臺灣博碩士論文加值系統

老鼠輔肌動蛋白(mouse alpha actinin 2, mACTN2)是屬於肌動蛋白結合蛋白質的家族成員之一，此蛋白質由894個胺基酸所組成，它廣泛表現於心臟、肌肉、肺及腦細胞中。我之前實驗室的研究顯示，mACTN2和它的家族蛋白成員皆含有與核受體結合的LXXLL motif，且作為核受體的輔活化子，並可與p160的輔活化子醣皮質素受體結合蛋白1的羧端結合，以扮演二級核受體活化轉錄的角色。由先前的實驗得知mACTN2本身具有特殊的出核序列(nuclear exporting sequence, NES )可參與本身在細胞核內外運輸的調控，也發現被運輸到細胞核外的mACTN2本身的蛋白質的穩定性也受到影響，再加上最近的研究指出calpain會裂解a-actinin。本研究主要探討mACTN2在細胞核或細胞質分佈時其蛋白質穩定性是否受到calpain的影響。根據實驗結果推論，calpain會參與裂解細胞質中的mACTN2而降低其在細胞中的穩定性，但似乎不是通過所謂保留性之裂解序列。進一步利用calpain抑制劑而降低calpain蛋白質表現的方式，以透過阻斷calpain功能方式而能減緩細胞核內mACTN2蛋白質被降解的速率。由本論文結果可知，利用mACTN2結構序列上出(或入)細胞核訊號以改變其在細胞內的分佈，進而改變其蛋白質之穩定程度而影響其在細胞內功能之表現。

Mouse alpha actinin 2 (mACTN2) belongs to the spectrin protein superfamily, which is composed of 894 residues and expresses in the heart, skeletal muscle, lung, and brain. Our previous study demonstrated that mACTN2 and its superfamily member contain LXXLL motif, the Nuclear receptor (NR) binding motif, and serve as a coactivator for nuclear receptor, mACTN2 also bind to the C-terminal region of GRIP1 (glucocorticoid receptor interacting protein 1) for the secondary nuclear receptor functions. Our previous study identified the NES (nuclear export signals) of mACTN2 for the regulation of its nucleo-cytoplasmic trafficking which was correlated with its protein stability in the cytoplasmic subcellular localization. The recent research suggested calpain could cleavage alpha actinin. In this thesis, I further examined calpain whether was able to affect nuclear or cytolasmic mACTN2 protein stability. My data demonstrated that calpain primarily cleavaged cytoplasmic mACTN2 and then decreased its protein stability via non-conserved calpain cleavage sequence. In addition to the calpain inhibitor, the stabilization of cytoplasmic mACTN2 protein, mediated through the decrease of endogenous calpain to suppress that rate of cleavage. Finally, the differential role of nuclear localization sequence and NES in the mACTN2 might determine its subcellular localization, protein stability, and subsequent endogenous functions.

目錄
目錄 I
表目錄 Ⅲ
圖目錄 Ⅳ
縮寫表 Ⅴ
中文摘要 Ⅵ
英文摘要 Ⅶ
第一章 緒論 1
第二章 實驗材料與方法 12
第一節 實驗材料 12
第二節 實驗方法 15
第三章 結果 27
第一節 預測 mACTN2可能為calpain的標的 27
第二節 Calpain主要表現於細胞質當中 27
第三節 mACTN2蛋白質降解透過 calpain 28
第四節 抑制mACTN2蛋白的降解是透過降低 calpain蛋白質的表現 29
第五節 mACTN2蛋白質可能受到其他蛋白質降解系統調控 30
第四章 討論 32
第一節 細胞質中的mACTN2蛋白質透過calpain被降解 32
第二節 不同的僅表現於細胞質之mACTN2受到calpain的影響 32
第三節 Calpain抑制劑的抑制機轉 33
第四節 是否有其他蛋白參與calpain影響mACTN2蛋白質的穩定性 33
第五節 是否calpain與mACTN2之間具有直接的鍵結 34
參考文獻 49


















表目錄
表一 各物種的-actinin序列比對 35
表二 已知的calpain的標的 36
















圖目錄
圖一 mACTN2的功能基團與NES、NLS訊號 37
圖二 一般蛋白質利用Importin 系統的細胞核質運輸模式和Leptomycin B作用機轉 38
圖三 calpain的序列與功能基團 39
圖四 hACTN1與mACYN2序列比對圖及calpain預測切位 40
圖五 mACTN2序列的PEST分析結果 41
圖六 calpain細胞分布的鑑定 42
圖七 mACTN2蛋白質表現量降低是透過calpain 43
圖八 MG101減緩calpain引起的mACTN2蛋白質減少 44
圖九 MG101抑制calpain蛋白質的表現進而使其功能降低 45
圖十 加入鈣離子和過氧化氫無法活化calpain使細胞質中mACTN2減少 46
圖十一 加入caffeine活化calpain造成細胞質中mACTN2減少 47
圖十二 推測mACTN2被calpain裂解導致蛋白質不穩定的機轉 48

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