Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxicshift reprogramming and has glyoxalase activity. We show thatsubstoichiometric concentrations of Hsp31 can abrogate aggrega-tion of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of -synuclein ( Syn), a target of the chaperoneactivity of human DJ-1, an Hsp31 homolog. We demonstrate thatHsp31 is able to suppress the in vitro fibrillization or aggregation of Syn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of Syn cytoplasmic foci, and yeast cells were rescued from Syn-generated proteotoxicity upon Hsp31overexpression. Moreover, we showed that Hsp31 protein levels are increased byH2O2, in the diauxic phase of normal growth con-ditions, and in cells under Syn-mediated proteotoxic stress. Weshow that Hsp31 chaperone activity and not the methylglyoxalaseactivity or the autophagy pathway drives the protective effects.Wealso demonstrate reduced aggregation of the Sup35 prion domain,PrD-Sup35, as visualized by fluorescent protein fusions. In addi-tion, Hsp31 acts on its substrates prior to the formation of largeaggregates because Hsp31 does not mutually localize with prionaggregates, and it prevents the formation of detectable in vitro Syn fibrils. These studies establish that the protective role ofHsp31 against cellular stress is achieved by chaperone activity thatintervenes early in the protein misfolding process and is effectiveona wide spectrum of substrate proteins, including Synandprion proteins

Targeting the Hsp90 C-terminus: Development of Novel Allosteric Modulators of Chaperone Function

Heat shock protein 90 (Hsp90) is a conserved, abundant, multi-domain protein that functions as a molecular chaperone to maintain protein homeostasis. Hsp90 has emerged as a promising target for a multitude of disease states due to its ability to inhibit maturation and stabilization of multiple client proteins simultaneously. Traditional Hsp90 inhibitors have functioned through two main mechanism of action: competing with N-terminal nucleotide binding to disrupt ATP hydrolysis, or binding to the C-terminal nucleotide binding site to disrupt dimerization of Hsp90. In the present study, we report NSC145366 as a novel, allosteric modulator of Hsp90’s function. We report that NSC145366 binds to the Hsp90 C-terminal domain to increase oligomerization resulting in allosteric inhibition of N-terminal ATPase activity. Interestingly, an ATP pulldown assay and fluorescence polarization indicate that NSC145366 is not competing with ATP binding at either domain. Molecular docking suggests that our compound is binding at a novel site located in proximity to the C-terminal dimer interface. Treatment of LNCaP prostate tumor cells resulted in selective degradation of client proteins BRCA1 and AR without induction of a compensatory heat shock response. Minimal structure activity analysis was completed that demonstrated inhibitory capacity of NSC145366 and its structural analogs is not driven by the bisphenol A core, and that symmetry in the terminal moieties is not necessary for Hsp90 inhibition. Due to its novel mechanism of action and structure, NSC145366 offers a unique method to modulate Hsp90 function in cells and could improve the utility of Hsp90 targeted probes and future therapeutics. In addition, follow up studies were conducted using NSC695801, NSC113237, and NSC255109. These compounds had activity in the NCI-60 cancer cell panel that correlates to that of NSC145366 and other known N-terminal Hsp90 inhibitors. We demonstrate that NSC695801 and NSC113237 bind to the Hsp90 C-terminus and inhibit Hsp90’s in vitro chaperone function and ATPase activity. Based on this, and their broad-spectrum activity in the NCI-60 cancer cell panel, investigation into their effects on heat shock network modulation is warranted. These unique probe molecules provide additional insight into multiple mechanisms by which Hsp90’s function can be modulated, laying the foundation for advanced SAR and development of future anti-cancer therapeutics