear at the present time; they may be highly specialized or may turn out to be critical in a broad range of cellular processes involving chaperones. Although some of the chaperones clearly are important in preventing protein aggregation, there is as yet no good evidence that chaperones play a role significant in the opposite side of this equation, namely, in solubilizing protein aggregates, although it would seem likely that this may in fact be a function of some chaperones. Even at the level of the specific mechanisms of chaperone function, there are many controversial aspects, and those in the field know there have been some quite rancorous discussions over competing mechanisms.