The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release.

The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.

Involvement of microphthalmia in the inhibition of melanocyte lineage differentiation and of melanogenesis by agouti signal protein.

In mouse follicular melanocytes, production of eumelanins (brown-black pigments) and pheomelanins (yellow-brownish pigments) is under the control of two intercellular signaling molecules that exert opposite actions, alpha-melanocyte-stimulating hormone (alphaMSH) which preferentially increases the synthesis of eumelanins, and agouti signal protein (ASP) whose expression favors the production of hair containing pheomelanins. In this study, we report that ASP does not only affect mature melanocytes but can also inhibit the differentiation of melanoblasts. We show that both alphaMSH and forskolin promote the differentiation of murine melanoblasts into mature melanocytes and that ASP inhibits this process. We present evidence that the expression of a specific melanogenic transcription factor, microphthalmia, and its binding to an M box regulatory element, is inhibited by ASP. We also show that, in B16 murine melanoma cells, ASP inhibits alphaMSH-stimulated expression of tyrosinase, tyrosine-related proteins 1 and 2 through an inhibition of the transcription activity of their respective promoters. Further, ASP inhibits alphaMSH-induced expression of the microphthalmia gene and reduces the level of microphthalmia in the cells. Our data demonstrate that ASP can regulate both melanoblast differentiation and melanogenesis, pointing out the key role of microphthalmia in the control of these processes.