HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii

We report on the molecular and biochemical characterization of HEAT SHOCK PROTEIN 90C (HSP90C), one of the three Hsp90 chaperones encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that HSP90C is a plastidic protein. In the chloroplast, HSP90C was localized to the soluble stroma fraction, but also to thylakoids and low-density membranes containing inner envelopes. HSP90C is expressed under basal conditions and is strongly induced by heat shock and moderately by light. In soluble cell extracts, HSP90C was mainly found to organize into dimers, but also into complexes of high molecular mass. Also, heterologously expressed HSP90C was mainly found in dimers, but tetramers and fewer monomers were detected, as well. HSP90C exhibits a weak ATPase activity with a Km for ATP of approximately 48 microM and a kcat of approximately 0.71 min(-1). This activity was inhibited by the Hsp90-specific inhibitor radicicol. In coimmunoprecipitation experiments, we found that HSP90C interacts with several proteins, among them plastidic HSP70B. The cellular concentration of HSP70B was found to be 2.9 times higher than that of HSP90C, giving a 4.8:1 stoichiometry of HSP70B monomers to HSP90C dimers. The strong inducibility of HSP90C by heat shock implies a role of the chaperone in stress management. Furthermore, its interaction with HSP70B suggests that, similar to their relatives in cytosol and the endoplasmic reticulum, both chaperones might constitute the core of a multichaperone complex involved in the maturation of specific client proteins, e.g. components of signal transduction pathways.     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

The region adjacent to the highly immunogenic site and shielded by the middle domain is responsible for self-oligomerization/client binding of the HSP90 molecular chaperone

We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity. The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule. The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304). Particularly, residues 341-350 were critical on oligomerization. On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule. Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain. Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain. With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546.     

Leishmania donovani P23 protects parasites against HSP90 inhibitor-mediated growth arrest

In Leishmania donovani, the HSP90 chaperone complex plays an essential role in the control of the parasite’s life cycle, general viability and infectivity. Several of the associated co-chaperones were also shown to be essential for viability and/or infectivity to mammalian cells. Here, we identify and describe the co-chaperone P23 and distinguish its function from that of the structurally related small heat shock protein HSP23. P23 is expressed constitutively and associates itself with members of the HSP90 complex, i.e. HSP90 and Sti1. Viable P23 gene replacement mutants could be raised and confirmed as null mutants without deleterious effects on viability under a variety of physiological growth conditions. The null mutant also displays near-wild-type infectivity, arguing against a decisive role played by P23 in laboratory settings. However, the P23 null mutant displays a marked hypersensitivity against HSP90 inhibitors geldanamycin and radicicol. P23 also appears to affect the radicicol resistance of a HSP90 Leu₃₃-Ile mutant described previously. Therefore, the annotation of L. donovani P23 as HSP90-interacting co-chaperone is confirmed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0595-y) contains supplementary material, which is available to authorized users.     

The chloroplast DnaJ homolog CDJ1 of Chlamydomonas reinhardtii is part of a multichaperone complex containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.     

Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.     

Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie

Scrapie is a transmissible spongiform encephalopathy in sheep and goats. Susceptibility to this neurodegenerative disease is mainly controlled by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to scrapie. On the basis of several studies in Alzheimer and different transmissible spongiform encephalopathy models, HSP90AA1 was chosen as a putative positional and functional candidate gene that might be involved in the polygenic variance mentioned above. In the present work, the ovine HSP90AA1 gene including the promoter and other regulatory regions has been isolated and characterized. Several sequence polymorphisms have also been identified. FISH-mapping localized the HSP90AA1 gene on ovine chromosome OAR19q24dist, which was confirmed by linkage analysis. This chromosome region has been shown to include a quantitative trait loci (QTL) for scrapie incubation period in sheep. Expression analyses were carried out in spleen and cerebellum samples. No differences in the expression of the HSP90AA1 gene were found in any of these tissues (p > 0.05) between control and infected animal samples. Nevertheless, association analyses revealed that several polymorphisms in the 5′ and 3′ regions of the HSP90AA1 gene were differentially distributed among animals with different responses to scrapie infection. Thus, results presented here support the hypothesis that HSP90AA1 could be a positional and functional candidate gene modulating the response to scrapie in sheep. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined HSP90 and kinase inhibitor therapy: Insights from The Cancer Genome Atlas

The merging of knowledge from genomics, cellular signal transduction and molecular evolution is producing new paradigms of cancer analysis. Protein kinases have long been understood to initiate and promote malignant cell growth and targeting kinases to fight cancer has been a major strategy within the pharmaceutical industry for over two decades. Despite the initial success of kinase inhibitors (KIs), the ability of cancer to evolve resistance and reprogram oncogenic signaling networks has reduced the efficacy of kinase targeting. The molecular chaperone HSP90 physically supports global kinase function while also acting as an evolutionary capacitor. The Cancer Genome Atlas (TCGA) has compiled a trove of data indicating that a large percentage of tumors overexpress or possess mutant kinases that depend on the HSP90 molecular chaperone complex. Moreover, the overexpression or mutation of parallel activators of kinase activity (PAKA) increases the number of components that promote malignancy and indirectly associate with HSP90. Therefore, targeting HSP90 is predicted to complement kinase inhibitors by inhibiting oncogenic reprogramming and cancer evolution. Based on this hypothesis, consideration should be given by both the research and clinical communities towards combining kinase inhibitors and HSP90 inhibitors (H90Ins) in combating cancer. The purpose of this perspective is to reflect on the current understanding of HSP90 and kinase biology as well as promote the exploration of potential synergistic molecular therapy combinations through the utilization of The Cancer Genome Atlas.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0604-1) contains supplementary material, which is available to authorized users.     

Inhibition of adjuvant arthritis by a DNA vaccine encoding human heat shock protein 60

Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-gamma secretion to HSP60 and, concomitantly, to down-regulation of IFN-gamma secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFbeta1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.     

The hexameric structures of human heat shock protein 90

Background

The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood.

Principal Findings

Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90.

Conclusions

While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis.     

RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice

A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.     

Molecular identification of Rickettsia parkeri infecting Amblyomma triste ticks in an area of Argentina where cases of rickettsiosis were diagnosed

Specimens of the hard tick Amblyomma triste were found infected with Rickettsia parkeri in an area of Argentina (General Lavalle, Buenos Aires Province) where cases of human illness attributed to this microorganism have been reported. Molecular detection of R. parkeri was based on polymerase chain reactions that amplify a ca. 400-bp fragment of the 23S-5S intergenic spacer and a ca. 500-bp fragment of the gene encoding a 190-kDa outer membrane protein. Three (6.97%) of 43 A. triste ticks were determined to be positive for R. parkeri. These results provide strong evidence that A. triste is the vector of R. parkeri in the study area. The findings of this work have epidemiological relevance because human parasitism by A. triste ticks has been frequently recorded in some riparian areas of Argentina and Uruguay and new cases of R. parkeri rickettsiosis might arise in the South American localities where humans are exposed to the bites of this tick species.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.