Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1

Complex conformational dynamics are essential for function of the dimeric molecular cha- perone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimer- ization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90

Bioinformatic Analysis and Post-Translational Modification Crosstalk Prediction of Lysine Acetylation

Recent proteomics studies suggest high abundance and a much wider role for lysine acetylation (K-Ac) in cellular functions. Nevertheless, cross influence between K-Ac and other post-translational modifications (PTMs) has not been carefully examined. Here, we used a variety of bioinformatics tools to analyze several available K-Ac datasets. Using gene ontology databases, we demonstrate that K-Ac sites are found in all cellular compartments. KEGG analysis indicates that the K-Ac sites are found on proteins responsible for a diverse and wide array of vital cellular functions. Domain structure prediction shows that K-Ac sites are found throughout a wide variety of protein domains, including those in heat shock proteins and those involved in cell cycle functions and DNA repair. Secondary structure prediction proves that K-Ac sites are preferentially found in ordered structures such as alpha helices and beta sheets. Finally, by mutating K-Ac sites in silico and predicting the effect on nearby phosphorylation sites, we demonstrate that the majority of lysine acetylation sites have the potential to impact protein phosphorylation, methylation, and ubiquitination status. Our work validates earlier smaller-scale studies on the acetylome and demonstrates the importance of PTM crosstalk for regulation of cellular function

Excessive HDAC activation is critical for neurodegeneration in the rd1 mouse

Inherited retinal degenerations, collectively termed retinitis pigmentosa (RP), constitute one of the leading causes of blindness in the developed world. RP is at present untreatable and the underlying neurodegenerative mechanisms are unknown, even though the genetic causes are often established. Acetylation and deacetylation of histones, carried out by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, affects cellular division, differentiation, death and survival. We found acetylation of histones and probably other proteins to be dramatically reduced in degenerating photoreceptors in the rd1 human homologous mouse model for RP. Using a custom developed in situ HDAC activity assay, we show that overactivation of HDAC classes I/II temporally precedes photoreceptor degeneration. Moreover, pharmacological inhibition of HDACs I/II activity in rd1 organotypic retinal explants decreased activity of poly-ADP-ribose-polymerase and strongly reduced photoreceptor cell death. These findings highlight the importance of protein acetylation for photoreceptor cell death and survival and propose certain HDAC classes as novel targets for the pharmacological intervention in RP

Synergistic Antitumor Effects of Novel HDAC Inhibitors and Paclitaxel In Vitro and In Vivo

Preclinical studies support the therapeutic potential of histone deacetylases inhibitors (HDACi) in combination with taxanes. The efficacy of combination has been mainly ascribed to a cooperative effect on microtubule stabilization following tubulin acetylation. In the present study we investigated the effect of paclitaxel in combination with two novel HDACi, ST2782 or ST3595, able to induce p53 and tubulin hyperacetylation. A synergistic effect of the paclitaxel/ST2782 (or ST3595) combination was found in wild-type p53 ovarian carcinoma cells, but not in a p53 mutant subline, in spite of a marked tubulin acetylation. Such a synergistic interaction was confirmed in additional human solid tumor cell lines harboring wild-type p53 but not in those expressing mutant or null p53. In addition, a synergistic cytotoxic effect was found when ST2782 was combined with the depolymerising agent vinorelbine. In contrast to SAHA, which was substantially less effective in sensitizing cells to paclitaxel-induced apoptosis, ST2782 prevented up-regulation of p21WAF1/Cip1 by paclitaxel, which has a protective role in response to taxanes, and caused p53 down-regulation, acetylation and mitochondrial localization of acetylated p53. The synergistic antitumor effects of the paclitaxel/ST3595 combination were confirmed in two tumor xenograft models. Our results support the relevance of p53 modulation as a major determinant of the synergistic interaction observed between paclitaxel and novel HDACi and emphasize the therapeutic interest of this combination

Comparative analysis of novel and conventional Hsp90 inhibitors on HIF activity and angiogenic potential in clear cell renal cell carcinoma: implications for clinical evaluation

<p>Abstract</p> <p>Background</p> <p>Perturbing Hsp90 chaperone function targets hypoxia inducible factor (HIF) function in a von Hippel-Lindau (VHL) independent manner, and represents an approach to combat the contribution of HIF to cell renal carcinoma (CCRCC) progression. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in halting the progression of advanced CCRCC.</p> <p>Methods</p> <p>Here we evaluated a novel next generation small molecule Hsp90 inhibitor, EC154, against HIF isoforms and HIF-driven molecular and functional endpoints. The effects of EC154 were compared to those of the prototypic Hsp90 inhibitor 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589.</p> <p>Results</p> <p>The findings indicate that EC154 is a potent inhibitor of HIF, effective at doses 10-fold lower than 17-AAG. While EC154, 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589 impaired HIF transcriptional activity, CCRCC cell motility, and angiogenesis; these effects did not correlate with their ability to diminish HIF protein expression. Further, our results illustrate the complexity of HIF targeting, in that although these agents suppressed HIF transcripts with differential dynamics, these effects were not predictive of drug efficacy in other relevant assays.</p> <p>Conclusions</p> <p>We provide evidence for EC154 targeting of HIF in CCRCC and for LBH589 acting as a suppressor of both HIF-1 and HIF-2 activity. We also demonstrate that 17-AAG and EC154, but not LBH589, can restore endothelial barrier function, highlighting a potentially new clinical application for Hsp90 inhibitors. Finally, given the discordance between HIF activity and protein expression, we conclude that HIF expression is not a reliable surrogate for HIF activity. Taken together, our findings emphasize the need to incorporate an integrated approach in evaluating Hsp90 inhibitors within the context of HIF suppression.</p

Targeting Huntington’s disease through histone deacetylases

Huntington’s disease (HD) is a debilitating neurodegenerative condition with significant burdens on both patient and healthcare costs. Despite extensive research, treatment options for patients with this condition remain limited. Aberrant post-translational modification (PTM) of proteins is emerging as an important element in the pathogenesis of HD. These PTMs include acetylation, phosphorylation, methylation, sumoylation and ubiquitination. Several families of proteins are involved with the regulation of these PTMs. In this review, I discuss the current evidence linking aberrant PTMs and/or aberrant regulation of the cellular machinery regulating these PTMs to HD pathogenesis. Finally, I discuss the evidence suggesting that pharmacologically targeting one of these protein families the histone deacetylases may be of potential therapeutic benefit in the treatment of HD

The Hsp90 mosaic: a picture emerges

Hsp90s, molecular chaperones critically involved in many essential cellular processes, were the focus of a recent international conference held in Seeon, Germany. The scope of the conference ranged from structural and mechanistic insights all the way to medical applications