細胞體外雙股DNA斷裂修復蛋白質XRCC4及去乙醯酵素SIRT2複合體特異性交互作用之研究

Study of the specific protein-protein interaction of the XRCC4-SIRT2 in vitro

黃意翔(Yi-Shiang Huang)

指導教授 ： 李玉梅

共同指導教授 ： 賴建勳

國立臺灣大學/生命科學院/生化科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02472

摘要

XRCC4為哺乳動物DNA修復系統中重要的蛋白質，細胞喪失XRCC4會造成對游離化輻射的高度敏感，並且無法執行V(D)J重組作用。XRCC4與LigaseIV複合體在雙股斷裂之非同源末端結合修復中，執行最後的連接作用。已知XRCC4對於穩定LigaseIV有重要貢獻，並且可以活化LigaseIV之連接酶活性。
酵母菌Sir2為一種需要NAD+的蛋白質去乙醯化酵素，可調節諸如基因默化、DNA修復以及卡路里限制使壽命延長之生物功能。人類SIRT2屬於酵母菌Sir2家族之一員，哺乳動物的Sir2相似物被稱做Sirtuin，歸屬於NAD+-dependent Histone deacetylase class III，其去乙醯之酵素活性與催化機制已被詳細研究。
本實驗室前人經由並列親合純化結合質譜技術，發現以XRCC4作為誘餌蛋白，成功釣取包含SIRT2在內的數種結合蛋白。再應用親合沉澱技術，證實在活體內SIRT2確實為XRCC4的結合蛋白。此外，經由刪除分析發現XRCC4的181-193區域對於SIRT2的結合有重要影響，同時此區域也是XRCC4與LigaseIV結合的重要區域，進一步以點突變分析位於此區域的氨基酸，發現離胺酸188位置對於SIRT2的結合扮演重要角色。此外，核內的XRCC4與核外的SIRT2在有絲分裂前期prophase/prometaphase結合，在活體內的實驗證據顯示XRCC4與SIRT2結合可使G2/M DNA damage checkpoint及spindle assembly checkpoint之運作正常維持。
為更進一步地確認XRCC4與SIRT2結合的專一性與親和力，利用細菌過量表現並純化之組胺酸標記及GST融合蛋白，分別以GST-pull down assay及表面電漿共振技術確認XRCC4與SIRT2具有直接的物理接觸，此外，兩者結合並不會干擾XRCC4與LigaseIV的結合，顯示一種非競爭性之結合模型。另外，經由表面電漿共振技術發現，SIRT2亦會直接與LigaseIV結合，三者可形成穩定之複合物結構。
為探討具酵素功能之SIRT2是否對於XRCC4 188位置之離胺酸進行化學修飾作用，利用定位突變方式分別將該離胺酸置換成麩醯胺或精胺酸，試圖模仿XRCC4離胺酸188乙醯化與去乙醯化，是否造成其對SIRT2之親和力之改變。結果顯示離胺酸188乙醯化與否，對於親和力並無顯著影響。
此外，將SIRT2酵素催化中心組胺酸187應用定位突變方式將其置換成丙胺酸或酪胺酸，以模擬SIRT2酵素中心部位喪失催化活性，但仍然具有蛋白質結合功能之情況。結合分析之結果顯示組胺酸187置換成丙胺酸或酪胺酸，均對其與XRCC4之結合有相當程度之影響。
經由本研究發現，SIRT2與XRCC4在活體外可專一性結合，亦可直接與LigaseIV形成複合體；而對XRCC4與SIRT2分別使用點突變技術之結合試驗，則說明XRCC4與SIRT2的結合，應屬於單純之蛋白質交互作用，而非酵素與受質之關係；使用連續結合試驗，顯示SIRT2結合於先形成的XRCC4-LigaseIV複合物上，三者形成穩定之複合體結構。

關鍵字

XRCC4 ； SIRT2 ； 交互作用

並列摘要

XRCC4 is an essential protein in the mammalian DNA repair system. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination. In the DNA double strand break (DSB) repair pathway, XRCC4-LigaseIV complex execute the final DNA end-joining reaction. XRCC4 has been known greatly stabilizing LigaseIV, and can stimulate the ligase activity of the DNA LigaseIV.
SIRT2 is a member of the yeast silent information regulator 2 (Sir2) enzyme families. Yeast Sir2 functions as an NAD+-dependent protein deacetylase, and conducts biological functions such as genomic silencing, DNA repair, life span in calorie restriction. Human SIRT2 is a member of yeast Sir2 family. Mammalian Sir2 ortholog is called Sirtuin and classified as NAD+-dependent Histone deacetylase class III. The enzyme activity and the catalytic mechanism of yeast Sir2 were clearly reported.
Based on the tandem affinity purification combining with the mass spectrometry technique, we previously identified several XRCC4 association proteins including SIRT2. Using the affinity precipitation technique, we corfirmed SIRT2 binds to XRCC4 in vivo. By serial deletion mutation analysis data, XRCC4(181-193) plays an important role in both SIRT2 and LigaseIV binding. From further point mutation experiments on the residues of this region, the lysine 188 shows a crucial role on SIRT2 binding. Binding of the nuclear XRCC4 and cytosoilc SIRT2 occurs in the prophase/prometaphase during mitosis. Other in vivo experiments concluded this binding may be attributed to the normal function of the G2/M DNA damage checkpoint and the spindle assembly checkpoint.
In order to further confirm the specificity and the binding affinity between SIRT2 and XRCC4, I overexpressed the His-tagged and GST-fusion proteins in bacterial system, purified them and use the surface plasmon resonance and the affinity precipitation techniques to perform binding assays. Data show that SIRT2 directly binds to XRCC4 and their binding behavior does not interfere the known binding of XRCC4 and LigaseIV. It therefore concludes a non-competing binding model.
In order to investigate whether the lysine 188 residue in the XRCC4 is chemically modified by the enzyme SIRT2 or not, I apply site-directed mutagenesis to alter this lysine to either arginine or glutamine, which mimicking the non-acetylating or fully acetylating of lysine residue. Data show the affinity is not altered whether the lysine residue is acetylated or not.
I also performed site-direct mutagenesis on the catalytic center, histidine 187, of the enzyme SIRT2. This residue is replaced by alanine or tyrosine to simulate the dead enzyme reserving protein-protein interaction function. From the data of binding assay, the affinity to XRCC4 of the mutant SIRT2(H187Y) and SIRT2(H187A) shows a certain level of decrease.
My reaserch shows that SIRT2 can directly and specifically binds to XRCC4 in vitro, and can directly form a complex with LigaseIV. From the binding assay performing on the mutants, I conclude that the binding of the SIRT2 and XRCC4 is belonging to a general protein-protein interaction, not an enzyme-substrate relationship. From the evidence obtained by tandem binding assay, it shows SIRT2 bind the pre-forming XRCC4-LigaseIV complex and form a stable complex structure.

並列關鍵字

XRCC4 ； SIRT2 ； interaction

參考文獻

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