Tubocapsenolide A targets C-terminal cysteine residues of HSP90 to exert the anti-tumor effect.

Heat shock protein 90 (HSP90) is a chaperone protein that has been shown to regulate cancer progression. As a result, HSP90 has emerged as an attractive target for cancer therapy. Tubocapsenolide A (TA) is an anti-tumor component isolated from Tubocapsicum anomalum. Although the anti-tumor activity of TA was considered to be related to HSP90, the binding site and deep anti-tumor mechanisms still need to be elucidated. In this study, we found that TA is a covalent inhibitor of HSP90, which inhibits HSP90 ATPase activity without blocking ATP binding. Further studies indicated that TA targets the C-terminal Cys521 site, which led to HSP90 partial oligomerization and hindered its anti-aggregation and refolding activity. The damage of the chaperone activity disrupted the interaction between HSP90 and its cochaperone CDC37 as well as its client proteins, thereby inducing cell cycle arrest and apoptosis. Moreover, TA was found to have therapeutic effects on the xenograft tumor model by inducing the degradation of HSP90 client proteins. Together, our results identified HSP90 as the direct target of TA for mediating the anti-tumor activity. TA could serve as a lead compound for developing novel HSP90 C-terminal covalent inhibitors with binding site different from the ATP-binding domain.

The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells.

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including ß-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR-/- mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR-/- mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.

Fatty acids influence the efficacy of lutein in the modulation of α-crystallin chaperone function: Evidence from selenite induced cataract rat model.

BACKGROUND: Loss of α-crystallin chaperone function results in the lens protein aggregation leading to cataract. In this study, we evaluated the efficacy of micellar lutein with different fatty acids in modulating α-crystallin chaperone function under selenite cataract conditions. METHODS: Cataract was induced in rat pups by giving sodium selenite (25 µM/kg body weight) by IP. Lutein [(L), 1.3 µmol/kg body weight)] was given day before and five days after selenite injection as a micelle with 7.5 mM linoleic acid (LA), or 7.5 mM eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) or 7.5 mM oleic acid (OA). Lens α-crystallins was purified, and its chaperone function and integrity was assessed. Cholesterol, calcium, calpain-2, procaspase-3, and expression of α-A and ß-B1 crystallin in the lens of cataract and micellar lutein administered rats were evaluated. RESULTS: Cataract induction significantly (p < 0.05) decreased lens α-crystallin chaperone function. Cataract rats had increased cholesterol and calcium level, increased the expression of calpain-2, and α-A and ß-B1 crystallin, and reduced the pro-caspase-3 level in the lens. However, micellar lutein administration significantly (p < 0.05) protected client proteins from aggregation via the modulation of calcium-dependent calpain-2 protease activity. The chaperone function of lens α-crystallins in rats administered micellar lutein with EPA + DHA was found to be highest when compared to OA and LA. CONCLUSIONS: Micellar lutein with unsaturated fatty acids beneficially modulates α-crystallin chaperone function. Among the fatty acids tested, micellar lutein with EPA + DHA exhibited superior effects, thereby offering a promising strategy for cataract management.

Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A.

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A's cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A's PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A's lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A's secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A's ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A's refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A's PM-translocation in tumor cells, making them sensitive to radiation therapy.

DNA-binding and repressor function are prerequisites for the turnover of the tomato heat stress transcription factor HsfB1.

HsfB1 is a central regulator of heat stress (HS) response and functions dually as a transcriptional co-activator of HsfA1a and a general repressor in tomato. HsfB1 is efficiently synthesized during the onset of HS and rapidly removed in the course of attenuation during the recovery phase. Initial results point to a complex regime modulating HsfB1 abundance involving the molecular chaperone Hsp90. However, the molecular determinants affecting HsfB1 stability needed to be established. We provide experimental evidence that DNA-bound HsfB1 is efficiently targeted for degradation when active as a transcriptional repressor. Manipulation of the DNA-binding affinity by mutating the HsfB1 DNA-binding domain directly influences the stability of the transcription factor. During HS, HsfB1 is stabilized, probably due to co-activator complex formation with HsfA1a. The process of HsfB1 degradation involves nuclear localized Hsp90. The molecular determinants of HsfB1 turnover identified in here are so far seemingly unique. A mutational switch of the R/KLFGV repressor motif's arginine and lysine implies that the abundance of other R/KLFGV type Hsfs, if not other transcription factors as well, might be modulated by a comparable mechanism. Thus, we propose a versatile mechanism for strict abundance control of the stress-induced transcription factor HsfB1 for the recovery phase, and this mechanism constitutes a form of transcription factor removal from promoters by degradation inside the nucleus.

Functional characterization of natural variants found on the major stress inducible 70-kDa heat shock gene, HSPA1A, in humans.

In this report, we investigated the effects of natural single nucleotide polymorphisms on the function of HSPA1A, the major stress-inducible Hsp70 gene in humans. We first established that all mutant proteins retain their ability to hydrolyze ATP, but three of them had a significantly lower rate of ATP hydrolysis as compared to the wild-type (WT) protein. We also used Isothermal Titration Calorimetry and found that although all mutants bind to protein substrate with dissociation constants similar to the WT protein, four of them had increased reaction entropies. We also tested whether these mutations affect the ability of HSPA1A to refold heat-denatured luciferase. These assays revealed that one mutation resulted in significantly lower levels while a second one resulted in higher levels of the refolded enzyme. We then determined whether the mutations affected the ability of HSPA1A to prevent apoptosis caused by poly-glutamine carrying huntingtin proteins. This assay determined that three of the mutations caused increased cell apoptosis as compared to the WT. Our results reveal that although none of these naturally occurring mutations exists on positions of known function, some alter the molecular chaperone activities of HSPA1A most probably by affecting the allosteric communication between its two major domains.

Interaction of α-crystallin with some small molecules and its effect on its structure and function.

BACKGROUND: α-Crystallin acts like a molecular chaperone by interacting with its substrate proteins and thus prevents their aggregation. It also interacts with various kinds of small molecules that affect its structure and function. SCOPE OF REVIEW: In this article we will present a review of work done with respect to the interaction of ATP, peptide generated from lens crystallin and other proteins and some bivalent metal ions with α-crystallin and discuss the role of these interactions on its structure and function and cataract formation. We will also discuss the interaction of some hydrophobic fluorescence probes and surface active agents with α-crystallin. MAJOR CONCLUSIONS: Small molecule interaction controls the structure and function of α-crystallin. ATP and Zn+2 stabilize its structure and enhance chaperone function. Therefore the depletion of these small molecules can be detrimental to maintenance of lens transparency. However, the accumulation of small peptides due to protease activity in the lens can also be harmful as the interaction of these peptides with α-crystallin and other crystallin proteins in the lens promotes aggregation and loss of lens transparency. The use of hydrophobic probe has led to a wealth of information regarding the location of substrate binding site and nature of chaperone-substrate interaction. Interaction of surface active agents with α-crystallin has helped us to understand the structural stability and oligomeric dissociation in α-crystallin. GENERAL SIGNIFICANCE: These interactions are very helpful in understanding the mechanistic details of the structural changes and chaperone function of α-crystallin. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

S-nitrosylation switches the Arabidopsis redox sensor protein, QSOX1, from an oxidoreductase to a molecular chaperone under heat stress.

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin.

α-crystallin is a structurally essential small heat shock protein (sHSP) with a chaperone-like activity which maintains transparency of the lenticular tissues during a period of time that is as long as human life. α-crystallin is a multimeric protein consisting of αA and αB subunits, with 57 % homology. The CRYAB gene on chromosome 11 encodes human αB-crystallin (αB-Cry), which contains 175 amino acid residues. In the current study, the cataractogenic mutations R12C, P20R, R69C, and double mutations R12C/P20R and R12C/P20R were embedded into the human CRYAB gene. Following successful expression in the prokaryotic system and purification, a number of spectroscopic techniques, gel electrophoresis, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to assess the role of these mutations on the structure, amyloidogenicity, and biological function of human αB-Cry. The created mutations caused significant changes in the structure, and oligomeric state of human αB-Cry. These mutations, particularly R12C, R12C/P20R, and R12C/R69C, dramatically enhanced the tendency of this protein for the amyloid fibril formation and reduced its chaperone-like activity. Since double mutations R12C/P20R and R12C/P20R were able to intensely change the protein's structure and chaperone function, it can be suggested that they may play a destructive role in a cumulative manner. Our findings indicated that the simultaneous presence of two pathogenic mutations may have a cumulative destructive impacts on the structure and function of human αB-Cry and this observation is likely related to the disease severity of the mutated proteins.

The Molecular Chaperone Mechanism of the C-Terminal Domain of Large-Size Subunit Catalases.

Large-size subunit catalases (LSCs) have an additional C-terminal domain (CT) that is structurally similar to Hsp31 and DJ-1 proteins, which have molecular chaperone activity. The CT of LSCs derives from a bacterial Hsp31 protein. There are two CT dimers with inverted symmetry in LSCs, one dimer in each pole of the homotetrameric structure. We previously demonstrated the molecular chaperone activity of the CT of LSCs. Like other chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in bacteria and fungi. Here, we analyze the mechanism of the CT of LSCs as an unfolding enzyme. The dimeric form of catalase-3 (CAT-3) CT (TDC3) of Neurospora crassa presented the highest activity as compared to its monomeric form. A variant of the CAT-3 CT lacking the last 17 amino acid residues (TDC3Δ17aa), a loop containing hydrophobic and charged amino acid residues only, lost most of its unfolding activity. Substituting charged for hydrophobic residues or vice versa in this C-terminal loop diminished the molecular chaperone activity in all the mutant variants analyzed, indicating that these amino acid residues play a relevant role in its unfolding activity. These data suggest that the general unfolding mechanism of CAT-3 CT involves a dimer with an inverted symmetry, and hydrophobic and charged amino acid residues. Each tetramer has four sites of interaction with partially unfolded or misfolded proteins. LSCs preserve their catalase activity under different stress conditions and, at the same time, function as unfolding enzymes.

Structural Basis of Parasitic HSP90 ATPase Inhibition by Small Molecules.

Protozoan parasites are responsible for several harmful and widespread human diseases that cause high morbidity and mortality. Currently available treatments have serious limitations due to poor efficiency, strong adverse effects, and high cost. Hence, the identification of new targets and the development of specific drug therapies against parasitic diseases are urgent needs. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that plays a key role in parasite survival during the various differentiation stages, spread over the vector insect and the human host, which they undergo during their life cycle. The N-terminal domain (NTD) of HSP90, containing the main determinants for ATPase activity, represents the most druggable domain for inhibitor targeting. The molecules investigated on parasite HSP90 are mainly developed from known inhibitors of the human counterpart, and they have strong limitations due to selectivity issues, accounting for the high conservation of the ATP-binding site between the parasite and human proteins. The current review highlights the recent structural progress made to support the rational design of new molecules able to effectively block the chaperone activity of parasite HSP90.

Structural determinants of multimerization and dissociation in 2-Cys peroxiredoxin chaperone function.

Peroxiredoxins (PRDXs) are abundant peroxidases present in all kingdoms of life. Recently, they have been shown to also carry out additional roles as molecular chaperones. To address this emerging supplementary function, this review focuses on structural studies of 2-Cys PRDX systems exhibiting chaperone activity. We provide a detailed understanding of the current knowledge of structural determinants underlying the chaperone function of PRDXs. Specifically, we describe the mechanisms which may modulate their quaternary structure to facilitate interactions with client proteins and how they are coordinated with the functions of other molecular chaperones. Following an overview of PRDX molecular architecture, we outline structural details of the presently best-characterized peroxiredoxins exhibiting chaperone function and highlight common denominators. Finally, we discuss the remarkable structural similarities between 2-Cys PRDXs, small HSPs, and J-domain-independent Hsp40 holdases in terms of their functions and dynamic equilibria between low- and high-molecular-weight oligomers.

Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone.

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: The biochemical pathomechanism underlying cataract and myopathy development.

In human αB-crystallin (αB-Cry), the highly conserved residues arginine 69 (R69) and aspartate 109 (D109) are located within a critical motif of α-crystallin domain (ACD), contributing to the subunit interactions and oligomeric assembly. Recently, two missense mutations (R69C and D109H) in human αB-Cry have been reported to cause congenital cataract and myopathy disorders. We used various spectroscopic techniques, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), gel electrophoresis and transmission electron microscopy (TEM) to show how these mutations cause significant changes in structure, amyloidogenic feature and biological function of human αB-Cry. These pathogenic mutations resulted in the important alterations of the secondary, tertiary and oligomeric (quaternary) structures of human αB-Cry. The missense mutations were also capable to significantly increase the amyloidogenic propensity of human αB-Cry and to diminish the chaperone-like activity of this protein. The above mentioned changes were observed more noticeably after D109H mutation. The detrimental effects of D109H mutation may be due to the loss of salt bridge with R120 in the dimeric interface, flagging the anti-aggregation ability of αB-Cry chaperone. In conclusion, the R69C and D109H mutations displayed a significant damaging effect on the structure and chaperone function of human αB-Cry which could be considered as their biochemical pathomechanisms in development of congenital cataract and myopathy disorders.

Penicisulfuranol A, a novel C-terminal inhibitor disrupting molecular chaperone function of Hsp90 independent of ATP binding domain.

The goal of this study is to explore the mechanism of a heat shock protein 90 (Hsp90) C-terminal inhibitor, Penicisulfuranol A (PEN-A), for cancer therapy. PEN-A was produced by a mangrove endophytic fungus Penicillium janthinellum and had a new structure with a rare 3H-spiro [benzofuran-2, 2'-piperazine] ring system. PEN-A caused depletion of multiple Hsp90 client proteins without induction of heat shock protein 70 (Hsp70). Subsequently, it induced apoptosis and inhibited xerograph tumor growth of HCT116 cells in vitro and in vivo. Mechanism studies showed that PEN-A was bound to C-terminus of Hsp90 at the binding site different from ATP binding domain. Therefore, it inhibited dimerization of Hsp90 C-terminus, depolymerization of ADH protein by C-terminus of Hsp90, and interaction of co-chaperones with Hsp90. These inhibitory effects of PEN-A were similar to those of novobiocin, an inhibitor binding to interaction site for ATP of C-terminus of Hsp90. Furthermore, our study revealed that disulfide bond was essential moiety for inhibition activity of PEN-A on Hsp90. This suggested that PEN-A may be bound to cysteine residues near amino acid region which was responsible for dimerization of Hsp90. All results indicate that PEN-A is a novel C-terminal inhibitor of Hsp90 and worthy for further study in the future not only for drug development but also for unraveling the bioactivities of Hsp90.

Global Dynamics of Yeast Hsp90 Middle and C-Terminal Dimer Studied by Advanced Sampling Simulations.

The Hsp90 protein complex is one of the most abundant molecular chaperone proteins that assists in folding of a variety of client proteins. During its functional cycle it undergoes large domain rearrangements coupled to the hydrolysis of ATP and association or dissociation of domain interfaces. In order to better understand the domain dynamics comparative Molecular Dynamics (MD) simulations of a sub-structure of Hsp90, the dimer formed by the middle (M) and C-terminal domain (C), were performed. Since this MC dimer lacks the ATP-binding N-domain it allows studying global motions decoupled from ATP binding and hydrolysis. Conventional (c)MD simulations starting from several different closed and open conformations resulted in only limited sampling of global motions. However, the application of a Hamiltonian Replica exchange (H-REMD) method based on the addition of a biasing potential extracted from a coarse-grained elastic network description of the system allowed much broader sampling of domain motions than the cMD simulations. With this multiscale approach it was possible to extract the main directions of global motions and to obtain insight into the molecular mechanism of the global structural transitions of the MC dimer.

Probing the structure-function relationship of Mycobacterium leprae HSP18 under different UV radiations.

Ultraviolet radiation, an effective sterilizing source, rapidly kills the causative organism (Mycobacterium leprae) of leprosy. But, the reasons behind this quick death are not clearly understood. Also, the impact of UV radiation on the antigen(s) which is/are responsible for the survival of this pathogen is still unknown. Many reports have revealed that M. leprae secrets a major immunodominant antigen, namely HSP18, whose chaperone function plays an important role in the growth and survival of this pathogen under various environmental insults. However, the effect of UV radiation on its structure and chaperone function is still unclear. Therefore, we have taken a thorough attempt to understand these two aspects of HSP18 under different UV radiations (UVA/UVB/UVC; doses: 1-50 J/cm2). Our study revealed that its chaperone function is decreased significantly with increasing doses of various UV radiations. These different UV irradiations perturb only its tertiary structure and induce tryptophan and tyrosine photo-oxidation to N-formyl kynurenine, kynurenine and dityrosine. Such photo-oxidation promotes the subunit cross-linking within a HSP18 oligomer, lowers the surface hydrophobicity and thermostability of the protein. All these factors together damage/reduce the chaperone function of HSP18 which may be an important factor behind the rapid death of M. leprae under UV exposure.

An alternative splice variant of human αA-crystallin modulates the oligomer ensemble and the chaperone activity of α-crystallins.

In humans, ten genes encode small heat shock proteins with lens αA-crystallin and αB-crystallin representing two of the most prominent members. The canonical isoforms of αA-crystallin and αB-crystallin collaborate in the eye lens to prevent irreversible protein aggregation and preserve visual acuity. α-Crystallins form large polydisperse homo-oligomers and hetero-oligomers and as part of the proteostasis system bind substrate proteins in non-native conformations, thereby stabilizing them. Here, we analyzed a previously uncharacterized, alternative splice variant (isoform 2) of human αA-crystallin with an exchanged N-terminal sequence. This variant shows the characteristic α-crystallin secondary structure, exists on its own predominantly in a monomer-dimer equilibrium, and displays only low chaperone activity. However, the variant is able to integrate into higher order oligomers of canonical αA-crystallin and αB-crystallin as well as their hetero-oligomer. The presence of the variant leads to the formation of new types of higher order hetero-oligomers with an overall decreased number of subunits and enhanced chaperone activity. Thus, alternative mRNA splicing of human αA-crystallin leads to an additional, formerly not characterized αA-crystallin species which is able to modulate the properties of the canonical ensemble of α-crystallin oligomers.