鈣離子活化 calpain 蛋白酶水解 RanGAP1 之分子機制與其生物意義

Abstract

Ran GTP 酶活化蛋白質 (簡稱 RanGAP1) 是一個對於細胞核與細胞質之間物質的運輸 (nucleocytoplasmic transport) 相當重要的蛋白質，同時也是第一個發現會被類泛素小型修飾因子 SUMO (small-ubiquitin-like modifier) 所修飾的蛋白質。過去研究已知 SUMO 修飾能使細胞質中的 RanGAP1 聚集至細胞核邊緣 (nulcear rim)，藉此維持正常的細胞核內外物質運輸，然而對於 RanGAP1 是否有其他轉譯後修飾與生物功能仍尚未知曉。在本篇論文中，我們證實當鈣離子流入胞內時 (intracellular Ca2+ influx) 會使 RanGAP1 發生限制性的蛋白酶水解，並可能藉由此現象調控核質間的物質運輸。為了探究此現象，我們首先利用鈣離子螯合物，發現其能減弱此水解現象，顯示 RanGAP1 水解確實由鈣離子所調控。接著，透過生化、藥理以及基因調控等方式，我們證實 RanGAP1 水解現象是透過鈣離子所活化的一種半胱氨酸蛋白酶 (cysteine protease) calpain 2蛋白酶所主導。經由將純化的RanGAP1蛋白質與 calpain 2 蛋白酶反應所得之 RanGAP1 水解產物進行蛋白質 N 端定序 (N-terminal sequencing)，進一步確認 calpain 2 蛋白酶在 RanGAP1 上的兩個主要切位：其一是位於 RanGAP1 N 端的第 60 個胺基酸蘇氨酸 (Threonin) 與第 61 個胺基酸纈草胺酸 (Valine) 之間，其二則位於 RanGAP1 酸性區塊 (acidic domain) 與 C 端交界處的第 427 個胺基酸絲氨酸 (Serine) 與第428 個胺基酸絲氨酸之間。我們進一步發現 calpain 2 蛋白酶對 RanGAP1 的水解作用會導致 RanGAP1 自細胞核邊緣離開，很可能是經由移除 C 端可被 SUMO 修飾的區塊；我們也觀察到經 calpain 2 蛋白酶切去 N 端的 RanGAP1 亦會改變其胞內位置而聚集至細胞核內，也許是經由移除 N 端的岀核訊息 (nuclear export signal)。综合本篇研究結果，我們歸納出一個新的 RanGAP1 蛋白質轉譯後修飾機制：透過鈣離子活化 calpain 2 蛋白酶所調控的 RanGAP1 蛋白酶水解現象。此現象不僅改變 RanGAP1 在胞內的位置，也極可能因而影響到 RanGAP1 所調控的核質運輸，關於此鈣離子調控之 RanGAP1 水解現象的生物功能與其生理意義皆有待更深入的探討。

The GTPase activating protein 1 of Ran (RanGAP1), which plays a vital role in nucleocytoplasmic transport, is the first modification substrate for small-ubiquitin-like-modifier (SUMO) identified. SUMOylation targets RanGAP1 to nuclear rim and has been suggested as an important regulation for its nucleocytoplasmic transport function. Nevertheless, other post-translational modification(s) on RanGAP1 and their influences on cellular function(s) of RanGAP1 remained largely unknown. In this thesis, we demonstrated that intracellular Ca2+ influx induces limited proteolysis on RanGAP1 and thereby potentially modulates nucleocytoplasmic transport of substrate proteins. First, this proteolytic event could be attenuated by Ca2+ chelators, indicating the involvement of Ca2+ in RanGAP1 proteolysis. Using biochemical, pharmacological and genetic approaches, we showed that calpain 2, a Ca2+-dependent cysteine protease, is mainly responsible for this Ca2+-mediated RanGAP1 cleavage. Through in vitro calpain 2 cleavage assay with recombinant RanGAP1proteins, and followed with the N-terminal sequencing, two major calpain 2-cleaved sites were identified: (i) Thr60-Val61 on N’-terminal leucine-rich-repeat and (ii) Ser427-Ser428 between acidic domain and C’-terminal tail. Further studies indicated that calpain 2-mediated proteolytic cleavages of RanGAP1 potentially caused its translocation from nuclear rim to cytoplasm, probably through proteolytic removal of its C’-terminus where SUMOylation occurred. Similarly, the N’-terminal truncation of RanGAP1 by calpain 2 seemed to delocalize RanGAP1 to nucleus possibly due to lost of domains containing nuclear export sequence (NES). In sum, our results suggest a potential proteolytic modification of RanGAP1 via calpain 2 and its influences on cellular locations of RanGAP1 and nucleocytoplasmic transport of reporter substrate proteins. The implications of our study for the RanGAP1-mediated biological functions therefore need further investigation.