Cellular Handling of Protein Aggregates by Disaggregation Machines.

Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.

Escherichia coli small heat shock protein IbpA plays a role in regulating the heat shock response by controlling the translation of σ32.

Small heat shock proteins (sHsps) act as ATP-independent chaperones that prevent irreversible aggregate formation by sequestering denatured proteins. IbpA, an Escherichia coli sHsp, functions not only as a chaperone but also as a suppressor of its own expression through posttranscriptional regulation, contributing to negative feedback regulation. IbpA also regulates the expression of its paralog, IbpB, in a similar manner, but the extent to which IbpA regulates other protein expressions is unclear. We have identified that IbpA down-regulates the expression of many Hsps by repressing the translation of the heat shock transcription factor σ32. The IbpA regulation not only controls the σ32 level but also contributes to the shutoff of the heat shock response. These results revealed an unexplored role of IbpA to regulate heat shock response at a translational level, which adds an alternative layer for tightly controlled and rapid expression of σ32 on demand.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.

Regulation of small heat-shock proteins by hetero-oligomer formation.

Small heat-shock proteins (sHsps) compose the most widespread family of molecular chaperones. The human genome encodes 10 different sHsps (HspB1-10). It has been shown that HspB1 (Hsp27), HspB5 (αB-crystallin), and HspB6 (Hsp20) can form hetero-oligomers in vivo However, the impact of hetero-oligomerization on their structure and chaperone mechanism remains enigmatic. Here, we analyzed hetero-oligomer formation in human cells and in vitro using purified proteins. Our results show that the effect of hetero-oligomer formation on the composition of the sHsp ensembles and their chaperone activities depends strongly on the respective sHsps involved. We observed that hetero-oligomer formation between HspB1 and HspB5 leads to an ensemble that is dominated by species larger than the individual homo-oligomers. In contrast, the interaction of dimeric HspB6 with either HspB1 or HspB5 oligomers shifted the ensemble toward smaller oligomers. We noted that the larger HspB1-HspB5 hetero-oligomers are less active and that HspB6 activates HspB5 by dissociation to smaller oligomer complexes. The chaperone activity of HspB1-HspB6 hetero-oligomers, however, was modulated in a substrate-specific manner, presumably due to the specific enrichment of an HspB1-HspB6 heterodimer. These heterodimeric species may allow the tuning of the chaperone properties toward specific substrates. We conclude that sHsp hetero-oligomerization exerts distinct regulatory effects depending on the sHsps involved.

The noncanonical small heat shock protein HSP-17 from Caenorhabditis elegans is a selective protein aggregase.

Small heat shock proteins (sHsps) are conserved, ubiquitous members of the proteostasis network. Canonically, they act as "holdases" and buffer unfolded or misfolded proteins against aggregation in an ATP-independent manner. Whereas bacteria and yeast each have only two sHsps in their genomes, this number is higher in metazoan genomes, suggesting a spatiotemporal and functional specialization in higher eukaryotes. Here, using recombinantly expressed and purified proteins, static light-scattering analysis, and disaggregation assays, we report that the noncanonical sHsp HSP-17 of Caenorhabditis elegans facilitates aggregation of model substrates, such as malate dehydrogenase (MDH), and inhibits disaggregation of luciferase in vitro Experiments with fluorescently tagged HSP-17 under the control of its endogenous promoter revealed that HSP-17 is expressed in the digestive and excretory organs, where its overexpression promotes the aggregation of polyQ proteins and of the endogenous kinase KIN-19. Systemic depletion of hsp-17 shortens C. elegans lifespan and severely reduces fecundity and survival upon prolonged heat stress. HSP-17 is an abundant protein exhibiting opposing chaperone activities on different substrates, indicating that it is a selective protein aggregase with physiological roles in development, digestion, and osmoregulation.

N- and C-terminal regions of αB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation.

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril-fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.

The calcium-dependent protein kinase ZmCDPK7 functions in heat-stress tolerance in maize.

Global warming poses a serious threat to crops. Calcium-dependent protein kinases (CDPKs)/CPKs play vital roles in plant stress responses, but their exact roles in plant thermotolerance remains elusive. Here, we explored the roles of heat-induced ZmCDPK7 in thermotolerance in maize. ZmCDPK7-overexpressing maize plants displayed higher thermotolerance, photosynthetic rates, and antioxidant enzyme activity but lower H2 O2 and malondialdehyde (MDA) contents than wild-type plants under heat stress. ZmCDPK7-knockdown plants displayed the opposite patterns. ZmCDPK7 is attached to the plasma membrane but can translocate to the cytosol under heat stress. ZmCDPK7 interacts with the small heat shock protein sHSP17.4, phosphorylates sHSP17.4 at Ser-44 and the respiratory burst oxidase homolog RBOHB at Ser-99, and upregulates their expression. Site-directed mutagenesis of sHSP17.4 to generate a Ser-44-Ala substitution reduced ZmCDPK7's enhancement of catalase activity but enhanced ZmCDPK7's suppression of MDA accumulation in heat-stressed maize protoplasts. sHSP17.4, ZmCDPK7, and RBOHB were less strongly upregulated in response to heat stress in the abscisic acid-deficient mutant vp5 versus the wild type. Pretreatment with an RBOH inhibitor suppressed sHSP17.4 and ZmCDPK7 expression. Therefore, abscisic acid-induced ZmCDPK7 functions both upstream and downstream of RBOH and participates in thermotolerance in maize by mediating the phosphorylation of sHSP17.4, which might be essential for its chaperone function.

Acid-denatured small heat shock protein HdeA from Escherichia coli forms reversible fibrils with an atypical secondary structure.

The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a "reversible" form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its "molecular chaperone" activity) and the formation of inactive fibrillary deposits (its "fibrillogenic" activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail.

HSPB5 engages multiple states of a destabilized client to enhance chaperone activity in a stress-dependent manner.

Small heat shock proteins (sHSPs) delay protein aggregation in an ATP-independent manner by interacting with client proteins that are in states susceptible to aggregation, including destabilized states related to cellular stress. Up-regulation of sHSPs under stress conditions supports their critical role in cellular viability. Widespread distribution of sHSPs in most organisms implies conservation of function, but it remains unclear whether sHSPs implement common or distinct mechanisms to delay protein aggregation. Comparisons among various studies are confounded by the use of different model client proteins, different assays for both aggregation and sHSP/client interactions, and variable experimental conditions used to mimic cellular stress. To further define sHSP/client interactions and their relevance to sHSP chaperone function, we implemented multiple strategies to characterize sHSP interactions with α-lactalbumin, a model client whose aggregation pathway is well defined. We compared the chaperone activity of human αB-crystallin (HSPB5) with HSPB5 variants that mimic states that arise under conditions of cellular stress or disease. The results show that these closely related sHSPs vary not only in their activity under identical conditions but also in their interactions with clients. Importantly, under nonstress conditions, WT HSPB5 delays client aggregation solely through transient interactions early in the aggregation pathway, whereas HSPB5 mutants that mimic stress-activated conditions can also intervene at later stages of the aggregation pathway to further delay client protein aggregation.

Plant small heat shock proteins - evolutionary and functional diversity.

Small heat shock proteins (sHSPs) are an ubiquitous protein family found in archaea, bacteria and eukaryotes. In plants, as in other organisms, sHSPs are upregulated by stress and are proposed to act as molecular chaperones to protect other proteins from stress-induced damage. sHSPs share an 'α-crystallin domain' with a ß-sandwich structure and a diverse N-terminal domain. Although sHSPs are 12-25 kDa polypeptides, most assemble into oligomers with ≥ 12 subunits. Plant sHSPs are particularly diverse and numerous; some species have as many as 40 sHSPs. In angiosperms this diversity comprises ≥ 11 sHSP classes encoding proteins targeted to the cytosol, nucleus, endoplasmic reticulum, chloroplasts, mitochondria and peroxisomes. The sHSPs underwent a lineage-specific gene expansion, diversifying early in land plant evolution, potentially in response to stress in the terrestrial environment, and expanded again in seed plants and again in angiosperms. Understanding the structure and evolution of plant sHSPs has progressed, and a model for their chaperone activity has been proposed. However, how the chaperone model applies to diverse sHSPs and what processes sHSPs protect are far from understood. As more plant genomes and transcriptomes become available, it will be possible to explore theories of the evolutionary pressures driving sHSP diversification.

Proteinaceous Transformers: Structural and Functional Variability of Human sHsps.

The proteostasis network allows organisms to support and regulate the life cycle of proteins. Especially regarding stress, molecular chaperones represent the main players within this network. Small heat shock proteins (sHsps) are a diverse family of ATP-independent molecular chaperones acting as the first line of defense in many stress situations. Thereby, the promiscuous interaction of sHsps with substrate proteins results in complexes from which the substrates can be refolded by ATP-dependent chaperones. Particularly in vertebrates, sHsps are linked to a broad variety of diseases and are needed to maintain the refractive index of the eye lens. A striking key characteristic of sHsps is their existence in ensembles of oligomers with varying numbers of subunits. The respective dynamics of these molecules allow the exchange of subunits and the formation of hetero-oligomers. Additionally, these dynamics are closely linked to the chaperone activity of sHsps. In current models a shift in the equilibrium of the sHsp ensemble allows regulation of the chaperone activity, whereby smaller oligomers are commonly the more active species. Different triggers reversibly change the oligomer equilibrium and regulate the activity of sHsps. However, a finite availability of high-resolution structures of sHsps still limits a detailed mechanistic understanding of their dynamics and the correlating recognition of substrate proteins. Here we summarize recent advances in understanding the structural and functional relationships of human sHsps with a focus on the eye-lens αA- and αB-crystallins.

Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart.

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity in vitro, the in vivo functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αBa and αBb We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in αBa than αBb mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.

HspB1 and Hsc70 chaperones engage distinct tau species and have different inhibitory effects on amyloid formation.

The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.

Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130.

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated ß-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity.

Proteostasis, or protein homeostasis, encompasses the maintenance of the conformational and functional integrity of the proteome and involves an integrated network of cellular pathways. Molecular chaperones, such as the small heat shock proteins (sHsps), are key elements of the proteostasis network that have crucial roles in inhibiting the aggregation of misfolded proteins. Failure of the proteostasis network can lead to the accumulation of misfolded proteins into intracellular and extracellular deposits. Deposits containing fibrillar forms of α-synuclein (α-syn) are characteristic of neurodegenerative disorders including Parkinson's disease and dementia with Lewy bodies. Here we show that the sHsp Hsp27 (HSPB1) binds to α-syn fibrils, inhibiting fibril growth by preventing elongation. Using total internal reflection fluorescence (TIRF)-based imaging methods, we show that Hsp27 binds along the surface of α-syn fibrils, decreasing their hydrophobicity. Binding of Hsp27 also inhibits cytotoxicity of α-syn fibrils. Our results demonstrate that the ability of sHsps, such as Hsp27, to bind fibrils represents an important mechanism through which they may mitigate cellular toxicity associated with aberrant protein aggregation. Fibril binding may represent a generic mechanism by which chaperone-active sHsps interact with aggregation-prone proteins, highlighting the potential to target sHsp activity to prevent or disrupt the onset and progression of α-syn aggregation associated with α-synucleinopathies.

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp-substrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles.

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.