Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp).

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17-29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the ß7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

Could Small Heat Shock Protein HSP27 Be a First-Line Target for Preventing Protein Aggregation in Parkinson's Disease?

Small heat shock proteins (HSPs), such as HSP27, are ubiquitously expressed molecular chaperones and are essential for cellular homeostasis. The major functions of HSP27 include chaperoning misfolded or unfolded polypeptides and protecting cells from toxic stress. Dysregulation of stress proteins is associated with many human diseases including neurodegenerative diseases, such as Parkinson's disease (PD). PD is characterized by the presence of aggregates of α-synuclein in the central and peripheral nervous system, which induces the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and in the autonomic nervous system. Autonomic dysfunction is an important non-motor phenotype of PD, which includes cardiovascular dysregulation, among others. Nowadays, the therapies for PD focus on dopamine (DA) replacement. However, certain non-motor symptoms with a great impact on quality of life do not respond to dopaminergic drugs; therefore, the development and testing of new treatments for non-motor symptoms of PD remain a priority. Since small HSP27 was shown to prevent α-synuclein aggregation and cytotoxicity, this protein might constitute a suitable target to prevent or delay the motor and non-motor symptoms of PD. In the first part of our review, we focus on the cardiovascular dysregulation observed in PD patients. In the second part, we present data on the possible role of HSP27 in preventing the accumulation of amyloid fibrils and aggregated forms of α-synuclein. We also include our own studies, highlighting the possible protective cardiac effects induced by L-DOPA treatment through the enhancement of HSP27 levels and activity.

The N terminus of the small heat shock protein HSPB7 drives its polyQ aggregation-suppressing activity.

Heat shock protein family B (small) member 7 (HSPB7) is a unique, relatively unexplored member within the family of human small heat shock proteins (HSPBs). Unlike most HSPB family members, HSPB7 does not oligomerize and so far has not been shown to associate with any other member of the HSPB family. Intriguingly, it was found to be the most potent member within the HSPB family to prevent aggregation of proteins with expanded polyglutamine (polyQ) stretches. How HSPB7 suppresses polyQ aggregation has remained elusive so far. Here, using several experimental strategies, including in vitro aggregation assay, immunoblotting and fluorescence approaches, we show that the polyQ aggregation-inhibiting activity of HSPB7 is fully dependent on its flexible N-terminal domain (NTD). We observed that the NTD of HSPB7 is both required for association with and inhibition of polyQ aggregation. Remarkably, replacing the NTD of HSPB1, which itself cannot suppress polyQ aggregation, with the NTD of HSPB7 resulted in a hybrid protein that gained anti-polyQ aggregation activity. The hybrid NTDHSPB7-HSPB1 protein displayed a reduction in oligomer size and, unlike WT HSPB1, associated with polyQ. However, experiments with phospho-mimicking HSPB1 mutants revealed that de-oligomerization of HSPB1 alone does not suffice to gain polyQ aggregation-inhibiting activity. Together, our results reveal that the NTD of HSPB7 is both necessary and sufficient to bind to and suppress the aggregation of polyQ-containing proteins.

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

Previously, we reported that elevated serum levels of heat shock protein 27 (HSP27) are predictive of a lower risk of having a heart attack, stroke, or death from cardiovascular disease. Moreover, augmenting HSP27 (or the murine ortholog, HSP25) attenuated experimental atherogenesis, reduced inflammation, and lowered cholesterol levels. Recently, we noted that HSP27 activates NF-κB via TLR-4, resulting in attenuation of plaque inflammation; however, the precise anti-atherosclerosis mechanisms mediated by extracellular HSP27 are incompletely understood. Our purpose in this study was to investigate the existence of HSP27 in extracellular vesicles (EVs) and whether HSP27 elicited atheroprotective effects on target cells. Here, we provide evidence that HSP27 localizes to EVs derived from THP-1 cells using transmission electron microscopy (TEM) and immunogold labeling, Western blotting, ELISA, and fluorescence-activated cell sorting. TEM imaging indicated that HSP27 is found at the exosomal membrane. Multiple reactor monitor-mass spectrometric analysis of large vesicles, which included microparticles and exosomes, isolated from human plasma, also led to detection of HSP27 using the unique signature peptide, R.LFDQAFGLPR.L. Studies using THP-1 and human embryonic kidney cells show that HSP27-laden exosomes significantly stimulated NF-κB activation ( P < 0.001) and release of IL-10 ( P < 0.0001), suggesting that HSP27 may be important exosomal cargo with beneficial anti-inflammatory effects.-Shi, C., Ulke-Lemée, A., Deng, J., Batulan, Z., O'Brien, E. R. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

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.

Small Heat Shock Proteins and Human Neurodegenerative Diseases.

The review discusses the role of small heat shock proteins (sHsps) in human neurodegenerative disorders, such as Charcot-Marie-Tooth disease (CMT), Parkinson's and Alzheimer's diseases, and different forms of tauopathies. The effects of CMT-associated mutations in two small heat shock proteins (HspB1 and HspB8) on the protein stability, oligomeric structure, and chaperone-like activity are described. Mutations in HspB1 shift the equilibrium between different protein oligomeric forms, leading to the alterations in its chaperone-like activity and interaction with protein partners, which can induce damage of the cytoskeleton and neuronal death. Mutations in HspB8 affect its interaction with the adapter protein Bag3, as well as the process of autophagy, also resulting in neuronal death. The impact of sHsps on different forms of amyloidosis is discussed. Experimental studies have shown that sHsps interact with monomers or small oligomers of amyloidogenic proteins, stabilize their structure, prevent their aggregation, and/or promote their specific proteolytic degradation. This effect might be due to the interaction between the ß-strands of sHsps and ß-strands of target proteins, which prevents aggregation of the latter. In cooperation with the other heat shock proteins, sHsps can promote disassembly of oligomers formed by amyloidogenic proteins. Despite significant achievements, further investigations are required for understanding the role of sHsps in protection against various neurodegenerative diseases.

The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins.

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/αA-crystallin, and HspB5/αB-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ∼1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4-6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.

Specific sequences in the N-terminal domain of human small heat-shock protein HSPB6 dictate preferential hetero-oligomerization with the orthologue HSPB1.

Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site ∼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins.

Dimer-monomer equilibrium of human HSP27 is influenced by the in-cell macromolecular crowding environment and is controlled by fatty acids and heat.

Small heat shock protein 27 (HSP27) is an essential element of the proteostasis network in human cells. The HSP27 monomer coexists with the dimer, which can bind unfolded client proteins. Here, we evaluated the in-cell dimer-monomer equilibrium and its relevance to the binding of client proteins in a normal human vascular endothelial cell line. When cells were treated with a membrane-permeable crosslinker, the protein existed primarily as a free monomer (27 kDa) with a markedly smaller percentage of dimer (54 kDa), hetero-conjugates, and minor smear-like bands. When the protein was crosslinked in a cell-free lysate, two of the hetero-conjugates that were crosslinked in live cells were also detected, but the dimer and other complexes were absent. However, when cells were pretreated with fatty acid (FA) and/or heat (42.5 °C), dissociation of the dimer was selectively prevented and two types of covalently linked dimers were increased. These changes occurred most prominently in cells treated with docosahexaenoic acid (DHA) and heat, which appeared to intensify the heat resistance of the cell. Both the formation of covalently linked dimers and heat resistance were prevented by N-acetylcysteine. By contrast, nearly all of the free monomers in the lysate converted to disulfide bond-linked dimers by a simple, long incubation at 4 °C. These results strongly suggest that the monomer-dimer equilibrium of HSP27 was inversed between the in-cell and cell-free systems. Temperature- and amphiphile-regulated dimerization was restricted probably due to the low hydration of the in-cell crowding environment.

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

Small Heat-shock Proteins Prevent α-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation.

The aggregation of α-synuclein (α-syn) into amyloid fibrils is associated with neurodegenerative diseases, collectively referred to as the α-synucleinopathies. In vivo, molecular chaperones, such as the small heat-shock proteins (sHsps), normally act to prevent protein aggregation; however, it remains to be determined how aggregation-prone α-syn evades sHsp chaperone action leading to its disease-associated deposition. This work examines the molecular mechanism by which two canonical sHsps, αB-crystallin (αB-c) and Hsp27, interact with aggregation-prone α-syn to prevent its aggregation in vitro Both sHsps are very effective inhibitors of α-syn aggregation, but no stable complex between the sHsps and α-syn was detected, indicating that the sHsps inhibit α-syn aggregation via transient interactions. Moreover, the ability of these sHsps to prevent α-syn aggregation was dependent on the kinetics of aggregation; the faster the rate of aggregation (shorter the lag phase), the less effective the sHsps were at inhibiting fibril formation of α-syn. Thus, these findings indicate that the rate at which α-syn aggregates in cells may be a significant factor in how it evades sHsp chaperone action in the α-synucleinopathies.

Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry.

The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins.

The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch.

Heat shock protein 27 (HSP27), functioning as a stress induced protective protein, has been reported to participate in various biological processes, including apoptosis, thermal protection, and virus infection. In this study, a HSP27-like gene from the seawater fish sea perch, designated as LjHSP27, was characterized. The 1361 bp full-length cDNA of LjHSP27 encoded a 221 amino acid protein containing a conserved α-crystallin domain, two variable amino- and carboxy-terminal extensions, a WD/EPF motif, two serine phosphorylation sites, and two putative actin binding regions. Phylogenetic analysis showed that LjHSP27 shared the closest genetic relationship with HSP27 of the Asian seabass Lates calcarifer. LjHSP27 mRNA was ubiquitously expressed in all tissues examined, but significantly up-regulated in spleen and kidney and down-regulated in brain post red spotted grouper nervous necrosis virus (RGNNV) infection. In vitro, LjHSP27 transcript was remarkably reduced post RGNNV infection, but rapidly increased after polyinosinic-polycytidylic acid treatment. Up-regulation and down-regulation of LjHSP27 inhibited and promoted RGNNV replication in cultured LJB cells, respectively. Luciferase assay indicated that LjHSP27 could enhance the promoter activities of zebrafish interferon (IFN)1 and IFN3, suggesting its potential role in innate immune responses. Moreover, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, as indicated by the up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes, while KNK437 caused down-regulation of LjHSP27 dramatically led to opposite results, suggesting that LjHSP27 might exert its anti-RGNNV activities by regulating the apoptosis signaling pathway. Our results would provide a new insight into the underlying molecular mechanism of HSP and RGNNV interaction.

Methylglyoxal and Small Heat Shock Proteins.

Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.

The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity.

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-ß peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.

Molecular Chaperones in the Pathogenesis of Amyotrophic Lateral Sclerosis: The Role of HSPB1.

Genetic discoveries in amyotrophic lateral sclerosis (ALS) have a significant impact on deciphering molecular mechanisms of motor neuron degeneration but, despite recent advances, the etiology of most sporadic cases remains elusive. Several cellular mechanisms contribute to the motor neuron degeneration in ALS, including RNA metabolism, cellular interactions between neurons and nonneuronal cells, and seeding of misfolded protein with prion-like propagation. In this scenario, the importance of protein turnover and degradation in motor neuron homeostasis gained increased recognition. In this study, we evaluated the role of the candidate gene HSPB1, a molecular chaperone involved in several proteome-maintenance functions. In a cohort of 247 unrelated Italian ALS patients, we identified two variants (c.570G>C, p.Gln190His and c.610dupG, p.Ala204Glyfs* 6). Functional characterization of the p.Ala204Glyfs* 6 demonstrated that the mutant protein alters HSPB1 dynamic equilibrium, sequestering the wild-type protein in a stable dimer and resulting in a loss of chaperone-like activity. Our results underline the relevance of identifying rare but pathogenic variations in sporadic neurodegenerative diseases, suggesting a possible correlation between specific pathomechanisms linked to HSPB1 mutations and the associated neurological phenotype. Our study provides additional lines of evidence to support the involvement of HSPB1 in the pathogenesis of sporadic ALS.

Reinventing Hsp90 Inhibitors: Blocking C-Terminal Binding Events to Hsp90 by Using Dimerized Inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone (90 kDa) that functions as a dimer. This protein facilitates the folding, assembly, and stabilization of more than 400 proteins that are responsible for cancer development and progression. Inhibiting Hsp90's function will shut down multiple cancer-driven pathways simultaneously because oncogenic clients rely heavily on Hsp90, which makes this chaperone a promising anticancer target. Classical inhibitors that block the binding of adenine triphosphate (ATP) to the N-terminus of Hsp90 are highly toxic to cells and trigger a resistance mechanism within cells. This resistance mechanism comprises a large increase in prosurvival proteins, namely, heat shock protein 70 (Hsp70), heat shock protein 27 (Hsp27), and heat shock factor 1 (HSF-1). Molecules that modulate the C-terminus of Hsp90 are effective at inducing cancer-cell death without activating the resistance mechanism. Herein, we describe the design, synthesis, and biological binding affinity for a series of dimerized C-terminal Hsp90 modulators. We show that dimers of these C-terminal modulators synergistically inhibit Hsp90 relative to monomers.

The preferential heterodimerization of human small heat shock proteins HSPB1 and HSPB6 is dictated by the N-terminal domain.

Small heat shock proteins are ATP-independent molecular chaperones. Their function is to bind partially unfolded proteins under stress conditions. In vivo, members of this chaperone family are known to preferentially assemble together forming large, polydisperse heterooligomers. The exact molecular mechanisms that drive specific heteroassociation are currently unknown. Here we study the oligomers formed between human HSPB1 and HSPB6. Using small-angle X-ray scattering we could characterize two distinct heterooligomeric species present in solution. By employing native mass spectrometry we show that such assemblies are formed purely from heterodimeric building blocks, in line with earlier cross-linking studies. Crucially, a detailed analysis of truncation variants reveals that the preferential association between these two sHSPs is solely mediated by their disordered N-terminal domains.

Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities.

Previous studies have identified peptides in the 'crystallin-domain' of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors.

Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site-Specifically Modified with Argpyrimidine.

Non-enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification.