Hsp27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c

Abstract

Mitochondrial cytochrome c release in response to pro-apoptotic signals leads to the formation of a cytochrome c/Apaf-1/procaspase-9 complex (the apoptosome) and resultant activation of caspase-9 and caspase-3. Here we demonstrate that the molecular chaperone, Hsp27, inhibits this cytochrome c-mediated activation of caspase-3. Immunodepeletion of Hsp27 from cytochrome c-activated cytosols resulted in decreased caspase activity. Furthermore, immunoprecipitation of Hsp27 resulted in the coprecipitation of both cytochrome c and procaspase-3. In reciprocal experiments, immunoprecipitation of both procaspase-3 and cytochrome c resulted in coprecipitation of Hsp27, indicating two independent interactions. These results point to Hsp27 mediating its inhibition of procaspase-3 activation through its ability to sequester both cytochrome c and procaspase-3, and thus prevent the correct formation/function of the apoptosome complex.

Figure 1

Induction of Hsp27 is associated with inhibition of cytochrome c-mediated activation of caspase-3. (A) Cells were maintained at 37°C or heat shocked at 42°C to induce Hsp27. After recovery cells were either further heat shocked at 44°C to induce apoptosis (T) or retained as controls (C). Equal amounts of protein were subjected to SDS-PAGE followed by Western blot analysis with specific antibodies against Hsp27 and PARP. (B) Cytosols prepared from control and thermotolerant cells were measured for DEVDase activity after the addition of cytochrome c/dATP.

Figure 2

Hsp27 interferes with the formation of the apoptosome complex. (A) Western blot analysis of Hsp27 in thermotolerant and cells transiently transfected with Hsp27 gene. (B) Cytosols from thermotolerant and transfected cells were assayed for cleavage of DEVD-AMC after the addition of cytochrome c/dATP. (C) Jurkat-neo or Jurkat-Hsp27 cell cytosols were immunodepleted of Hsp27 prior to the addition of cytochrome c/dATP (left panel) or after the addition of cytochrome c/dATP (right panel). The immunodepleted extracts were assayed for cleavage of the fluorogenic substrate DEVD-AMC.

Figure 3

Hsp27 associates with both procaspase-3 and cytochrome c. (A) Jurkat-Hsp27 cell lysates were immunoprecipitated with anti-Hsp27, anti-cytochrome c, anti-caspase-3 (CPP32), or anti caspase-9, followed by Western blotting using antibody to Hsp27. (B) Western blot analysis using antibodies against caspase-3 (pl7) and cytochrome c, of proteins immunoprecipitated from cell lysate with anti-Hsp27, anti-cytochrome c, or anti-caspase-3.