減弱果蠅神經系統中酪氨酸亞硫酸基轉移酶基因表現量對蛋白質亞硫酸化功能之探討

Abstract

蛋白質的亞硫酸化是很重要的蛋白質後修飾之一，而酪氨酸亞硫酸基轉移酶(tyrosylprotein sulfotransferase)負責將腺苷3’-磷酸-5'-磷醯硫酸 (PAPS)的亞硫酸基轉移到許多特定分泌性或膜上蛋白質的酪氨酸上。目前發現許多帶有亞硫酸化酪氨酸的蛋白質參與了不同的生理過程，其中包含凝血反應、白血球貼附血管細胞、趨化素的訊息傳遞、以及人類免疫缺陷病毒(HIV)感染細胞。至今對於亞硫酸化蛋白質體以及酪氨酸亞硫酸機轉移酶本身所調控的生物功能都仍然不清楚。根據基因序列以及基因微陣列(gene microarray)的分析發現在果蠅體內僅存在單一個酪氨酸亞硫酸基轉移酶基因，並且此基因的訊息核醣核酸(mRNA)在果蠅頭部及腦部有很高的表現量。利用了GAL4-UAS系統將果蠅神經系統中的酪氨酸亞硫酸基轉移酶進行基因表現減弱(knockdown)。由二維蛋白質電泳以及數位影像分析可以發現有四個蛋白質受到正調控(up-regulation)，而有十八個蛋白質受到負調控(down-regulation)。利用質譜儀針對這二十二個蛋白質做身分鑑定僅鑑別出九個蛋白質，而他們大多與代謝以及氧化壓力(oxidative stress)有關。銅鋅超氧化歧化酶(superoxide dismutase [Cu-Zn])是一種抗氧化的酵素，其主要負責清除體內的自由基，而此酵素在神經系統中酪氨酸亞硫酸機轉移酶進行基因減弱的果蠅中有顯著的負調控。利用巴拉刈(paraquat)進行氧化耐受度分析實驗，發現這些基因減弱的果蠅擁有顯著較長的生存率。這些結果有助於進一步了解蛋白質上酪氨酸的亞硫酸化在神經系統中所扮演的角色以及可能所參與的生理調控機制。

Protein tyrosine sulfation, catalyzed by tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications. TPST transfers the moiety of sulfuryl group from adenosine 3’-phosphate 5’-phosphosulfate (PAPS), to the hydroxyl group of specific tyrosine residues of various secreted and membrane-bound proteins overspread the eukaryotes. Tyrosine-sulfated proteins are known to mediate many physiological processes including coagulation, leukocyte adhesion, chemokine signaling, and HIV infection. At present, the sulfoproteomics and TPST-regulated biological functions remain largely unknown. A single TPST gene whose mRNA was highly expressed in the head and brain of Drosophila melanogaster was identified following the analyses of genomic sequences and gene microarray. TPST gene was knockdown specifically in the nervous system of D. melanogaster using GAL4-UAS system. 2-D electrophoresis and digital image analysis indicated that 4 proteins were up-regulated and 18 proteins were down-regulated. Among them, 9 proteins were identified by MS spectrum and most of them were involved in the metabolism and oxidative stress. Superoxide dismutase [Cu-Zn], an antioxidant enzyme against reactive oxygen species, was mainly down-regulated in the neuron-specific TPST knockdown flies. Oxidative stress assay was examined with paraquat treatment, and surprisingly the result showed the knockdown flies had remarkably longer survival time (1.32-fold). These results are important to comprehend the biological roles and regulatory actions of protein tyrosine sulfation in the nervous system.