探討NEDDylation接合酵素Ubc12及黏合酵素Rbx1之間交互作用之介面

Abstract

類泛素分子Nedd8是一種在演化上被高度保留的真核生物蛋白質。它會透過一種稱為NEDDylation的轉譯後修飾方式被黏接至目標蛋白質上。NEDDylation的過程和泛素化（ubiquitination）的過程相當類似，都需經活化酵素（activating enzyme, E1），接合酵素（conjugating enzyme, E2），以及黏合酵素（ligase, E3）等三種酵素的作用。許多人類疾病都和NEDDylation受到不當的調控有關，因此NEDDylation路徑中的各個成員都具有成為藥物治療目標的潛力。 泛素黏合酵素（ubiquitin ligase）中最大的一個家族CRLs (Cullin–RING ubiquitin E3 ligases) 是以Cullin以及RING (really interesting new gene) 蛋白質為核心的黏合酵素。Rbx1是一種含有RING的蛋白質且在CRLs中有重要的功能。Rbx1扮演NEDDylation黏合酵素的角色將Nedd8黏接至cullin後會活化CRLs的功能：Rbx1首先和Neddylation接合酵素Ubc12交互作用後將Nedd8黏接至cullin並活化CRLs，而後位於已活化CRLs內的Rbx1會再和泛素化接合酵素（ubiqitination E2）交互作用以進行目標蛋白質的泛素化（ubiqitination）。Rbx1因此是一種雙重功能的黏合酵素，在CRLs的NEDDylation以及接踵而至的目標蛋白質泛素化中切換其角色。此角色切換的機制目前尚不清楚。為了解答此問題，精確定出Rbx1和泛素化接合酵素的交互作用介面以及Rbx1和NEDDylation接合酵素的交互作用介面並加以比較是優先的工作。 在本研究中，酵母菌雙雜合實驗（yeast two-hybrid system）指出Ubc12的獨特N端延伸以及His88，Pro121，Val122等三個胺基酸對於Ubc12以及Rbx1間的交互作用是重要的。Rbx1的Ile54，Pro95，以及Leu96等三個胺基酸也經由酵母菌雙雜合實驗證明對於其與Ubc12間的交互作用是重要的。我們進一步使用桿狀病毒-昆蟲細胞蛋白質表現系統 (baculovirus-insect cell expression system)來說明Rbx1中此三個胺基酸的突變對於cullin NEDDylation的影響。我們還需要更多的實驗才能夠揭露Rbx1和泛素化接合酵素交互作用及NEDDylation接合酵素交互作用時的介面的全貌以及兩者間相異之處。

Neural precursor cell expressed, developmentally down-regulated 8 (Nedd8) is a highly conserved eukaryotic ubiquitin-like protein. It is conjugated to its substrates in a process known as NEDDylation which comprises an E1-E2-E3 enzymatic cascade similar to ubiquitination. Dysregulated NEDDylation is implicated in the pathogenesis of many human diseases and the components of the NEDDylation pathway may accordingly be of potential therapeutic importance. Cullin–RING ubiquitin E3 ligases (CRLs) are the most prominent class of ubiquitin-ligases. Rbx1/Roc1, a RING finger protein, functions as an important component of CRLs. Modification of cullins by Nedd8 has been shown to activate CRLs and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. In other words, Rbx1 interacts first with Ubc12, the NEDDylation E2, to neddylate cullins and activate CRLs. Rbx1 then, in the context of activated CRL holo-enzyme, interacts with the ubiquitination E2s to promote substrates ubiqutination. Rbx1 is thus a dual - functioning E3 and switches its role in the processes of CRLs NEDDylation and the following substrates ubiquitination. The role-switching mechanism of Rbx1 as a dual- functioning E3 remains elusive. To answer this question, deciphering the precise surfaces of Rbx1/ubiquitination E2 versus Rbx1/NEDDylation E2 interactions is a top priority. Here we report that His88, Pro121, Val122, and the unique N-terminal extension of Ubc12 are implicated in the interaction with Rbx1 in yeast two-hybrid system. Ile54, Pro95, and Leu96 of Rbx1 are also shown to be important for the interaction between Ubc12 and Rbx1 in yeast two-hybrid system. The implications of Rbx1 I54 / P95 / L96 in cullin NEDDylation were further demonstrated in baculovirus-insect cell system. More work is necessary to reveal the full picture of both Rbx1/ubiquitination E2 and Rbx1/NEDDylation E2 interaction surfaces.