嘧啶合成酶促進線蟲的計畫性細胞死亡

Abstract

計畫性細胞凋亡是一個受到高度調控且演化保留的機制，它在平衡細胞數量、維持細胞品質以及發育過程中扮演著重要的角色。在擁有固定細胞譜系的秀麗隱桿線蟲中，131顆細胞會在發育過程中以計畫性細胞凋亡的方式被移除，而這些細胞是藉由不對稱分裂所產生的，其中比較大的細胞會繼續進行分裂及分化，然而比較小的細胞會進行計畫性細胞凋亡。實驗室發現在計畫性細胞凋亡和不對稱分裂同時受損的線蟲中，尾巴會產生多餘的表皮細胞hyp8/9而形成球狀或桿狀突起的表現型，稱之為“tail defect”。利用此表現型，實驗室在線蟲具有不對稱分裂受損的情況下進行正向基因篩選 (forward genetic screen)以找尋調控計畫性凋亡的新基因，其中篩選出pyr-1突變株。pyr-1 基因與人類的cad (carbamoyl phosphate synthetase、aspartate transcarbamylase and dihydroorotase)是同源基因，其為一個嘧啶合成酶，負責催化嘧啶全程合成路徑(de novo synthesis pathway)中的前三個步驟。實驗室先前發現PYR-1具有促使細胞凋亡的功能，也證明了PYR-1作用於CED-3的下游或者是平行於CED-3去促進細胞凋亡。在此研究中，我證明了CED-3會截切PYR-1，並找到其切位為PYR-1的第1664個天門冬氨酸。然而，我也發現此截切對於PYR-1促進細胞凋亡的功能是不必要的。藉由補充PYR-1參與步驟的最終產物，L-二氫乳清酸(L-dihydroorotate)，我發現它可以回復pyr-1及grp-1雙重突變株的tail defect表現型。我因此更進一步地利用RNA干擾的方式來抑制在嘧啶全程合成路徑中接續PYR-1的酵素dhod-1，並發現此抑制也可以在grp-1突變株造成tail defect表現型。綜上所述，我的研究指出PYR-1及DHOD-1的酵素活性對於促進細胞凋亡扮演著重要的角色，並提供了一個新的計畫性細胞凋亡調控機制。

Programmed cell death (PCD) is a highly regulated and conserved process that balances the cell number, maintains cell quality and participates in morphogenesis during development in multicellular organisms. In Caenorhabditis elegans (C. elegans), a free-living roundworm with an invariant cell lineage, 131 cells are programmed to die by apoptosis during development and these cells are generated by asymmetric cell divisions (ACDs). Impairment of both PCD and ACD results in extra differentiated cells in specific cell lineages, such as hyp8/9 hypodermal cells. Extra hyp8/9 hypodermal cells cause a ball or rod-like tail morphology, which is called “tail defect”. A forward genetic screen was then conducted in the grp-1(gm350) mutant, which presents defective ACD, to identify new PCD genes. pyr-1, one of isolated mutations, is the C. elegans homolog of cad (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) and is a pyrimidine synthetase which catalyzes the first three steps of the de novo pyrimidine synthesis pathway. Previous study shows that PYR-1 acts downstream of or in parallel to CED-3 to promote programmed cell death. In this study, I demonstrate that CED-3 cleaves PYR-1 at 1664th aspartate in vitro and in vivo. However, substitution of 1664th aspartate for alanine does not abolish the pro-apoptotic function of pyr-1, showing that the CED-3-mediated cleavage is not required for its cell death-promoting activity. By supplement of the final product of PYR-1, L-dihydroorotate, I find that L-dihydroorotate can rescue the tail defect of pyr-1 mutant in the grp-1 mutant. Moreover, knockdown of dhod-1, which catalyzes the next enzymatic step downstream of pyr-1, can phenocopy the PCD defect of pyr-1 to cause the tail defect in the grp-1(gm350) mutant. My study reveals that the enzymatic activity of PYR-1 and DHOD-1 in the pyrimidine de novo synthesis pathway is important for PCD and provides a novel mechanism for the regulation of PCD.