Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90.

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.

Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species.

OBJECTIVES: Invasive mould infections are associated with a high mortality rate and the emergence of MDR moulds is of particular concern. Calcineurin and its chaperone, the heat shock protein 90 (Hsp90), represent an important pathway for fungal virulence that can be targeted at different levels. We investigated the antifungal activity of compounds directly or indirectly targeting the Hsp90-calcineurin axis against different mould species. METHODS: The in vitro antifungal activity of the anticalcineurin drug FK506 (tacrolimus), the Hsp90 inhibitor geldanamycin, the lysine deacetylase inhibitor trichostatin A and the Hsp70 inhibitor pifithrin-μ was assessed by the standard broth dilution method against 62 clinical isolates of Aspergillus spp. and non-Aspergillus moulds (Mucoromycotina, Fusarium spp., Scedosporium spp., Purpureocillium/Paecilomyces spp. and Scopulariopsis spp.) RESULTS: FK506 had variable antifungal activity against different Aspergillus spp. and was particularly active against Mucor spp. Geldanamycin had moderate antifungal activity against Fusarium spp. and Paecilomyces variotii. Importantly, trichostatin A had good activity against the triazole-resistant Aspergillus ustus and the amphotericin B-resistant Aspergillus terreus as well as the MDR Scedosporium prolificans. Moreover, trichostatin A exhibited synergistic interactions with caspofungin against A. ustus and with geldanamycin against Rhizopus spp. for which none of the other agents showed activity. Pifithrin-μ exhibited little antifungal activity. CONCLUSIONS: Targeting the Hsp90-calcineurin axis at different levels resulted in distinct patterns of susceptibility among different fungal species. Lysine deacetylase inhibition may represent a promising novel antifungal strategy against emerging resistant moulds.