哺乳類動物細胞中SUMO結合受質之蛋白質體學研究

The Proteomics Study of the SUMO-conjugated Substrates in Mammalian Cells

劉邦宇(Pang-Yu Liu)

指導教授 ： 張世宗

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10691

摘要

SUMO (samll ubiquitin-like modifier) 由於與泛素具有相似結構與類似的酵素作用系統而得名。SUMO化 (SUMOylation) 是由E1活化酶、E2銜接酶和E3黏合酶三種酵素依序進行活化、銜接與黏合的步驟，以有效調控生物體內之蛋白質的SUMO化修飾作用。相較於絕大多數經泛素修飾的蛋白質所導向的蛋白酶體降解，SUMO化在細胞中已被發現有更多樣且範圍廣泛的生理功能，如訊息傳遞、蛋白質穩定性、酵素活性調控，以及細胞內分子運送等，因而使其相關的研究受到高度的重視。
本論文利用蛋白質體學技術分析細胞內尚未被發現之SUMO化受質，同時並結合金屬逆境之研究，探討逆境下是否會誘導細胞內SUMO化修飾作用的改變。實驗結果顯示，砷逆境會誘導COS-7細胞內SUMO之耦合蛋白質明顯的上升；而鋅與鎘處理細胞後則沒有觀察到SUMO化有提升的現象。此外，經質譜儀鑑定結果發現19S蛋白解體次單元Rpt3與Rpt5可能為SUMO之受質。利用真核細胞HeLa或大腸桿菌的SUMO化系統，進一步證實了Rpt3會受SUMO-1修飾，而Rpt5則會受SUMO-1與SUMO-2共同修飾。本研究發現蛋白解體可能直接受SUMO化修飾而調控，顯示SUMO與泛素-蛋白解體兩個系統間有更為複雜的交互作用機制，這將是未來值得深入研究的方向之一。

關鍵字

SUMO化修飾作用 ； 蛋白質體學 ； 金屬逆境 ； 蛋白解體

並列摘要

SUMO (small ubiquitin-like modifier) has similar molecular structure and undergoes similar conjugation mechanism with ubiquitn. SUMOylation is a sequential catalytic enzyme cascase, which includes E1 activation, E2 conjugation and E3 ligation, for effectively regulating SUMOylated proteins in vivo. In contrast to the fact that most ubiquitinated proteins undergo proteasomal degradation, it has been shown that SUMOylated proteins are involved in a wide range of physiological functions, including signal transduction, protein stability, enzyme activity and localization. Due to this interesing difference, more and more research has been done on sumoylation.
In this study, the proteomics technique has been applied to analyze the unidentified SUMOlyated substrates in mammalian cells. To understand that whether the heavy metal stress can change the level of SUMOylated protein, cells were treated with As, Zn and Cd under various conditions. The results demonstrate that arsenic stress induces SUMO-2 sumoylation in COS-7 cells, however, this phenomemon is missing in Zn and Cd treatment . The data of mass spectrometry analysis showed that Rpt3 and Rpt5, whitch are both of proteasomal 19S subunits, are possibly SUMO SUMOylated. Furthermore, I found that Rpt3 undergoes SUMO-1 SUMOylation and Rpt5 undergoes SUMO-1 and SUMO-2 co-SUMOylation by using gene transfection in HeLa cells and E. coli sumoylation system.

並列關鍵字

SUMO ； Proteomics ； Heavy metal stress ； Proteasome ； Rpt

參考文獻

Aposhian HV, Zakharyan RA, Avram MD, Sampayo-Reyes A, Wollenberg ML (2004) A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species. Toxicol Appl Pharmacol 198: 327-335

Bae DS, Gennings C, Carter WH, Jr., Yang RS, Campain JA (2001) Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes. Toxicol Sci 63: 132-142

Bardag-Gorce F, Venkatesh R, Li J, French BA, French SW (2004) Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared. Life Sci 75: 585-597

Baumeister W, Walz J, Zuhl F, Seemuller E (1998) The proteasome: paradigm of a self-compartmentalizing protease. Cell 92: 367-380

Bayer P, Arndt A, Metzger S, Mahajan R, Melchior F, Jaenicke R, Becker J (1998) Structure determination of the small ubiquitin-related modifier SUMO-1. J Mol Biol 280: 275-286