Proteasomes and molecular chaperones: cellular machinery responsible for folding and destruction of unfolded proteins

Molecular chaperones recognize proteins of non-native structure, prevent them from irreversible intracellular aggregation, and then act with regulatory co-chaperones in the conversion of proteins to be properly folded and in a functional state. However, not every non-native protein is folded successfully. Those proteins that are not accurately folded/ refolded are then directed to the ubiquitin-proteasome system (UPS) for destruction. Both chaperones and proteasomes act jointly together for selective removal of proteins with aberrant structure so as to keep protein homeostasis in cells. Though the precise nature of the cooperative linkage between chaperone and UPS pathways remains largely elusive so far, accumulating evidence from in vivo and in vitro studies shed some light on the molecular mechanisms that link proteasomes and molecular chaperones. This review focuses on how unfolded proteins are handled by these two machineries.     

Getting a grip on non-native proteins

It is an underappreciated fact that non-native polypeptides are prevalent in the cellular environment. Native proteins have the folded structure, assembled state and cellular localization required for activity. By contrast, non-native proteins lack function and are particularly prone to aggregation because hydrophobic residues that are normally buried are exposed on their surfaces. These unstable entities include polypeptides that are undergoing synthesis, transport to and translocation across membranes, and those that are unfolded before degradation. Non-native proteins are normal, biologically relevant components of a healthy cell, except in cases in which their misfolding results from disease-causing mutations or adverse extrinsic factors. Here, we explore the nature and occurrence of non-native proteins, and describe the diverse families of molecular chaperones and coordinated cellular responses that have evolved to prevent their misfolding and aggregation, thereby maintaining quality control over these potentially damaging protein species.     

Roles of molecular chaperones in cytoplasmic protein folding

Newly synthesized polypeptide chains must fold and assemble into unique three-dimensional structures in order to become functionally active. In many cases productive folding depends on assistance from molecular chaperones, which act in preventing off-pathway reactions during folding that lead to aggregation. The inherent tendency of incompletely folded polypeptide chains to aggregate is thought to be strongly enhanced$L in vivo *I$Lby the high macromolecular concentration of the cellular solution, resulting in crowding effects, and by the close proximity of nascent polypeptide chains during synthesis on polyribosomes. The major classes of chaperones acting in cytoplasmic protein folding are the Hsp70s and the chaperonins. Hsp70 chaperones shield the hydrophobic regions of nascent and incompletely folded chains, whereas the chaperonins provide a sequestered environment in which folding can proceed unimpaired by intermolecular interactions between non-native polypeptides. These two principles of chaperone action can function in a coordinated manner to ensure the efficient folding of a subset of cytoplasmic proteins.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

The ubiquitin-proteasome system in cardiac dysfunction

Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin-proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease.     

From the cradle to the grave: molecular chaperones that may choose between folding and degradation

Molecular chaperones are known to facilitate cellular protein folding. They bind non-native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy-dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.     

Contribution of molecular chaperones to protein folding in the cytoplasm of prokaryotic and eukaryotic cells

While it is clear that many unfolded proteins can attain their native state spontaneously in vitro, the efficiency of such folding is usually limited to conditions far removed from those encountered within cells. Two properties of the cellular environment are expected to enhance strongly the propensity of incompletely folded polypeptides to misfold and aggregate: the crowding effect caused by the high concentration of macromolecules, and the close proximity of nascent polypeptide chains emerging from polyribosomes. However, in the living cell, non-productive protein folding is in many, if not most, cases prevented by the action of a highly conserved set of proteins termed molecular chaperones. In the cytoplasm, the Hsp70 (heat-shock protein of 70 kDa) and chaperonin families of molecular chaperones appear to be the major contributors to efficient protein folding during both normal conditions and adverse conditions such as heat stress. Hsp70 chaperones recognize and shield short, hydrophobic peptide segments in the context of non-native polypeptides and probably promote folding by decreasing the concentration of aggregation-prone intermediates. In contrast, the chaperonins interact with and globally enclose collapsed folding intermediates in a central cavity where efficient folding can proceed in a protected environment. For a number of proteins, folding requires the co-ordinated action of both of these molecular chaperones.     

Insights into chaperonin function from studies on archaeal thermosomes

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.     

Protein folding assisted by chaperones

Molecular chaperones are one of the most important cell defense mechanisms against protein aggregation and misfolding. These specialized proteins bind non-native states of other proteins and assist them in reaching a correctly folded and functional conformation. Chaperones also participate in protein translocation by membranes, in the stabilization of unstable protein conformers and regulatory factors, in the delivery of substrates for proteolysis and in the recovery of proteins from aggregates.     

Molecular chaperones

Chaperones are unique remodeling proteins that participate in a great number of intracellular processes and are involved in the correction of protein structure, the prevention of the aggregation of misfolded proteins, the destruction of protein aggregates, and also the unfolding of native protein targets for their translocation across a membrane. In addition to this, chaperones assist in the dismantling of active oligomers into inactive unfolded monomers for their subsequent proteolytic degradation and the assembly of folded subunits into protein assemblies and specific complexes. Data on the structure and functioning of molecular chaperones from five basic families are summarized in the review.     

ProOmpA contains secondary and tertiary structure prior to translocation and is shielded from aggregation by association with SecB protein.

Escherichia coli protein export involves cytosolic components termed molecular chaperones which function to stabilize precursors for membrane translocation. It has been suggested that chaperones maintain precursor proteins in a loosely folded state. We now demonstrate that purified proOmpA in its translocation component conformation contains both secondary and tertiary structure as analyzed by circular dichroism and intrinsic tryptophan fluorescence. Association with one molecular chaperone, SecB, subtly modulates the conformation of proOmpA and stabilizes it by inhibiting aggregation, permitting its translocation across inverted E.coli inner membrane vesicles. These results suggest that translocation competence does not simply result from the maintenance of an unfolded state and that molecular chaperones can stabilize precursor proteins by inhibiting their oligomerization.     

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.     

Protein folding in vitro and in the cellular environment

The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino-acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re-evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assisted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the "molten globule" state. However, very little is known about the recognition process.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

Protein folding and quality control in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least two main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasises more than ever that chaperones act in concert with co-factors and with each other.     

Some like it hot: the structure and function of small heat-shock proteins

Small heat-shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. Recent evidence suggests that they maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation. Some members of the sHsp family are inactive or only partially active under physiological conditions, and transition toward the active state is induced by specific triggers, such as elevated temperature. Release of substrate proteins bound to sHsps requires cooperation with ATP-dependent chaperones, suggesting that sHsps create a reservoir of non-native proteins for subsequent refolding.     

Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

Chaperones Rescue Luciferase Folding by Separating Its Domains

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.     

An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function.

p23 is a co-chaperone of the heat shock protein Hsp90. p23 binds to Hsp90 in its ATP-bound state and, on its own, interacts specifically with non-native proteins. In our attempt to correlate these functions to specific regions of p23 we have identified an unstructured region in p23 that maps to the C-terminal part of the protein sequence. This unstructured region is dispensible for interaction of p23 with Hsp90, since truncated p23 can still form complexes with Hsp90. In contrast, however, truncation of the C-terminal 30 amino acid residues of p23 affects the ability of p23 to bind non-native proteins and to prevent their non-specific aggregation. The isolated C-terminal region itself is not able to act as a chaperone nor is it possible to complement truncated p23 by addition of this peptide. These results imply that the binding site for Hsp90 is contained in the folded domain of p23 and that for efficient interaction of p23 with non-native proteins both the folded domain and the C-terminal unstructured region are required.