Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins

AbstractThe activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC–MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes

Gambogic Acid, a Natural Product Inhibitor of Hsp90

A high-throughput screening of natural product libraries identified (−)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2α kinase (HRI, an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors

Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.Peer reviewedBiochemistry and Molecular Biolog

Hsp90-dependent assembly of the DBC2/RhoBTB2-Cullin3 E3-ligase complex.

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes

GTP binding activity of DBC2 and its Rho domain.

<p>(<b>A</b>) Homology model of the DBC2 Rho domain was built using Swiss-model and the crystal structure of Rnd1 (2CLS) as a template. The 46 residues which are deleted when DBC2's Rho-domain is truncated to a Ras domain including the loss of the G5 loop <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090054#pone.0090054-Chang1" target="_blank">[32]</a> are colored in magenta. The switch II region is colored yellow with the R99 residue in green. GTPgammaS is shown as spheres with V191 colored in cyan and K27 in light blue. The image was rendered by PyMol, Delano scientific. (<b>B</b>) [³⁵S]- Flag-tagged DBC2 and CΔ210 were synthesized by TnT in the absence of any additions (no additions, N.A.). TnT RRLs containing empty vector were used as controls for non-specific binding (NS). [³⁵S]DBC2 was pulled down from RRL with GMP or GTP-linked agarose, washed with P100T and analyzed by SDS-PAGE, and autoradiography, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090054#s2" target="_blank">Materials and methods</a>. (<b>C</b>) [³⁵S]-Flag-tagged DBC2 was synthesized by TnT in the absence of any additions. The samples were diluted with 5 volumes of TBS (buffer) or TBS containing GTP (GTP) to give a final concentration of 20 mM GTP. After 45 min of incubation with GTP-agarose at 4°C, the samples were washed and the amount of [³⁵S]-DBC2 was determined by scintillation counting. The experiment was carried out in triplicate. (<b>D</b>) [³⁵S]-Labeled wild-type DBC2, DBC2 Rho domain (CΔ210), DBC2ΔRho (NΔ258), and the DBC2/R99G (R99G) mutant were synthesized by TnT, pulled down from RRL with GTP-linked agarose, and washed with P100T as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090054#s2" target="_blank">Materials and methods</a>. The amount of bound [³⁵S]-DBC2 constructs were quantified by scintillation counting and normalized for the number of Met present in each construct, and for [³⁵S]-Met non-specifically bound to the GTP-agarose. The data represent three independent bio-replicates including three technical replicates. The (***) denotes a significant difference based on a 95% confidence interval, P<0.05.</p

Model for the modulation of DBC2 GTP binding/activation by the Hsp90 chaperone machine.

<p>The domains of DBC2 domains are colored light green (Rho domain), gray (BTB1 domain), blue (BTB2 domain), and brown (putative RING domain). Hsp90-dependent binding of Cul3 (red) and CSN4 (brown) and other COP9 signalosome components (yellow-green) are used as example of DBC2-associating proteins. “Early”, “intermediate”, and “late” refer to complexes that are formed during Hsp90's ATPase cycle.</p

Interaction of DBC2 with the Hsp90 chaperone machine.

<p>(<b>A</b>) [³⁵S]-Labeled Flag-tagged DBC2 was synthesized by TnT in RRL and immuno-adsorbed with immobilized anti-Flag antibodies in the presence of 20 mM molybdate. Samples were washed and analyzed by SDS-PAGE and Western blotting for the co-adsorption of Hsp90 and Cdc37. (<b>B</b>) MCF7 cells were transfected with pcDNA3 plasmid expressing HA-tagged DBC2 or empty vector control. After 16 h cells were lysed, and immuno-adsorbed with immobilized anti-HA-antibody. Samples were washed and analyzed by SDS-PAGE and Western blotting for the co-adsorption of Hsp90. (<b>C</b>) [³⁵S]-Labeled Flag-tagged DBC2 was synthesized in the presence of geldanamycin (GA), or molybdate (MoO4), or their vehicle controls, water (no additions, NA) or DMSO, and immuno-adsorbed from RRL with immobilized anti-Flag antibodies, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090054#s2" target="_blank">Materials and methods</a>. TnT RRLs containing empty vector were used as controls for non-specific binding (NS). Immuno-adsorbed samples were washed with buffer containing low (100 mM NaCl) or high salt (500 mM NaCl). Samples were analyzed by SDS-PAGE, and autoradiography (bottom panel) or Western blotting for co-adsorption of endogenous Hsp90, Hsp70, HOP and Cdc37.</p

Western blots for DBC2-associated components from reticulocyte lysates identified by LC-MS/MS.

<p>Flag-tagged DBC2 was synthesized and immuno-adsorbed from RRL with anti-Flag antibody-agarose, washed with P100T, and analyzed by SDS-PAGE, and Western blotting for DBC2 and co-adsorption of Cul3 and CSN4 as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090054#s2" target="_blank">Materials and methods</a>.</p