Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain

Green fluorescent protein (GFP) has been used to report protein folding by correlating solubility with fluorescence. In a GFP fusion protein, an upstream aggregation-prone domain can disrupt de novo folding of the GFP domain in Escherichia coli, resulting in a loss of fluorescence. Previously, we showed that prevention of misfolding of the upstream aggregation-prone domain by a coupled folding and binding interaction during protein synthesis restored both GFP fluorescence and solubility. Since molecular chaperones often fold nascent polypeptides through a bind-and-release interaction, the question remains whether the chaperone interaction with the upstream aggregation-prone domain enhances GFP fluorescence. Here, we demonstrate that a significant increase in GFP fluorescence occurred only when appropriate chaperones that recognized the aggregation-prone protein and helped its folding were co-expressed. A possible correlation between GFP fluorescence and the productive folding by chaperones is proposed. This study may provide a general strategy for identifying chaperones specific for difficult-to-fold proteins.     

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.     

Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein.     

The molecular chaperone concept

Molecular chaperones are a ubiquitous family of cellular proteins which mediate the correct folding of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures. Known chaperones do not possess steric information for protein folding but inhibit unproductive folding and assembly pathways which would otherwise act as dead-end kinetic traps and produce incorrect structures. Chaperones function by binding specifically and non-covalently to interactive protein surfaces that are exposed transiently during cellular processes such as protein synthesis, protein transport across membranes, DNA synthesis, the recycling of clathrin cages, the assembly of organellar complexes from imported subunits, and stress responses. This binding is reversed under circumstances which favour correct interactions and in some cases ATP hydrolysis is involved in this reversal. Some chaperones bind specifically to a structural feature present in a wide range of unrelated proteins that is accessible only during the early stages of folding. The nature of this structural feature is unknown, but its identification is an important goal of current research. Knowledge of chaperone function may be important for the production of proteins for biotechnological purposes since in some cases chaperones may improve the yield of functional product. It is likely that chaperone diseases exist which result from the failure of certain proteins to fold correctly due to changes in chaperone structure.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Monitoring protein conformation along the pathway of chaperonin-assisted folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.     

The human HSP70 family of chaperones: where do we stand?

The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as “chaperokines” or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5′-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.     

Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only “early” by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed.     

The unique chaperone operon of Thermotoga maritima: cloning and initial characterization of a functional Hsp70 and small heat shock protein.

The hyperthermophilic eubacterium Thermotoga maritima possesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of T. maritima, one of the deepest-branching eubacteria known.     

A second-site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo

Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second-site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. Here we report evidence that su substitutions that rescue bacteriophage P22 temperature-sensitive-folding (tsf) coat protein variants function in a novel way. The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production. The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents. Phage-infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones. Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents. We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone-driven kinetic partitioning between aggregation-prone intermediates and the final assembled state. Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect.     

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.     

Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

A protein quality control system, consisting of molecular chaperones and proteases, controls the folding status of proteins and prevents the aggregation of misfolded proteins by either refolding or degrading aggregation-prone species. During severe stress conditions this protection system can be overwhelmed by high substrate load, resulting in the formation of protein aggregates. In such emergency situations, Hsp104/ClpB becomes a key player for cell survival, as it has the extraordinary capacity to rescue proteins from an aggregated state in cooperation with an Hsp70 chaperone system. The ring-forming Hsp104/ClpB chaperone belongs to the AAA+ protein superfamily, which in general drives the assembly and disassembly of protein complexes by ATP-dependent remodelling of protein substrates. A disaggregation activity was also recently attributed to other eubacterial AAA+ proteins, while such an activity has not yet been identified in mammalian cells. In this review, we report on new insights into the mechanism of protein disaggregation by AAA+ proteins, suggesting that these chaperones act as molecular crowbars or ratchets.     

Charge-Rich Regions Modulate the Anti-Aggregation Activity of Hsp90

Protein aggregation can have dramatic effects on cellular function and plays a causative role in many human diseases. In all cells, molecular chaperones bind to aggregation-prone proteins and hinder aggregation. The ability of a protein to resist aggregation and remain soluble in aqueous solution is linked to the physical properties of the protein. Numerous physical studies demonstrate that charged atoms favor solubility. We note that many molecular chaperones possess a substantial negative charge that may allow them to impart solubility on aggregation-prone proteins. Hsp90 is one such negatively charged molecular chaperone. The charge on Hsp90 is largely concentrated in two highly acidic regions. To investigate the relationship between chaperone charge and protein solubility, we deleted these charge-rich regions and analyzed the resulting Hsp90 constructs for anti-aggregation activity. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. The anti-aggregation role of the deleted charge-rich regions could be due to net charge or sequence-specific features. To distinguish these possibilities, we attached an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct. This charge rescue construct displayed effective anti-aggregation activity indicating that the net charge of Hsp90 contributes to its anti-aggregation activity.     

Assessment of substrate-stabilizing factors for DnaK on the folding of aggregation-prone proteins

Hydrophobic interactions between molecular chaperones and their nonnative substrates have been believed to be mainly responsible for both substrate recognition and stabilization against aggregation. However, the hydrophobic contact area between DnaK and its substrate proteins is very limited and other factors of DnaK for the substrate stabilization could not be excluded. Here, we covalently fused DnaK to the N-termini of aggregation-prone proteins in vivo. In the context of a fusion protein, DnaK has the ability to efficiently solubilize its linked proteins. The point mutation of the residue of DnaK critical for the substrate recognition and the deletion of the C-terminal substrate-binding domain did not have significant effect on the solubilizing ability of DnaK. The results imply that other factors of DnaK, distinct from the hydrophobic shielding of folding intermediates, also contributes to stabilization of its noncovalently bound substrates against aggregation. Elucidation of the nature of these factors would further enhance our understanding of the substrate stabilization of DnaK for expedited protein folding.     

The chaperonin folding machine

Chaperonins are versatile molecular machines that assist the folding of a wide range of substrate proteins. They harness an ATPase cycle to control access of non-native proteins to hydrophobic binding sites. ATP binding promotes large conformational changes that partially bury the hydrophobic sites and initiate the binding of a co-chaperonin, creating closed and open cavities. Non-native proteins progress towards the native fold during their confinement in these cavities, and are then released by the allosteric action of ATP.     

Inducible HSP70 Is Critical in Preventing the Aggregation and Enhancing the Processing of PMP22

Chaperones, also called heat shock proteins (HSPs), transiently interact with proteins to aid their folding, trafficking, and degradation, thereby directly influencing the transport of newly synthesized molecules. Induction of chaperones provides a potential therapeutic approach for protein misfolding disorders, such as peripheral myelin protein 22 (PMP22)-associated peripheral neuropathies. Cytosolic aggregates of PMP22, linked with a demyelinating Schwann cell phenotype, result in suppression of proteasome activity and activation of proteostatic mechanisms, including the heat shock pathway. Although the beneficial effects of chaperones in preventing the aggregation and improving the trafficking of PMP22 have been repeatedly observed, the requirement for HSP70 in events remains elusive. In this study, we show that activation of the chaperone pathway in fibroblasts from PMP22 duplication-associated Charcot–Marie–Tooth disease type 1A patient with an FDA-approved small molecule increases HSP70 expression and attenuates proteasome dysfunction. Using cells from an HSP70.1/3^−/− (inducible HSP70) mouse model, we demonstrate that under proteotoxic stress, this chaperone is critical in preventing the aggregation of PMP22, and this effect is aided by macroautophagy. When examined at steady-state, HSP70 appears to play a minor role in the trafficking of wild-type-PMP22, while it is crucial for preventing the buildup of the aggregation-prone Trembler-J-PMP22. HSP70 aids the processing of Trembler-J-PMP22 through the Golgi and its delivery to lysosomes via Rab7-positive vesicles. Together, these results demonstrate a key role for inducible HSP70 in aiding the processing and hindering the accumulation of misfolded PMP22, which in turn alleviates proteotoxicity within the cells.     

A Virally Encoded Chaperone Specialized for Folding of the Major Capsid Protein of African Swine Fever Virus

It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of African swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.     

Dynamics of trigger factor interaction with translating ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.