Helen M. Beere

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Stress management – heat shock protein-70 and the regulation of apoptosis

Apoptosis, molecularly regulated cell death, can be induced by a range of environmental, physical or chemical stresses, and is characterized by a sequence of precisely regulated events that culminate in the self-destruction of a cell. Fascinating biochemical and genetic parallels exist between the cell death pathways of different animal species. However, an even more highly conserved and evolutionarily ancient cellular response can be engaged as a consequence of stress, which functions to maintain cellular survival. This response is mediated by the heat-shock or stress proteins. This article discusses the functional interactions between the stress response and the apoptotic cell death pathway and how these might impact on determining cellular survival.

Characterization of Cytoplasmic Caspase-2 Activation by Induced Proximity

Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.

Apoptosis. Gone but not forgotten.

Cells that have died by apoptosis are taken up by phagocytic cells, and a receptor on the phagocytes that allows them to recognize the apoptotic cells has now been identified. Binding of ligand to the receptor triggers the release of anti-inflammatory cytokines, ensuring that the apoptotic cell has a lasting effect on phagocyte behaviour.

Necroptosis in development, inflammation and disease.

In the early 2000s, receptor-interacting serine/threonine protein kinase 1 (RIPK1), a molecule already recognized as an important regulator of cell survival, inflammation and disease, was attributed an additional function: the regulation of a novel cell death pathway that came to be known as necroptosis. Subsequently, the related kinase RIPK3 and its substrate mixed-lineage kinase domain-like protein (MLKL) were also implicated in the necroptotic pathway, and links between this pathway and apoptosis were established. In this Timeline article, we outline the discoveries that have helped to identify the roles of RIPK1, RIPK3, MLKL and other regulators of necroptosis, and how they interact to determine cell fate.

Bcl-x L does not inhibit the function of Apaf-1

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1–5 μM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9. Cell Death and Differentiation (2000) 7, 402–407

A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc

Cell number is regulated by maintaining a balance between cell proliferation and cell death through apoptosis. Key regulators of this balance include the oncogene product c-Myc, which promotes either entry into the cell cycle or apoptosis [1]. Although the mechanism of c-Myc-induced apoptosis remains unclear, it is susceptible to regulation by survival factors [2,3] and can proceed through the interaction of Fas ligand (FasL) with its receptor, Fas [4]. Activated T lymphocytes are eliminated by an apoptotic process known as activation-induced cell death (AICD), which requires the transcriptional induction of FasL expression [5-7] and sustained levels of c-Myc [8]. The FasL promoter can be driven by c-Myc overexpression, and functional inhibitors of Myc and its binding partner, Max, inhibit the transcriptional activity of the FasL promoter [9,10]. We identified a non-canonical binding site (ATTCTCT) for c-Myc-Max heterodimers in the FasL promoter, which, when mutated, abolished activity in response to c-Myc. Exchange of the canonical c-Myc responsive elements (CACGTG) in the ornithine decarboxylase (ODC) promoter [11] with the non-canonical sequence in the FasL promoter generated an ODC-FasL promoter that was significantly more responsive to c-Myc than the wild-type ODC promoter. Our findings identify a precise physiological role for c-Myc in the induction of apoptosis as a transcriptional regulator of the FasL gene.

Inhibition of Hsp90 via 17-DMAG induces apoptosis in a p53-dependent manner to prevent medulloblastoma

Elevated expression of HSP90 is observed in many tumor types and is associated with a limited clinical response. Targeting HSP90 using inhibitors such as 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin) has shown limited therapeutic success. HSP90 regulates the function of several proteins implicated in tumorigenesis although the precise mechanism through which 17-DMAG regulates tumor cell survival remains unclear. We observed a requirement for p53 in mediating 17-DMAG-induced cell death. The sensitivity of primary mouse embryonic fibroblasts and tumor cells to 17-DMAG-induced apoptosis depended on the p53 status. Wild-type MEFs underwent 17-DMAG-induced caspase-dependent cell death, whilst those lacking p53 failed to do so. Interestingly p53-dependent cell death occurred independently of Atm or Arf. Primary tumor cells derived from two models of murine medulloblastoma (Ptch1+/−;Ink4c−/− and p53FL/FL;Nestin-Cre+; Ink4c−/−) that retain and lack p53 function, respectively, displayed a dependence on functional p53 to engage 17-DMAG-induced apoptosis. Strikingly, 17-DMAG treatment in an allograft model of Ptch1+/−;Ink4c−/− but not p53FL/FL;Nestin-Cre+; Ink4c−/− tumor cells prevented tumor growth in vivo. Our data suggest that p53 status is a likely predictor of the sensitivity of tumors to 17-DMAG.

Fas ligand gene expression is directly regulated by stress-inducible heat shock transcription factor-1.

Heat shock transcription factor-1 (HSF-1) is the primary stress responsive transcription factor that regulates expression of heat shock proteins (Hsps) in response to elevated temperature. We show that the transcriptional activity of HSF-1 can also directly mediate hyperthermia-induced Fas ligand (FasL) expression in activated T cells. We identify a conserved region within the human FasL promoter spanning from –276 to –236 upstream of the translational start site that contains two 15 bp non-identical adjacent HSF-1-binding sites or heat shock elements (HSEs) separated by 11 bp. Both the distal HSE (HSE1) (extending from −276 to –262) and the proximal HSE (HSE2) (spanning from −250 to –236) consist of two perfect and one imperfect nGAAn pentamers. We show the direct binding of HSF-1 to these elements and that mutation of these sites abrogates the ability of HSF-1 to bind and drive promoter activity. HSF-1 associates with these elements in a cooperative manner to mediate optimal promoter activity. We propose that the ability of HSF-1 to mediate stress-inducible expression of FasL extends its classical function as a regulator of Hsps to encompass a function for this transcription factor in the regulation of immune function and homeostasis.

Killers or Clean-Up Crew

Recent characterization of a number of specific mitochondrial events, which may or may not precede the downstream activation of apoptotic caspases, has focused much attention on the absolute requirement for mitochondrial involvement in the irreversible commitment to cell death. Although the mitochondrial release of cytochrome-c appears essential for at least some caspase-mediated events, the mechanism of its release is not clear, particularly regarding a specific role for changes in mitochondrial membrane potential and permeability transition. Mitochondrial-independent activation of caspases via activation of the Fas—FasL pathway is certainly well defined, although a role for mitochondrial-mediated amplification of this cascade supports the concept that mitochondria may ultimately authorize cellular demise. Evidence that mitochondria represent a convergence point for a number of pro- and antiapoptotic signals is presented in the context of their dependence on caspase-mediated events.