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

Abstract

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-jB activation. PIDDosome activation is dependent on autoprocessing 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-jB 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.

The PIDDosome, DNA-damage-induced apoptosis and beyond

“…At protein level, western blot analysis of endogenous PIDD suggests that the different PIDD isoforms are expressed at quite equivalent levels, especially when one considers the PIDD-C fragment. 6,13,56 Upon DNA damage, only mRNA for isoform 3 is induced, while the two other isoforms are unaffected, as this isoform is unable to trigger apoptosis, this points again towards an important role for alternative PIDD functions upon DNA damage. 56 Recently, a fourth isoform (PIDD4) has been described that lacks the LRR domain.…”

“…Heat shock and DNA damage seem to be the best characterized PIDDosome stimuli, 4,9,[11][12][13] although there is still some debate as to whether heat shock really activates caspase-2.…”

“…However, although Hsp90 is required for PIDD autocleavage, stability and function, it is not a part of the final signaling complex. 13 PIDD and caspase-2 have been shown to be activated by heat shock, 9,11 and therefore it is possible that Hsp90 could actually function as a heat sensor. Many LRR-containing proteins, such as NLRs, require Hsp90 for their function and it has been suggested that chaperones support the less stable, but functionally advantageous LRRs in their client proteins.…”

“…In addition to its strictly chaperone function, Hsp90 plays a crucial role in PIDD activation and function. 9,13 Activation of PIDD is associated with Hsp90 release, resulting in signaling-competent PIDDosomes (such as nuclear PIDD) that no longer interact with Hsp90. Interestingly, Hsp90 interaction with PIDD is required for PIDD activation as measured by caspase-2 and NF-kB activation.…”

“…A search for such regulatory proteins led to the identification of heat-shock protein 90 (Hsp90) as a binding partner for PIDD. 13 In unstimulated cells, the Hsp90, as well as its co-chaperone p23, is bound to PIDD, and is essential for its stability, and optimal autoprocessing capacity by supporting its conformation (Figure 2). In addition to its strictly chaperone function, Hsp90 plays a crucial role in PIDD activation and function.…”