The formation of reactive oxygen species (ROS) and the change of the intracellular pH (pH i ) are common phenomena during apoptosis. How they are interconnected, however, is poorly understood. Here we show that numerous anticancer drugs and cytokines such as Fas ligand and tumour necrosis factor a provoke intracellular acidification and cause the formation of mitochondrial ROS. In parallel, we found that the succinate:ubiquinone oxidoreductase (SQR) activity of the mitochondrial respiratory complex II is specifically impaired without affecting the second enzymatic activity of this complex as a succinate dehydrogenase (SDH). Only in this configuration is complex II an apoptosis mediator and generates superoxides for cell death. This is achieved by the pH i decline that leads to the specific dissociation of the SDHA/SDHB subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required for the SQR activity.

Components of the TNFR1 complex are subject to dynamic ubiquitination that impacts on their effects as signalling factors. We have found that the ubiquitin-specific protease USP2a has a pivotal role in the decision for cell death or survival by the TNFR1 complex. This enzyme is a novel component of the TNFR1 complex that is recruited upon ligand binding and controls the signalling activity of the TNFR1-interacting protein RIP1 by removing its K63-linked ubiquitin chains. USP2a similarly de-ubiquitinates TRAF2, a ubiquitin-ligase recruited to the TNFR1 complex. During the TNF response the activity of USP2a on RIP1 and TRAF2 is required for the efficient reappearance of IjBa, which is essential to inactivate the anti-apoptotic transcription factor NF-jB. The effects of USP2a culminate in the conversion of the anti-apoptotic TNFR1 complex I into the pro-apoptotic TNFR1 complex II. Consequently, downregulation of USP2a promotes NF-jB activation and protects cells against TNF-induced cell death. Tumour necrosis factor (TNF) is a pleiotropic cytokine that can, through the TNF receptor 1 (TNFR1), elicit various cellular responses ranging from inflammation, cell proliferation, cell survival to apoptosis. The interaction of this cytokine with TNFR1 results in the recruitment of the adaptor TRADD to its intracellular death domain. RIP kinase 1 (RIP1), the ubiquitin ligase TNF receptor-associated factor 2 (TRAF2), and the inhibitors of apoptosis cIAP1 and cIAP2 then associate in the so-called complex I. 1 Following its recruitment to the complex I, RIP1 is ubiquitinated mainly by K63 ubiquitin chains. 2-5 The E3 ubiquitin ligase TRAF2 was the first to be described as being responsible for K63 ubiquitination of RIP1 5-7 together with the lipid shingosine-1-phosphate. 8 Other publications have shown that cIAP1/2 can attach K63 ubiquitin chains to RIP1 [9][10][11][12] or that TRAF2 and cIAPs cooperate for the ubiquitination of RIP1 6,13,14 . These ubiquitin chains bind and associate the TAB/TAK (TAK1/TAB2/TAB3) and NEMO/IKK (IKKa, b, g) kinases complex. 3,4,12,15 By proximity, TAK1 activates IKKb by phosphorylation, which in turn phosphorylates IkBa to promote its K48-linked polyubiquitination and degradation by the proteasome. The antiapoptotic transcription factor NF-kB is then free to translocate to the nucleus and activate gene transcription leading to survival of the cells. 7,[15][16][17][18][19][20] Upon internalisation of the complex and in the absence of K63 ubiquitination, RIP1 is able to form the so-called complex II with FADD and pro-caspase-8 to initiate apoptosis. 1,16 Hence, ubiquitin conjugation has a crucial role in allowing cells to decide between cell death and survival in the TNF signalling cascade. Although a number of ubiquitin ligases, that presumably modify components of the TNFR1 complex, have been identified, so far only two de-ubiquitinating proteases have functionally been found in the TNF signalling pathway that impact on cell death, A20 and CYLD (cylindromatosis tumour suppressor). 21-24 A20 is a ...

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