Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.

p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.Tumors can be distinguished from their nonmalignant counterparts by specific molecular signatures expressed in malignant cells and tumor vasculature. We explore such differences by identifying tumor-homing peptides from phage libraries that we screen in vivo (60). We recently showed (19) that the cellular receptor for one of our tumor-homing peptides is a protein variously known as p32, p33, gC1q receptor (gC1qR), or hyaluronic acid binding protein 1 (HABP1). This protein was originally isolated based on its copurification with the nuclear splicing factor SF-2 (37). However, it was subsequently shown to bind also the globular heads of complement component C1q (23), hyaluronic acid (10), and numerous other extracellular and intracellular proteins (24,28,33,42). Most recently it has been shown that p32 interacts with the long and short forms of the tumor suppressor ARF (30,56,57). Despite the numerous reports on p32 interaction partners, the role of these binding activities in the physiological function of the protein is unknown, and some investigators have proposed that p32 may be a chaperone protein (58,65).The p32 protein is primarily localized in the mitochondrial matrix (12, 46, 48) but has also been reported to be present in other subcellular locations (53). Some of the p32 protein can be at the cell surface, a location that appears to be specific for tumors (19). In this regard, p32 is similar to some other intracellular proteins that are also partially localized at the cell surface in tumor cells (8,49). In addition to the partial cell surface localization of p32, many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues (7, 19, 52, 59). Moreover, p32 is differentially expressed during the progression of epidermal carcinoma, accumulating in metastatic islands (25).We set out to modulate p32 expression in tumor cells to gain information on the role of this protein in cancer. We show here that p32 knockdown cells shift their metabolism from oxidative phosph...

a-Tocopheryl succinate (a-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of a-TOS has not been identified. Here, we show that a-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q P and Q D , respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of a-TOS compared to that of UbQ for the Q P and Q D sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of a-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to a-TOS. We propose that a-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

These results suggest that mitochondrial chaperonins HSP60 and HSP10 in combination or individually play an important role in maintaining mitochondrial integrity and capacity for ATP generation, which are the crucial factors in determining survival of cardiac myocytes undergoing ischemia/reperfusion injury.