人類四D腺嘌呤核苷二磷酸核醣化相似因子與其結合蛋白特性探討

Abstract

人類腺嘌呤核苷二磷酸核醣化相似因子4D（Arl4D）為二磷酸腺苷核醣基化因子（Arfs）小分子家族的一員。Arl4D與肌動蛋白的重組、粒線體的型態以及膜電位的調控功能有關。我們透過酵母菌雙雜交篩選法發現了Arl4D的新作用蛋白TACC3。TACC家族的蛋白會出現在中心體與周遭紡錘體，並且都是微管正端的微管結合蛋白。TACC3則與有絲分裂紡錘體的組織還有微管之生長有關。為了進一步釐清Arl4D與TACC3之間的作用，我們縮小觀察它們之間的結合位置。發現TACC結構域的N端是與Arl4D結合作用的重要片段。利用丙胺酸置換掃描的方法，我們發現了幾個不會和Arl4D作用的突變型TACC3。也藉由穀胱苷肽轉移酶融合蛋白進行試管結合實驗（in vitro binding assay）與共同免疫沉澱（co-immunoprecipitation）的方法證實。A2及A5突變型的TACC3的確失去與Arl4D結合的能力。雖然在細胞週期間期時看不出Arl4D與TACC3間有特別的共同座落位置，在有絲分裂期間，卻可以發現它們共同坐落在中心體與周遭紡錘體。降低Arl4D與TACC3的表現量則都會影響微管的重新生長，也告訴我們可能Arl4D與TACC3間的作用與微管之生長有重要性。 小分子三磷酸鳥苷蛋白酶家族之蛋白為單分子構造的，透過構型的改變能夠調控GTP與GDP的循環。然而Arl4A與Arl4D則被發現會自發的在生物體內外形成低聚合物。Arl4家族之蛋白質特徵在於N端有豆蔻酸化之修飾（myristoylation），與膜之結合有關，C端則有poly-basic的區域。本篇論文中，我們發現Arl4A與Arl4D不只能與自己結合，彼此之間也會結合在一起。Arl4A與Arl4D會高度的集中在細胞核周與細胞膜上。並且失去上膜能力的N端豆蔻酸化修飾突變的Arl4A G2A則可以被Arl4D帶到細胞膜上。低聚合化的Arl4在生理上扮演的功能值得我們進一步去釐清。

ADP-ribosylation factor-like protein 4D (Arl4D) is a member of Arf family small G proteins. Arl4D function in actin reorganization, mitochondrial morphology and membrane potential. We identify a novel interacting protein of Arl4D by yeast two-hybrid assay, Transforming Acidic Coiled-Coil Protein 3 (TACC3). TACC proteins can concentrate at the centrosomes and mitotic spindles and function as microtubule plus-end tracking proteins. TACC3 is associate with mitotic spindle organization and microtubule nucleation. To further confirm the interaction between Arl4D and TACC3, we narrow down the interaction region. N-terminal of TACC domain is the important region that Arl4D and TACC3 can interaction to each other. By alanine scanning mutagenesis, we found several Arl4D interaction-defect TACC3 mutants. These data are also confirm by in vitro binding assay and co-immunoprecipitation. TACC3 A2 and A5 loss the interaction ability to Arl4D. Although Arl4D and TACC3 show no specific co-localization in interphase cells, both Arl4D and TACC3 can localize to the centrosomes and spindles in mitotic cells. Knock down of Arl4D and TACC3 affect the microtubule regrowth, which the interaction between Arl4D and TACC3 may be important for microtubule nucleation. Small GTP-binding proteins are monomeric G proteins, which cycle through GTP- and GDP-bound states, according to the conformational changed. However, Arl4A and Arl4D was found to form oligomers in vitro and in vivo spontaneously. Arl4s is feature of their N-terminal myristoylation, which lead to membrane association and the C-terminal poly-basic region. In this thesis, we found Arl4A and Arl4D not only can interact with themselves, but also interact to each other. Arl4A and Arl4D show high co-localization at the perinuclear and plasma membrane. N-terminal myristoylation defect mutation Arl4A G2A can recruit to the membrane by Arl4D. The physiological function of the oligomerization of Arl4s is still a problem for us to clarify.