We identified Caspase‐8 as a new substrate for Src kinase. Phosphorylation occurs on Tyr380, situated in the linker region between the large and the small subunits of human Procaspase‐8, and results in downregulation of Caspase‐8 proapoptotic function. Src activation triggers Caspase‐8 phosphorylation on Tyr380 and impairs Fas‐induced apoptosis. Accordingly, Src failed to protect Caspase‐8‐defective human cells in which a Caspase‐8‐Y380F mutant is expressed from Fas‐induced cell death. Remarkably, Src activation upon EGF‐receptor stimulation triggers endogenous Caspase‐8 phosphorylation and prevents Fas‐induced apoptosis. Tyr380 is phosphorylated also in human colon cancers where Src is aberrantly activated. These data provide the first evidence for a direct role of tyrosine phosphorylation in the control of caspases and reveal a new mechanism through which tyrosine kinases inhibit apoptosis and participate in tumor progression.

Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death‐inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of ΔΨ _m and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c , apoptosis inducing factor, and caspase 9. Soluble factors released from GD3‐treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide‐induced ΔΨ _m dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo . Finally, enforced expression of bcl‐2 significantly prevents GD3‐induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl‐2 control.—Rippo, M. R., Malisan, F., Ravagnan, L., Tomassini, B., Condo, I., Costantini, P., Susin, S. A., Rufini, A., Todaro, M., Kroemer, G., Testi, R. GD3 ganglioside directly targets mitochondria in a bcl‐2‐controlled fashion. FASEB J. 14, 2047–2054 (2000)

The defective expression of frataxin causes the hereditary neurodegenerative disorder Friedreich's ataxia (FRDA). Human frataxin is synthesized as a 210 amino acid precursor protein, which needs proteolytic processing into mitochondria to be converted into the functional mature form. In vitro processing of human frataxin was previously described to yield a 155 amino acid mature form, corresponding to residues 56–210 (frataxin ^56–210 ). Here, we studied the maturation of frataxin by in vivo overexpression in human cells. Our data show that the main form of mature frataxin is generated by a proteolytic cleavage between Lys80 and Ser81, yielding a 130 amino acid protein (frataxin ^81–210 ). This maturation product corresponds to the endogenous frataxin detected in human heart, peripheral blood lymphocytes or dermal fibroblasts. Moreover, we demonstrate that frataxin ^81–210 is biologically functional, as it rescues aconitase defects in frataxin-deficient cells derived from FRDA patients. Importantly, our data indicate that frataxin ^56–210 can be produced in vivo when the primary 80–81 maturation site is unavailable, suggesting the existence of proteolytic mechanisms that can actively control the size of the mature product, with possible functional implications.

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.

Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich's ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly other human mitochondria-associated disorders.