Intra-mitochondrial degradation of Tim23 curtails the survival of cells rescued from apoptosis by caspase inhibitors

Abstract

Caspase inhibition can extend the survival of cells undergoing apoptosis beyond the point of mitochondrial outer membrane permeabilisation (MOMP), but this does not confer long-term protection because caspase-independent death pathways emerge. Here, we describe a novel mechanism of mitochondrial self-destruction in caspase-inhibited cells, whose hallmark is the degradation of Tim23, the essential pore-forming component of the TIM23 inner membrane translocase. We show that Tim23 degradation occurs in cycling and post-mitotic cells, it is caspase-independent but Bax/Bak dependent, and it follows cytochrome c release. The proteolytic degradation of Tim23 is induced by MOMP and is mitochondrion-autonomous, as it also occurs in isolated mitochondria undergoing permeability transition. Degradation of Tim23 is selective, as expression of several other inner membrane proteins that regulate respiratory chain function is unaffected, and is not autophagic, as it occurs similarly in autophagy-proficient and -deficient (Atg-5 knockout) cells. Depleting Tim23 with siRNA is sufficient to inhibit cell proliferation and prevent long-term survival, while expression of degradation-resistant Tim23-GFP in mitochondria delays caspaseindependent cell death. Thus, mitochondrial autodigestion of Tim23 joins the array of processes contributing to caspaseindependent cell death. Because mitochondrial biogenesis requires a functional protein-import machinery, preventing Tim23 degradation might, therefore, be essential for repairing damaged mitochondria in chronic degenerative diseases. Apart from providing cellular ATP, mitochondria are also integral components of proapoptotic signalling. During apoptosis, mitochondrial outer membrane permeabilisation (MOMP) causes release of apoptogenic factors such as cytochrome c from the intermembrane space (IMS) to the cytoplasm. Cytochrome c-mediated activation of caspases via Apaf-1 is a dominant regulator of apoptosis.2 Although pan-specific caspase inhibitors can extend survival considerably depending on the cell type and death stimulus, caspaseindependent mechanisms of death execution take over in the long term. This has been ascribed to the action of other mitochondrial apoptogenic factors, such as AIF or EndoG.2 In addition, autophagy, which is co-activated by many apoptotic stimuli, has pro-death activity when apoptosis execution is impaired. 3,4 Exploiting mechanisms that suppress mitochondrial dysfunction and its deleterious follow-on effects is an obvious starting point for the development of treatments against loss of irreplaceable cells during neurodegenerative diseases, stroke, or myocardial infarction. However, therapeutic strategies should also address ways to mend defective mitochondria, thereby hopefully achieving long-term protection. Therefore, it is important to assess whether damaged mitochondria retain a functional import machinery so that the supply of cytoplasmic proteins is maintained.Here we focus on Tim23, the eponymous constituent of the TIM23 complex that forms the crucia...

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“…Exactly why these complexes lose function is unclear but it might be due to degradation of one or several subunits of these multi-protein complexes. Post-mitochondrial permeabilisationdependent cleavage of TIM23, a crucial subunit of the mitochondrial inner membrane translocase complex, and degradation of cytochrome c also contributes to breakdown of mitochondrial function (Goemans et al, 2008;Ferraro et al, 2008) (Fig. 4).…”