線蟲嘧啶合成酶在計畫性細胞死亡中扮演的角色

Abstract

計畫性細胞死亡（Programmed cell death）是演化上高度保留的一個機制，在動物生長發育的過程中扮演了十分重要的角色。線蟲已被證明是用來研究計畫性細胞死亡很好的模型。過去研究已知線蟲計畫性細胞死亡中的執行步驟主要由egl-1、ced-9、ced-4及ced-3四個基因所調控，其中ced-3此蛋白質分解脢的活化更是直接造成細胞的死亡。然而，究竟ced-3是透過分解、截切哪些蛋白質來促使細胞凋亡至今仍不清楚。為了找出參與在計畫性細胞死亡的其他基因，我們利用遺傳篩選的方式篩選，並篩選出tp12突變株。tp12在胚胎發育過程中細胞屍體數相較於野生株少。tp12突變於基因pyr-1，pyr-1與人類的CAD (carbamoyl phosphate synthetase、aspartate transcarbamylase與dihydroorotase)是同源基因。CAD是一個三官能的蛋白質，含有3種酵素活性並參與合成嘧啶的速率決定步驟。在顯微鏡拍攝觀察突變株胚胎發育過程的實驗中，我們發現pyr-1(tp12)突變株在一個敏感的突變條件下(grp-1 mutant background)，有些特定細胞會不正常存活。這些不正常存活的細胞分別是，分泌細胞(excretory cell)的姨細胞(aunt cell)及表皮細胞(hyp8/9 cells)的姨細胞。我們也發現，pyr-1(tp12)突變株在grp-1這個敏感的突變條件下有多餘的分泌細胞和表皮細胞。這些結果暗示著不正常存活的姨細胞繼續分裂並分化成分泌細胞和表皮細胞，顯示pyr-1參與在分泌細胞及表皮細胞的姨細胞之凋亡中。另外，過去研究指出，老鼠的CAD是caspase的受質，於是我們好奇PYR-1是否也為CED-3的受質。在CED-3的截切實驗中，我們證實PYR-1能被CED-3截切，為CED-3的受質，並且發現CED-3的切位坐落於PYR-1的DHO 區域(domain)。透過CASVM切位預測軟體，我們找出兩個可能的CED-3切位：DLPD及EYID。綜合所有結果，我們假設PYR-1在被CED-3截切後產生能夠促進細胞死亡的功能，並且只影響到特定細胞如分泌細胞和表皮細胞之姨細胞的死亡。

Programmed cell death (PCD) is a conserved cellular process, which is important for animal development. C.elegans is one of the most commonly-used model organisms in the studies of PCD. Extensive studies in C.elegans show that four key genes, egl-1, ced-9, ced-4 and ced-3 control the execution of PCD, and the activation of caspase CED-3 leads to the demise of the cell. However, only few substrates of CED-3 have been identified thus far. To identify more genes that participate in PCD, we undertook a genetic screen and isolated the tp12 mutation. The tp12 mutant has reduced numbers of embryonic cell corpses as compared to those of wildtype. A genetic complementation test reveals that tp12 is an allele of pyr-1. PYR-1 is a homolog of the mammalian CAD, which stands for carbamoyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, and is known to control the rate-limiting step during pyrimidine biosynthesis. Using 4-dimentional microscopy analysis, we found that the pyr-1(tp12) mutant, in a sensitized background, shows frequent loss of specific cell corpses, the aunt cells of the hyp8/9 hypodermal cells and the excretory cell. Consistently, an extra hyp8/9 nucleus and excretory cell are observed in the pyr-1(tp12) mutant in a sensitized background. These results suggest that pyr-1 is involved in the death of the aunt cells of the hyp8/9 cells and the excretory cell. Furthermore, previous research showed that the PYR-1 homolog mammalian protein, CAD is a substrate of caspase 3, therefore we wonder whether PYR-1 is also a CED-3 substrate. In a CED-3 cleavage assay, we found that PYR-1 can be cleaved by CED-3, which indicated that PYR-1 is a CED-3 substrate. Moreover, the cleavage sites were shown to locate in the DHO domain of PYR-1. By using the CASVM(Server for SVM Prediction of Caspase Substrates Cleavage Sites), two potential CED-3 cleavage sites: DLPD and EYID were found. Together, we hypothesize that PYR-1 gains a proapoptotic function after being cleaved by CED-3, and functions in the programmed cell death of specific cells such as the aunt cell of excretory cell and hyp8/9 cells.