嘧啶合成酶在線蟲計畫性細胞死亡的嶄新功能

Abstract

計畫性細胞死亡（Programmed cell death）是演化上高度保留的一個機制，此機制對於動物生長發育是非常重要的。線蟲已被證明是用來研究計畫性細胞死亡很好的模型。儘管線蟲計畫性細胞死亡已有廣泛的研究，有幾個重要問題仍然懸而未決。例如：細胞死亡的時間是如何被調控的？ CED-3/Caspase-3促進細胞死亡的受質和調控因子是什麼？為了找出參與在計畫性細胞死亡的新基因，之前實驗室的成員利用grp-1突變株來進行遺傳篩選；發現當grp-1與執行或調控細胞死亡時間的基因雙重突變時，可導致高外顯率的tail defect，而各自的單一突變株則不會看到。我從此次遺傳篩選分離出tp12突變株；實驗觀察發現，tp12突變株會造成tail defect與在胚胎發育過程中細胞屍體數的減少。我利用單核苷酸多態性(single nucleotide polymorphism)與基因體缺失，將tp12定位在第二條染色體0.83∼1.02 cM的區間。接著，我利用在此區間內的染色體DNA進行germ-line transformation並結合次世代定序的結果顯示，tp12突變於基因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。實驗結果發現，提供額外嘧啶於培養基中可以挽救pyr-1突變株胚胎的死亡率，但無法挽救減少的細胞屍體或tail defect。這一結果表明了，嘧啶的缺乏，是造成胚胎的致命性的原因，而非影響細胞凋亡的缺陷的因素。有趣的是，最近的研究表明，哺乳動物的CAD是Caspase-3的受質，並且可與擁有caspase recruitment domain (CARD)的蛋白質相互作用。因此，PYR-1可能是CED-3/caspase的受質，或是與線蟲唯二擁有CARD 的蛋白質CED-3和CED-4有相互作用。總和來說，我的數據表明，PYR-1在計畫性細胞死亡中扮演著非常重要的角色，且這個角色與嘧啶的合成毫無關聯。

Programmed cell death (PCD) is an evolutionarily conserved process important for animal development. C. elegans has been established as an excellent model for studying PCD. Despite the extensive work in C. elegans PCD, several important questions remained unanswered. For example, how the timing of cell death is regulated and what are the CED-3/Caspase-3 substrates and regulators that promote cell death. To identify new genes involved in PCD, former members in the lab performed a forward genetic screen in a sensitized grp-1 mutant background. Double mutations of grp-1 and a gene in the execution or timing control of the cell-death process can cause high penetrance of a tail defect, whereas the respective single mutations do not. The tp12 mutant was isolated from the screen. I found that tp12 mutants have tail defects and reduced cell corpses during embryogenesis. My genetic mapping data using single nucleotide polymorphisms and deletions positioned tp12 in a 0.83~1.02 cM interval on chromosome II. I then cloned tp12 by germ-line transformation using genomic DNA in this interval, I found that genomic fragments containing pyr-1 can rescue the defects in tail morphology and enhance the number of cell corpses. Further sequencing analyses conclude that tp12 is an allele of pyr-1, the C. elegans homolog of CAD. CAD stands for carbamoyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase. CAD is a trifunctional protein containing three enzyme activities and controls the rate-limiting step for pyrimidine biosynthesis. I found that supplying additional pyrimidine to the medium could rescue the embryo lethality, but not the reduced cell corpses or extra surviving cells, of the tp12 mutants. These results show that embryo lethality is caused by pyrimidine deficient in the mutants whereas the cell death defect is not. Interestingly, recent studies showed that mammalian CAD is a substrate of Caspase-3 and can interact with proteins containing the caspase recruitment domain (CARD). Therefore, PYR-1 may be a substrate of CED-3/caspase or interacts with CED-3 and CED-4, the only two CARD-domain containing proteins in C. elegans. Together, our data shows that PYR-1 plays a crucial role in PCD and it is independent of pyrimidine biosynthesis.