Effect of <i>HCH1</i> or <i>AHA1</i> deletion on yeast growth.

*<p>- ipT22Ia appeared to have two copies of HCH1 (possibly due to chromosomal rearrangement or duplication) and was not tested.</p>**<p>- We were unable to obtain a <i>HCH1</i> deletion for this strain.</p

Co-chaperone competition in Hsp82p ATPase reactions.

<p>A. Stimulation of the ATPase activity of wildtype Hsp82p (2 µM) by increasing concentrations of Hch1p in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. B. Inhibition of Aha1p-stimulated ATPase rate by increasing concentrations of Sba1p (triangles) and Sti1p (squares). Dashed line represents intrinsic Hsp82p ATPase rate. Reactions contain 2 µM Hsp82p, 4 µM Aha1p and indicated concentrations of Sba1p or Sti1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). C. Effect of increasing concentrations of Hch1p on the Aha1p-stimulated ATPase activity of wildtype Hsp82p (2 µM) in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.</p

ATPase stimulation of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

<p>A. Stimulation of the ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Aha1p. B. Stimulation of the ATPase activity of wildtype Hsp82p (black bars), and Hsp82p^A587T (grey bars) by Hch1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.</p

Hch1p regulates Hsp90 inhibitor drug sensitivity in yeast.

<p>A. Deletion of <i>HCH1</i> confers resistance to Hsp90 inhibitor NVP-AUY922 in yeast expressing Hsp82p, Hsp82p^G313S or Hsp82p^A587T. Cells were grown overnight in YPD and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with indicated concentrations of NVP-AUY922. B. Overexpression of myc-tagged Hch1p results in hypersensitivity to Hsp90 inhibitor NVP-AUY922 in yeast. Cells were grown overnight in YPD supplemented with 200 mg/L G418 and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with 200 mg/L G418 and indicated concentrations of NVP-AUY922.</p

Overexpression of C-terminally myc-tagged Hch1p impairs growth of yeast expressing Hsp82p^A587T but not yeast expressing wildtype Hsp82p.

<p>A. Cells were grown overnight in YPD supplemented with 200 mg/L G418 and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with 200 mg/L G418 and grown for 2 days at 30, 35.5 and 37°C. B. Western analysis of yeast shown in 3A with anti-myc tag, anti-His tag and anti-actin antibodies. Levels of mycHch1p (lanes 2, 4, 6, and 8) in strains expressing Hsp82p (lanes 1–4), or Hsp82p^A587T (lanes 5–8) with (lanes 3,4, 7, 8) or without (lanes 1, 2, 5, 6) <i>HCH1</i> deletion. (Anti-myc antibody 9E10).</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Deletion of <i>HCH1</i>, but not <i>AHA1</i>, alleviates temperature sensitivity in <i>S. cerevisiae</i> expressing Hsp82p^G313S or Hsp82p^A587T as the sole source of Hsp90.

<p>Cells were grown overnight in YPD at 30°C and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD-agar plates. A. Viability of mutant strains (expressing untagged Hsp82p) grown at 30, 35.5 and 37°C on YPD agar plates; B. Viability of mutant strains (expressing His-tagged Hsp82p) grown at 30, 35.5 and 37°C on YPD agar plates. C. Western analysis of HisHsp82p expression versus actin in mutant strains.</p

Inhibition of Aha1p-stimulated ATPase activity of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

<p>A. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. ATPase rate for wildtype Hsp82p and Hsp82p^A587T shown on left axis (Reactions contained 2 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). ATPase rate for Hsp82p^G313S shown on right axis (reactions contained 5 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). B. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (circles), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. Reactions contained 2 µM Hsp82p, 10 µM Hch1p and indicated concentrations of Sba1p. C. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. D. Inhibition of Aha1p-stimulated ATPase activity of Hsp82p^G313S (grey bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. E. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. ATPase rates shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min).</p

Structure and sequence of Aha1p and Hch1p.

<p><b>A. Domain structure of the 350 amino acid Aha1p and the 153 amino acid Hch1p.</b> B. Alignment of Aha1p and Hch1p.</p

Image1_Hsp90 inhibition leads to an increase in surface expression of multiple immunological receptors in cancer cells.pdf

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1, two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells, suggesting it has a unique role in mediating the immune system’s response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment, immune checkpoint blockade therapy, which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients.</p