Caenorhabditis elegans ArsA同源基因的調控與其功能之探討

Abstract

砷是一種毒性物質且是已知的人類致癌物質。在原核生物中，砷的解毒機制有較詳盡的研究，當類金屬進入細胞時，ars operon中的ArsA蛋白受到激發且水解ATP，使三價砷和三價銻化合物轉移到ArsB蛋白運送出細胞。其中，人類中ArsA同源體也被證實具有水解ATP的活性，然而卻只受到三價砷的激發，而酵母菌中的ArsA同源體則是不受任何類金屬的激發。因此，探討Caenorhabditis elegans（C. elegans）中ArsA同源體的功能，能更加了解C. elegans中砷的解毒途徑和獲得演化上間接的證據。 本研究利用生物資訊方法預測出C. elegans中具有兩個ArsA蛋白的同源體，我們發現藉由RNA干擾技術抑制Ce-ArsA蛋白的產生，將會造成C. elegans對三價砷和三價銻的抵抗性降低，顯示Ce-ArsA蛋白扮演了重要抵抗類金屬的角色。此外，Ce-arsA-1上游基因的分析和轉殖C. elegans的實驗，顯示當C. elegans處於不同壓力的情況時，Ce-ArsA-1蛋白的表達量也會相對明顯地增加，推測這些壓力因子活化了Ce-arsA-1的轉錄機制。 本研究利用原核生物系統大量表達和純化Ce-ArsA-1蛋白，並利用呈色法檢測純化過的Ce-ArsA-1蛋白。結果顯示Ce-ArsA-1蛋白的確具有ATPase活性，並且可被三價砷和三價銻分別激發2.2和4.8倍，此實驗結果與原核中ArsA蛋白功能較為相似，並且由研究結果證實C. elegans中Ce-ArsA-1蛋白與原核中ArsA蛋白皆屬於相同類型的ATP水解蛋白。

Arsenic is a potent toxin and carcinogen. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid operon-encoded efflux systems. Two Caenorhabditis elegans genes, Ce-arsA-1 and Ce-arsA-2, that are homologous to the Escherichia coli (E. coli) arsA genes were identified and characterized in this study. The level of Ce-arsA-1 transcription was significantly affected by heat, metalloids and heavy metal exposure. When Ce-arsA-1 expression was inhibited using RNA interference (RNAi), lower resistance of arsenite and antimonite were observed. This suggests that Ce-ArsA-1 is required for arsenite and antimonite tolerance in C. elegans. We expressed and purified Ce-ArsA-1 protein, a nematode homologue of arsA encoding the ATPase component of E. coli arsenite/antimonite transporter. Purified MBP-Ce-ArsA-1 fusion protein was biochemically characterized, and its properties were compared with those of E. coli ArsA. The MBP-Ce-ArsA-1 exhibited a basal level of ATPase activity in the absence of arsenite or antimonite. Antimonite induced a 4.8 fold stimulation of ATPase activity, which was related to an increase in Vmax; arsenite induced 2.2 fold stimulation of ATPase activity. The results indicate that Ce-ArsA-1 is a distinct arsenite and antimonite-stimulated ATPase belonging to the same superfamily of ATPases represented by the E. coli ArsA protein.