人類腺嘌呤核苷二磷酸核醣化相似因子四與其結合蛋白之特性探討

Abstract

二磷酸腺苷核糖基化因子(Arfs)是一群會攜帶三磷酸鳥苷(GTP)的小分子家族，在細胞中調控著囊報的運輸和肌動蛋白的重組。與其構造相近的次家族為腺嘌呤核苷二磷酸核醣化相似因子(Arf-like 4, Arl4)，其中包含的成員有Arl4A，Arl4C和Arl4D，它們在生物發育期間是被嚴格調控且值得研究的。為了近一步得知他們功能，我們運用了酵母菌雙雜交篩選法發現了Arl4的新作用蛋白HYPK，它是一個會與導致亨丁頓舞蹈症的治病蛋白Htt作用的蛋白，並被報導可降低Htt引起的不正常堆積，同時有類似保護蛋白(chaperon)的特性。此外，HYPK可以與終端乙酰轉移酶共同作用，幫助剛做出的胜肽蛋白正確摺疊。為了釐清Arl4與HYPK的作用，我們在酵母菌雙雜交法中得知HYPK只特定與Arl4A和Arl4D，不與Arl4C作用，並發現HYPK尾端115-129是與Arl4結合的位置，用此片段我們繼續篩選出A1和A3兩個失去與Arl4結合能力的HYPK突變體。我們同時也驗證HYPK不偏好與活化態或非活化的態的Arl4作用。以上發現都藉由穀胱苷肽轉移酶 (GST) 融合蛋白進行試管結合實驗(in vitro binding assay)、共同免疫沉澱(IP)得到確認。接著也利用免疫螢光染色法在細胞中觀察到HYPK與Arl4A共同坐落於細胞膜上。功能探討部分，HYPK不僅在細菌系統裡可以加強Arl4A的表現量，更可以在COS-7細胞株與Arl4A一起促進細胞移動的能力。以上資訊告訴我們Arl4可能透過與尚未透徹的機制與HYPK作用而有更好的折疊，因而促進在細胞中的功能。 另一項研究主要在探討Arl4家族和磷酸肌醇(phosphoinositide)之間的相互關係。實驗室之前的研究顯示Arl4位在的細胞膜上累積許多特定的磷酸肌醇，因而好奇這樣的磷酸肌醇累積是否會影響Arl4家族對細胞膜的親和力。而我們用特殊的脂質磷酸酶系統降低細胞膜上特定的磷酸肌醇的含量，結果顯示Arl4家族駐於細胞膜上的能力並不受到這些特定的磷酸肌醇影響，此外，還意外發現Arl4家族有能力顯著地將實驗過程所使用的脂質磷酸酶INPP5E吸引到細胞膜上。利用免疫沉澱的分析，我們發現INPP5E可以直接與Arl4A作用。由於INPP5E被發現的主要功能為降低纖毛中特定磷酸肌醇的累積以幫助訊傳遞，所以我們在NIH/3T3細胞株抑制Arl4A的表現之後，發現INPP5E失去維持纖毛中特定磷酸肌醇的含量的能力。就這些結果顯示，INPP5E很有可能是Arl4A家族訊息下游新穎的作用分子，來共同調特定的生理功能，詳細的影響機制值得進一步探討。

ADP-ribosylation factors (Arfs) are small GTPases that control vesicle trafficking and actin remodeling. The Arf-like 4 (Arl4) proteins contain Arl4A, Arl4C, and Arl4D which belong to Arf subfamily, are tightly regulated in development and different tissues. To understand the function of Arl4s more clearly, we identified a novel interacting partner huntingtin yeast two hybrid protein K (HYPK) by yeast two-hybrid system. HYPK is primarily found to be one of interacting proteins of huntingtin (Htt), which its N-terminal CAG repeats can cause product aggregation, thus leading to Huntington disease. HYPK was reported to reduce aggregates and apoptosis induced by Htt and exhibited chaperone-like activity. Also, it was found to act with N-terminal acetyltransferase (NaA) to confer nascent proteins’ proper folding. To confirm the interaction between Arl4s and HYPK, we found that HYPK prefers to interact with Arl4A and Arl4D but not Arl4C in yeast two-hybrid system, and HYPK’s C-terminal domain 115-129 was further narrow downed to be important for interacting with Arl4s, which led to identification of A1 and A3 mutants. Accordingly, HYPK has not preference toward Arl4A Q79L or T51N forms. These data are consistent in in intro binding assay, and co-immunoprecipitation. Next, HYPK can co-localize with Arl4A WT, Q79L and T51N but not T34N and G2A forms on plasm membrane in COS-7 cells. To focus on the functions between Arl4s and HYPK, we found that HYPK can not only promote the abundancy of Arl4A in E. coli system, but also enhance cell migration in the presence of Arl4A. As a result, HYPK probably through assisting proper folding of Arl4A by unknown mechanism to improve the functions of Arl4A. Another study around Arl4s is focused on their interdependent relationship toward phosphoinositides (PIs). Our previous data showed that Arl4s localized membrane enriched with phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) and phosphatidylserine. Curious about whether depletion of PI(4,5)P2 could affect the ability of Arl4s’s membrane targeting, we use Pseudojanin (PJ) phosphatase chimera coupled with rapamycin induced membrane translocation to deplete PI(4,5)P2 on plasma membrane. Arl4s seemed not to be affected by lipid composition change but accidentally potentiated phosphatase INPP5E in PJ to attach to membrane and co-localize with Arl4s. Co-immunoprecipitation of INPP5E confirmed its direct interaction toward Arl4A in COS-7 cells. Moreover, knockdown of Arl4A disrupt INPP5E maintained low PI(4,5)P2 distribution in cilia lumen in NIH/3T3 cells. Put together, INPP5E the could be a newly identified effector down stream of Arl4s.