Distinct hsp70 Domains Mediate Apoptosis-inducing Factor Release and Nuclear Accumulation

Abstract

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-⌬EEVD) or ATPase function (hsp70-⌬ATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-⌬EEVD) or the ATP hydrolytic domain (hsp70-⌬ATPase) failed to prevent mitochondrial AIF release. Although hsp70-⌬EEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-⌬ATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.Disruption of the outer mitochondrial membrane releases toxic proteins, including cytochrome c and apoptosis-inducing factor (AIF) 3 that activate caspase-dependent and -independent pathways responsible for apoptotic cell death. In a prior study in our laboratory, neither a pancaspase inhibitor nor a specific caspase 3 inhibitor completely prevented apoptosis in renal cells exposed to metabolic inhibitors (1), suggesting that at least a portion of the observed apoptosis is mediated by non-caspase-dependent factors including AIF. Nuclear translocation of AIF activates endogenous endonucleases leading to degradation of native DNA into 50-kilobase fragments, peripheral chromatin condensation, and nuclear shrinkage (2-4) that is sufficient to cause apoptosis (5, 6). AIF mediates apoptosis in multiple cell lines after diverse insults including ischemia (7-10), oxidant stress (11), ethanol (12), or p53 exposure (8).AIF is synthesized in the cytosol as a 67-kDa precursor protein that contains a mitochondrial-localizing sequence (13,14). After being imported into mitochondria, the mitochondrial-localizing sequence is cleaved, resulting in the accumulation of the mature, 57-kDa AIF protein (13). Under normal circumstances AIF may facilitate electron transport, since it exhibits robust oxidoreductase activity (14). AIF-mediated cell death is a two-step process that involves initial release from the intramembranous mitochondrial space into the cytosol followed by nuclear uptake and accumulation (5,13,15,16).hsp70, an inducible cytoprotectant protein, antagonizes apoptosis by interfering with multiple ch...

Novel Mouse Model of Monocular Amaurosis Fugax

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Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

“…While we and others have observed that the Hsp72 substrate-binding domain inhibits apoptosis, others have shown that chaperone activity is required to regulate apoptotic signaling (Gotoh et al 2004;Mosser et al 2000;Ruchalski et al 2006). In each of these examples, Hsp72 was expressed under control of the inducible tetracycline promoter, while those studies that reported protection independent of chaperone activity used constitutive Hsp72 expression systems Sun et al 2006;Volloch et al 1999).…”

The BH3-mimetic ABT-737 targets the apoptotic machinery in cholangiocarcinoma cell lines resulting in synergistic interactions with zoledronic acid

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