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.     

Energy landscape remodeling mechanism of Hsp70-chaperone-accelerated protein folding

Hsp70 chaperone is one of the key protein machines responsible for the quality control of protein production in cells. Facilitating in vivo protein folding by counteracting misfolding and aggregation is the essence of its biological function. Although the allosteric cycle during its functional actions has been well characterized both experimentally and computationally, the mechanism by which Hsp70 assists protein folding is still not fully understood. In this work, we studied the Hsp70-mediated folding of model proteins with rugged energy landscape by using molecular simulations. Different from the canonical scenario of Hsp70 functioning, which assumes that folding of substrate proteins occurs spontaneously after releasing from chaperones, our results showed that the substrate protein remains in contacts with the chaperone during its folding process. The direct chaperone-substrate interactions in the open conformation of Hsp70 tend to shield the substrate sites prone to form non-native contacts, which therefore avoids the frustrated folding pathway, leading to a higher folding rate and less probability of misfolding. Our results suggest that in addition to the unfoldase and holdase functions widely addressed in previous studies, Hsp70 can facilitate the folding of its substrate proteins by remodeling the folding energy landscape and directing the folding processes, demonstrating the foldase scenario. These findings add new, to our knowledge, insights into the general molecular mechanisms of chaperone-mediated protein folding.     

Single-Molecule Force Spectroscopy of Protein Folding

The use of force probes to induce unfolding and refolding of single molecules through the application of mechanical tension, known as single-molecule force spectroscopy (SMFS), has proven to be a powerful tool for studying the dynamics of protein folding. Here we provide an overview of what has been learned about protein folding using SMFS, from small, single-domain proteins to large, multi-domain proteins. We highlight the ability of SMFS to measure the energy landscapes underlying folding, to map complex pathways for native and non-native folding, to probe the mechanisms of chaperones that assist with native folding, to elucidate the effects of the ribosome on co-translational folding, and to monitor the folding of membrane proteins.     

A circumventing role for the non-native intermediate in the folding of β-lactoglobulin

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ?o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.     

Protein <Emphasis Type="Italic">N</Emphasis>-glycosylation,protein folding,and protein quality control

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.     

Protein quality control in the endoplasmic reticulum

Protein folding and quality control in the endoplasmic reticulum (ER) are synchronized mechanisms ensuring that only properly folded proteins are integrated in the plasma membrane or secreted from the cell. These mechanisms act in close collaboration with the molecular machinery involved in retrograde-translocation and degradation of non-native proteins and with the ER-stress activated signalling systems. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. Protein misfolding can be detrimental to the cell and contributes to the disease mechanism in several inherited disorders, e.g. cystic fibrosis, familial hypercholesterolemia and diabetes insipidus. This review outlines the molecular mechanisms in protein quality control occurring in the ER, signalling caused by ER stress, and finally ER associated protein degradation.     

Improved Gō-like models demonstrate the robustness of protein folding mechanisms towards non-native interactions

The use of simple theoretical models has provided a considerable contribution to our present understanding of the means by which proteins adopt their native fold from the plethora of available unfolded states. A common assumption in building computationally tractable models has been the neglect of stabilizing non-native interactions in the class of models described as "Gō-like." The focus of this study is the characterization of the folding of a number of proteins via a Gō-like model, which aims to map a maximal amount of information reflecting the protein sequence onto a "minimalist" skeleton. This model is shown to contain sufficient information to reproduce the folding transition states of a number of proteins, including topologically analogous proteins that fold via different transition states. Remarkably, these models also demonstrate consistency with the general features of folding transition states thought to be stabilized by non-native interactions. This suggests that native interactions are the primary determinant of most protein folding transition states, and that non-native interactions lead only to local structural perturbations. A prediction is also included for an asymmetrical folding transition state of bacteriophage lambda protein W, which has yet to be subjected to experimental characterization.     

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.     

Diffusion-collision model study of misfolding in a four-helix bundle protein.

Proteins with complex folding kinetics will be susceptible to misfolding at some stage in the folding process. We simulate this problem by using the diffusion-collision model to study non-native kinetic intermediate misfolding in a four-helix bundle protein. We find a limit on the size of the pairwise hydrophobic area loss in non-native intermediates, such that burying above this limit creates long-lasting non-native kinetic intermediates that would disrupt folding and prevent formation of the native state. Our study of misfolding suggests a method for limiting the production of misfolded kinetic intermediates for helical proteins and could, perhaps, lead to more efficient production of proteins in bulk.     

A residue in helical conformation in the native state adopts a β-strand conformation in the folding transition state despite its high and canonical Φ-value

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state.     

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.     

Energetic Frustrations in Protein Folding at Residue Resolution: A Homologous Simulation Study of Im9 Proteins

Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.     

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 stability in the presence of osmolytes

Osmolytes are molecules whose function, among others, is to balance the hydrostatic pressure between the intracellular and extracellular compartments. Accumulation of osmolytes in a cell occurs in response to stress caused by changes in pressure, temperature, pH, or the concentration of inorganic salts. Osmolytes can prevent the denaturation of native proteins and promote the renaturation of unfolded proteins. Investigation of the roles of osmolyte in these processes is essential for our understanding of the mechanisms of protein folding and function in vivo. The large number of published reports that have been devoted to the effects of osmolytes on proteins are not always consistent with each other. In this review, an attempt is made to systemize the array of data on this subject and to consider the problem of protein folding and stability in osmolyte solutions from a single viewpoint.     

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.     

Effects of glycosylation on the structure and function of the extracellular chaperone clusterin

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.     

The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.     

Complex folding pathways in a simple beta-hairpin

The determination of the folding mechanisms of proteins is critical to understand the topological change that can propagate Alzheimer and Creutzfeld-Jakobs diseases, among others. The computational community has paid considerable attention to this problem; however, the associated time scale, typically on the order of milliseconds or more, represents a formidable challenge. Ab initio protein folding from long molecular dynamics simulations or ensemble dynamics is not feasible with ordinary computing facilities and new techniques must be introduced. Here we present a detailed study of the folding of a 16-residue beta-hairpin, described by a generic energy model and using the activation-relaxation technique. From a total of 90 trajectories at 300 K, three folding pathways emerge. All involve a simultaneous optimization of the complete hydrophobic and hydrogen bonding interactions. The first two pathways follow closely those observed by previous theoretical studies (folding starting at the turn or by interactions between the termini). The third pathway, never observed by previous all-atom folding, unfolding, and equilibrium simulations, can be described as a reptation move of one strand of the beta-sheet with respect to the other. This reptation move indicates that non-native interactions can play a dominant role in the folding of secondary structures. Furthermore, such a mechanism mediated by non-native hydrogen bonds is not available for study by unfolding and Gō model simulations. The exact folding path followed by a given beta-hairpin is likely to be influenced by its sequence and the solvent conditions. Taken together, these results point to a more complex folding picture than expected for a simple beta-hairpin.