| Co-Investigator(Kenkyū-buntansha) |
N.-SHIMADA Junko JUNTENDO UNIV., SCH.MED., ASSIST. PROFESSOR, 医学部, 講師 (20211964)
NARA Takeshi JUNTENDO UNIV., SCH.MED., ASSIST. PROFESSOR, 医学部, 講師 (40276473)
YOSHIDA Ayako JUNTENDO UNIV., SCH.MED., RESEARCHER, 医学部, 助手 (20343486)
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| Budget Amount *help |
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2004: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2003: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Research Abstract |
Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically upregulates cellular FLICE inhibitory protein(c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, post-transcriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T.cruzi- infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T.cruzi post-transcriptionally upregulates and exploits host c-FLIP for the inhibition of d
… More
eath-inducing signal, a mechanism that may allow parasites to persist in host cells. T.cruzi dihydroorotate dehydrogenase(DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T.cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that a euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, possessed the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase(ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E.gracilis, and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Evolutionary implications of the ACT/DHOD gene fusion are discussed. T.cruzi DHOD would therefore be a potentially good target for chemotherapy. Less
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