ATPase Domain Of Hsp70 Research Articles

Genome-wide identification of hsp40 genes in channel catfish and their regulated expression after bacterial infection.

Heat shock proteins (HSPs) consist of a large group of chaperones whose expression is induced by high temperature, hypoxia, infection and a number of other stresses. Among all the HSPs, Hsp40 is the largest HSP family, which bind to Hsp70 ATPase domain in assisting protein folding. In this study, we identified 57 hsp40s in channel catfish (Ictalurus punctatus) through in silico analysis using RNA-Seq and genome databases. These genes can be classified into three different types, Type I, II and III, based on their structural similarities. Phylogenetic and syntenic analyses provided strong evidence in supporting the orthologies of these HSPs. Meta-analyses of RNA-Seq datasets were conducted to analyze expression profile of Hsp40s following bacterial infection. Twenty seven hsp40s were found to be significantly up- or down-regulated in the liver after infection with E. ictaluri; 19 hsp40s were found to be significantly regulated in the intestine after infection with E. ictaluri; and 19 hsp40s were found to be significantly regulated in the gill following infection with F. columnare. Altogether, a total of 42 Hsp40 genes were regulated under disease situations involving three tissues and two bacterial infections. The significant regulated expression of Hsp40 genes after bacterial infection suggested their involvement in disease defenses in catfish.

Both the charged linker region and ATPase domain of Hsp90 are essential for Rad51-dependent DNA repair.

The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time.

Hsp70 Cochaperones HspBP1 and BAG-1M Differentially Regulate Steroid Hormone Receptor Function

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Identification and characterization of OsEBS, a gene involved in enhanced plant biomass and spikelet number in rice

Common wild rice (Oryza rufipogon Griff.) is an important genetic reservoir for rice improvement. We investigated a quantitative trait locus (QTL), qGP5-1, which is related to plant height, leaf size and panicle architecture, using a set of introgression lines of O. rufipogon in the background of the Indica cultivar Guichao2 (Oryza sativa L.). We cloned and characterized qGP5-1 and confirmed that the newly identified gene OsEBS (enhancing biomass and spikelet number) increased plant height, leaf size and spikelet number per panicle, leading to an increase in total grain yield per plant. Our results showed that the increased size of vegetative organs in OsEBS-expressed plants was enormously caused by increasing cell number. Sequence alignment showed that OsEBS protein contains a region with high similarity to the N-terminal conserved ATPase domain of Hsp70, but it lacks the C-terminal regions of the peptide-binding domain and the C-terminal lid. More results indicated that OsEBS gene did not have typical characteristics of Hsp70 in this study. Furthermore, Arabidopsis (Arabidopsis thaliana) transformed with OsEBS showed a similar phenotype to OsEBS-transgenic rice, indicating a conserved function of OsEBS among plant species. Together, we report the cloning and characterization of OsEBS, a new QTL that controls rice biomass and spikelet number, through map-based cloning, and it may have utility in improving grain yield in rice.

Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle

The Hsp70-interacting protein, Hip, cooperates with the chaperone Hsp70 in protein folding and prevention of aggregation. Hsp70 interacts with non-native protein substrates in an ATP-dependent reaction cycle regulated by J-domain proteins and nucleotide exchange factors (NEFs). Hip is thought to delay substrate release by slowing ADP dissociation from Hsp70. Here we present crystal structures of the dimerization domain and the tetratricopeptide repeat (TPR) domain of rat Hip. As shown in a cocrystal structure, the TPR core of Hip interacts with the Hsp70 ATPase domain through an extensive interface, to form a bracket that locks ADP in the binding cleft. Hip and NEF binding to Hsp70 are mutually exclusive, and thus Hip attenuates active cycling of Hsp70-substrate complexes. This mechanism explains how Hip enhances aggregation prevention by Hsp70 and facilitates transfer of specific proteins to downstream chaperones or the proteasome.

Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90

The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.

Detection of soluble BAG3 and anti-BAG3 antibodies in patients with chronic heart failure

Dear Editor, Bcl-2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones that interact with the ATPase domain of the heat shock protein HSP70 through the BAG domain (amino acids 110–124). BAG3 contains also a WW domain, a proline-rich repeat (PXXP) and two conserved Ile-Pro-Val motifs that mediate binding to other proteins.1 bag3 gene is constitutively expressed in a few normal cell types, including myocytes, and in several primary tumors, while its expression is inducible by stressors in other cell types. Many evidence indicate that BAG3 has a role in sustaining cell survival, through mechanisms that, depending on the cell context, rely on the ability of BAG3 to modulate levels or localization of apoptosis-regulating proteins, such as IKKγ, Bax or BRAF, in either an Hsp70-dependent or -independent fashion.1, 2, 3 BAG3 is expressed during cardiomyoblast differentiation and sustains myogenin expression. In cardiomyocytes, BAG3 protein localizes at the Z-disc and interacts with the actin capping protein, CapZβ1, stabilizing myofibril structure; BAG3 mutations can impair the Z-disc assembly and increase the sensitivity to stress-induced apoptosis. Mutations in bag3 gene have been associated with some forms of myofibrillar myopathy and dilated cardiomyopathy.4, 5, 6, 7 Interestingly, we detected BAG3 protein in supernatants of the rat cardiomyocyte cell line H9c2 and adult Human Cardiac Myocytes (HCMa) after 16 h of serum deprivation (Figure 1a). We then verified that BAG3 was present in extracellular vesicles isolated through a differential centrifugation procedure (Supplementary Figure S1A). To investigate the possibility that BAG3 could be released in vivo and become detectable in sera in conditions in which the heart is undergoing chronic stress, we analyzed sera from two patients affected by chronic heart failure (CHF). By western blot analysis, we detected a band recognized by anti-BAG3 antibody at the expected molecular weight in sera from patients but not from healthy donors (Figure 1b). We excised the band from a replica gel and subjected it to mass spectrometry, unmistakably identifying BAG3 (Supplementary Figure S1B). Furthermore, we found that sera from CHF patients recognized BAG3 protein in western blotting, using an anti-human IgG as secondary antibody (Supplementary Figure S1C). This result indicated the presence of anti-BAG3 antibodies in CHF patients' sera. To confirm this finding, we developed an ELISA test using recombinant BAG3 to coat plates and anti-human IgG to reveal and analyzed sera from 52 CHF patients (EF<45%), compared with sera from 84 healthy donors. As shown in Figure 1c (and in Supplementary Figure S1D), we detected significantly higher values of anti-BAG3 antibodies in patients' compared with controls' sera. These data suggest that upon cardiac stress cardiomyocytes release BAG3 and this in turn results in production of auto-antibodies. There is no correlation with NYHA scores and antibody levels at this stage but screening of a larger number of patients in the future might be necessary to reveal potential correlations. Figure 1 (a) Detection of BAG3 protein in supernatants from cultured cardiomyocytes. Human (HCMa) and rat (H9c2) cardiomyocytes, at 80% confluence, were incubated with or without 10% FBS for 16 h at 37 °C in a 5% ... These results describe for the first time an extracellular BAG3 (eBAG3) released by stressed cardiomyocytes. As BAG3 lacks the consensus signal required for secretion via ER–Golgi pathway, it is likely to be released by the non-classical secretory pathway.8 eBAG3 release by stressed cardiomyocytes appears to result in production of auto-antibodies that could potentially be used as a biomarker for CHF patients, in combination with other already established markers. The presence of anti-BAG3 antibodies in CHF patients' sera indicates that released BAG3 can activate the immune system, and might therefore exert positive or negative functional effects on cardiac function, depending on the context. Future studies are required to clarify the biological roles of BAG3 and anti-BAG3 antibodies in CHF, and the utility of anti-BAG3 antibodies as a tool contributing to the study of the disease.

Rapid Protein–Ligand Costructures from Sparse NOE Data

An efficient way to rapidly generate protein-ligand costructures based on solution-NMR using sparse NOE data combined with selective isotope labeling is presented. A docked model of the 27 kDa N-terminal ATPase domain of Hsp90 bound to a small molecule ligand was generated using only 21 intermolecular NOEs, which uniquely defined both the binding site and the orientation of the ligand. The approach can prove valuable for the early stages of fragment-based drug discovery.

Abstract 2768: IDH1057, A novel, synthetic, small molecule inhibitor of heat shock protein 90(Hsp90)

Abstract Backgroud: The molecular chaperone Heat shock protein 90(Hsp90) governs the stabilization and activity of an array of over 100 ‘client proteins’, many of which are key signaling molecules involved in cell proliferation, survival, and transformation. A significant number of these client proteins are oncogenic in nature, including the protein kinases Her2, C-Raf, B-Raf, and AKT. We report here that IDH1057 is structurally distinct from a geldanamycin derivative, tanespimycin(17-AAG) as a novel and potent small molecule Hsp90 inhibitor that binds to the NH2-terminal ATP-binding pocket of Hsp90. Results: IDH1057 exhibited superior binding affinity for the ATPase domain of Hsp90(IDH1057, human Hsp90α FP IC50&amp;lt;150 nM) compared to 17-AAG(IC50 =456.3 nM) in vitro. IDH1057 also markedly inhibited proliferation in a broad panel of human tumor cell lines(IC50α100 nM) and depleted Her2 in SKBR3 breast cancer cells(IC50&amp;lt;50 nM). 10 NSCLC cell lines were relatively sensitive to IDH1057, irrespective of EGFR, Her2 or KRAS mutational status. IDH1057 was also highly effective against cell lines that displayed resistance to erlotinib. IDH1057 efficiently inhibited cell proliferation in 8 breast cancer cell lines including triple-negative breast cancer(TNBC) cell lines such as BT-20, Hs578T and MDA-MB-231. Exposure to IDH1057 led to a rapid and dramatic degradation of key signaling molecules in BT474 cells. Based on these results, we examined in vivo anti-tumor activity of IDH1057 in BT474 breast cancer xenografts. Following once daily oral dosing of IDH1057 at 200mg/kg on a weekly 5-day-on and 2-day-off schedule for 4 weeks, this compound showed statistically significant tumor growth inhibition(TGI=84.5%) and was well-tolerated with no observable body weight loss. A2780 ovarian cancer xenografts in mice treated orally with IDH1057 also resulted in potent tumor growth inhibition. More importantly, IDH1057 displayed favorable oral PK profiles in mice. Additionally, we performed in vitro ADME assays to characterize a new series of small molecule inhibitors. IDH1057 exhibited high metabolic stability in liver microsomes from human, mouse and beagle(≥60%). Five human CYP isoforms were not significantly inhibited by IDH1057 at 10uM. IDH1057 had moderate permeability in MDCK permeability assay(Papp=4.7x10−6 cm/sec). Conclusion: Our results indicate that IDH1057 is a novel and fully synthetic Hsp90 inhibitor with strong anti-tumor activity in vitro and in vivo. IDH1057 is a promising and orally active drug candidate for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2768. doi:1538-7445.AM2012-2768

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

BackgroundHeat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.Principal FindingsHere we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.SignificanceOur study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

Long-Term Exercises Increase the Concentration of HspBP1, a Co-Chaperone of 70-KDa Heat Shock Protein

Extracellular concentration of heat shock protein (Hsp) with a molecular weight of 70 kDa (Hsp70) rapidly increases in the serum in response to stress and returns to the basal level during recovery. Further regulation of its blood concentration is unclear. A possible regulator is HspBP1, a protein binding Hsp70. Binding to ATPase domain of Hsp70, HspBP1 inactivates it, thus acting as a factor of nucleotide exchange. Blood sera from athletes were examined at the beginning and end of the last mesocycle of the training period by two-staged immunoaffinity test system. The concentration of HspBP1 increased with decreasing Hsp70 concentration under conditions of long-term training. Presumably, the dynamics of Hsp70 and HspBP1 concentrations can serve as the test for evaluating the adaptation potential.

Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain.

The Sacsin Repeating Region (SRR): A Novel Hsp90-Related Supra-Domain Associated with Neurodegeneration

Protein supra-domains are defined as recurring arrangements of two or three domains present adjacent to each other along a polypeptide chain. Such combinations have novel functions beyond those of the individual partner domains that compose them, which can exist in isolation. Here, we describe a new type of large supra-domain (∼360 residues) in which one of the component partners (∼200 residues) appears to be incapable of existing in a context other than immediately adjacent to the C-terminus of the well-characterized Hsp90-like ATPase domain. We found that this supra-domain has a broad phylogenetic distribution, with examples in Archaea, Bacteria, and Eukarya. There is strong selective pressure for this arrangement to occur as part of repeated regions of unprecedented length. We identified multiple strategies of convergent evolution to attain such configurations. In humans, this supra-domain is present in triplicate at the N-terminus of the protein sacsin (4579 residues), mutated in the neurodegenerative disorder known as spastic ataxia of Charlevoix–Saguenay, and thus, we termed it “sacsin repeating region” (SRR). Biochemical characterization demonstrated that SRRs possess ATPase activity, which appears to be a requirement for sacsin function, as a disease-causing mutation leads to an alternate conformation completely incapable of hydrolyzing ATP. We also found evidence of a convergent evolutionary strategy to place SRRs in proteins containing C-terminal J domains, which we demonstrated here to be capable of stimulating the intrinsic ATPase activity of Hsp70. Our sequence and biochemical analyses indicate that SRRs necessitate nucleotide hydrolysis for their function, provided by the common Hsp90 ATPase domain, which, when coupled to the unique adjacent sequence, may give rise to a novel activity related to protein quality control.

Hsp70 Interacts with the Retroviral Restriction Factor TRIM5α and Assists the Folding of TRIM5α

Tripartite motif (TRIM) protein TRIM5alpha has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5alpha is not known. In this study, we showed that transiently overexpressed TRIM5alpha causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5alpha suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5alpha-binding partner. The interaction between Hsp70 and TRIM5alpha was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5alpha-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5alpha, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5alpha-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5alpha. Studies using a series of TRIM5alpha deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5alpha structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5alpha, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5alpha in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5alpha-induced cellular insults.

Abstract A217: Comparison of long-term pharmacodynamic actions of the synthetic small molecule HSP90 inhibitor AT13387 in multiple xenograft models

Abstract AT13387 is a novel small molecule inhibitor of HSP90. a member of a family of molecular chaperones. Previously we highlighted an association between the high affinity binding of AT13387 to the N-terminal ATPase domain of HSP90 and the duration of target inhibition in tumor cell lines in vitro. Further, AT13387 was shown to inhibit HSP90 and deplete client proteins in tumor xenografts longer than other, lower affinity inhibitors in the class. Here we have expanded the investigation to a wider number of tumor cell lines and to in vivo xenograft models and demonstrate that AT13387 has an extended pharmacodynamic action in tumors compared to other HSP90 inhibitors. We reason that the cumulative effects of these properties allow for less frequent dosing thus maximising efficacy whilst minimising systemic exposure and the potential for side effects. This study reports extended inhibition of HSP90 by AT13387 in a wider range of tumor cell lines in vitro. A 24hr exposure of A375 (melanoma) cells to AT13387 suppressed the expression of client proteins for 72 hrs or more. However in other cell lines such as NCI-H1975 (lung) and BT474 (breast), the suppression of client proteins by AT13387 was found to last in excess of 7 days. The pharmacodynamic action of AT13387 in vivo has been compared with that of 17-AAG and SNX-5422 in A375 and NCI-H1975 xenografts in nude mice. Following a single dose of each agent, we have investigated and compared the time course of the suppression of levels of several client proteins (e.g. AKT, CDK4) and the phosphorylation of key growth/survival signalling components (e.g. pERK, pS6, pAKT). These effects were rapidly induced in tumors following treatment with AT13387 and levels remained suppressed for up to 96 hrs. The durability of the AT13387 effects was significantly greater than for the other competitor compounds. Investigation of tumor growth in these models demonstrated that the longer pharmacodynamic action of AT13387 ensured that efficacy could be maintained on a once weekly schedule, whereas such a schedule for the other agents resulted in a significant loss of their anti-tumor effects. These data provide further support for the potential benefit of long acting HSP90 inhibitors as a way of maintaining anti-tumor effects whilst minimising potential for undesirable effects associated with systemic exposure. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A217.

ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the glucocorticoid receptor

The 70-kDa heat shock protein (Hsp70) is involved in providing the appropriate conformation of various nuclear hormone receptors, including the glucocorticoid receptor (GR). The Bcl-2 associated athanogene 1M (Bag-1M) is known to downregulate the DNA binding by the GR. Also, Bag-1M interacts with the ATPase domain of Hsp70 to modulate the release of the substrate from Hsp70. In this study, we demonstrate that ATP hydrolysis enhances Bag-1M-mediated inhibition of the DNA binding by the GR. However, the inhibitory effect of Bag-1M was abolished when the intracellular ATP was depleted. In addition, a Bag-1M mutant lacking the interaction with Hsp70 did not influence the GR to bind DNA, suggesting the interaction of Bag-1M with Hsp70 in needed for its negative effect. These results indicate that ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the GR and Hsp70 is a mediator for this process.

Inhibitor Design for Human Heat Shock Protein 70 ATPase Domain by Pharmacophore-based in silico Screening

The 70 kDa heat-shock protein (Hsp70) involved in various cellular functions, such as protein folding, translocation and degradation, regulates apoptosis in cancer cells. Recently, it has been reported that the green tea flavonoid (−)-epigallocatechin 3-gallate (EGCG) induces apoptosis in numerous cancer cell lines and could inhibit the anti-apoptotic effect of human Hsp70 ATPase domain (hATPase). In the present study, docking model between EGCG and hATPase was determined using automated docking study. Epi-gallo moiety in EGCG participated in hydrogen bonds with side chain of K71 and T204, and has metal chelating interaction with hATPase. Hydroxyl group of catechin moiety also participated in metal chelating hydrogen bond. Gallate moiety had two hydrogen bondings with side chains of E268 and K271, and hydrophobic interaction with Y15. Based on this docking model, we determined two pharmacophore maps consisted of six or seven features, including three or four hydrogen bonding acceptors, two hydrogen bonding donors, and one lipophilic. We searched a flavonoid database including 23 naturally occurring flavonoids and 10 polyphenolic flavonoids with two maps, and myricetin and GC were hit by map I. Three hydroxyl groups of B-ring in myricetin and gallo moiety of GC formed important hydrogen bonds with hATPase. 7-OH of A-ring in myricetin and OH group of catechin moiety in GC are hydrogen bond donors similar to gallate moiety in EGCG. From these results, it can be proposed that myricetin and GC can be potent inhibitors of hATPase. This study will be helpful to understand the mechanism of inhibition of hATPase by EGCG and give insights to develop potent inhibitors of hATPase.

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

Distinct binding sites for the ATPase and substrate-binding domain of human Hsp70 on the cell surface of antigen presenting cells

Hsp70 has high potential as an immune-adjuvant molecule: it mediates cytokine expression and maturation of antigen presenting cells (APCs) and also elicits a cytotoxic T-lymphocyte (CTL) response to antigenic peptides. How Hsp70 interacts with APCs is only poorly understood. Various surface proteins have been implicated in binding Hsp70 but their role in antigen presentation has remained controversial. The specific aim of this work was to determine the binding and uptake of human full-length Hsp70 as well as its separate ATPase (N70) and substrate-binding domains (C70) by APCs. Using laser scanning microscopy and FACS analysis, we established the existence of at least two distinct receptors for Hsp70, which are localized to distinct microdomains of the APC membrane. These receptors interact with the N70 and C70 domains of Hsp70, respectively. This observation was supported by the finding of a substantial portion of Hsp70 and C70, but not N70, in a detergent resistant membrane fraction. Accordingly, C70 and N70 did not compete with each other for binding. The bound proteins were rapidly internalized, with N70 and C70 localizing to separate endosomal compartments. Similarly, internalized free and peptide-loaded Hsp70 segregated rapidly within the cell. Efficient cross presentation of antigenic peptide bound to Hsp70 or C70 was demonstrated with the B3Z read out system. Consequently, the interaction of C70 with its putative receptor seems to be responsible for Hsp70-mediated cross presentation. Future studies should make use of C70 in identifying the uptake receptor of Hsp70–peptide complexes. In addition we could observe a stimulation of uptake of free peptide by preincubation with Hsp70 and N70, but not C70, whereas an Hsp-dependent cytokine secretion could not be detected. Consequently, by employing the individual domains it may be possible to distinguish between the different outcomes of Hsp70 treatment, like immune stimulation, DC maturation and antigen-specific responses.