Inhibition of Embryonic HSP 90 Function Promotes Variation of Cold Tolerance in Zebrafish

Accumulating evidence indicates that heat shock protein 90 (HSP90) plays essential roles in modulation of phenotypic plasticity in vertebrate development, however, the roles of HSP90 in modulation of cold tolerance capacity in fish are still unclear. In the present study, we showed that transient inhibition of embryonic HSP90 function by a chemical inhibitor or low conductivity stress promoted variation of cold tolerance capacity in adult zebrafish. Further work showed that embryonic HSP90 inhibition enhanced cold tolerance in adult zebrafish could be transmitted to their offspring. RNA-seq data showed that embryonic HSP90 inhibition enhanced cold tolerance involves variation of gene expression related to proteasome, lysosome, autophagy, and ribosome. Experiments with zebrafish ZF4 cells showed that two differentially expressed genes atg9b and psmd12 were up-regulated by radicicol treatment and provided protective roles for cells under cold stress, indicating that up-regulation of autophagy and proteasome function contributes to enhanced cold tolerance. The present work sheds a light on the roles of HSP90 in regulation of phenotypic plasticity associated with thermal adaptation in fish.

The Proteasome Governs Fungal Morphogenesis via Functional Connections with Hsp90 and cAMP-Protein Kinase A Signaling

ABSTRACT Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis. For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis. IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.

Combinatorial Inhibition of mTORC2 and Hsp90 Leads to a Distinctly Effective Therapeutic Strategy in Malignant Pheochromocytoma

Background: Malignant pheochromocytoma (mPCC) is an uncommon tumor with poor prognosis, and no effective therapeutic strategy exists as yet. Discovering new and effective therapeutic strategies to improve prognosis is an urgent need. Objective: To investigate whether a combinatorial inhibition of both mTORC2 and Hsp90 in PC12 cells could lead to a distinct anti-tumor effect in vitro and in vivo that was greater than the inhibition of mTORC2 or Hsp90 alone. Methods: Targeting mTORC2 was assessed by knockdown of Rictor using shRNA, and 17-AAG was used to inhibit Hsp90 function. Results: Combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vitro that was greater than the inhibition of mTORC2 or Hsp90 alone. Inhibiting Hsp90 specifically could inhibit tumor growth of sh-Rictor cells in vivo, suggesting that the combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vivo. Western blotting has shown that both p-Akt Ser473 and p-Akt Thr450 showed significantly decreased expression after targeting mTORC2, while p-Akt Thr308 did not. However, all three different p-AKTs, including p-Akt Ser473, p-Akt Thr450 and p-Akt Thr308, showed a significantly decreased expression in combinatorial inhibition of both mTORC2 and Hsp90. Collectively, it revealed that combinatorial inhibition of mTORC2 and Hsp90 could destabilize the Akt signaling. Conclusion: Our results demonstrated that combinatorial inhibition of mTORC2 and Hsp90 could increase their anti-tumor effect and destabilize the Akt signaling in PC12 cells, suggesting a combinatorial inhibition of both mTORC2 and Hsp90 which might be an effective therapeutic strategy for mPCC.

Targeting the MYC Oncogene in Burkitt Lymphoma through HSP90 Inhibition

Overexpression of the MYC oncogene is a key feature of many human malignancies including Burkitt lymphoma. While MYC is widely regarded to be a promising therapeutic target, a clinically effective MYC inhibitor is still elusive. Here, we report an alternative strategy, targeting MYC indirectly through inhibition of the HSP90 machinery. We found that inhibition of HSP90 function reduces MYC expression in human Burkitt lymphoma through suppression of MYC transcription and destabilization of MYC protein, thereby diminishing the proliferation of tumor cells. Consistently, treatment of Burkitt lymphoma cell lines with HSP90 inhibitors (17-AAG or 17-DMAG) was accompanied by downregulation of canonical MYC target genes. Combination treatment with 17-DMAG and the proteasome inhibitor, MG-132, led to accumulation of MYC protein, indicating that upon HSP90 inhibition, MYC is degraded by the proteasome. Using co-immunoprecipitation, we furthermore demonstrated a direct interaction between MYC and HSP90, indicating that MYC is an HSP90 client protein in Burkitt lymphoma. Together, we report here the use of HSP90 inhibitors as an alternative approach to target the MYC oncogene and its network in Burkitt lymphoma.