Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5
Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5
复制标题
膜蛋白细胞外近膜柄区域的可及性对于病毒泛素连接酶 K5 底物识别的重要性
DOI：
10.1042/bcj20220288
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发表时间：
2022
影响因子：
4.1
通讯作者：
Kanamoto Taisei
中科院分区：
文献类型：
--
作者：
Kajikawa Mizuho;Hata Mizuki;Ishimura Maho;Imaizumi Nanae;Kimura Minako;Miyano Kei;Ose Toyoyuki;Asai Daisuke;Ishido Satoshi;Kanamoto Taisei
卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌病毒，可潜伏感染B细胞并在免疫功能低下的患者中引起恶性肿瘤。KSHV在感染KSHV的细胞中利用两种病毒E3泛素连接酶K3和K5介导多泛素化依赖性的几种参与免疫系统的宿主膜蛋白的下调。虽然K3和K5是同一家族的成员，具有相似的结构拓扑，但K3和K5具有不同的底物特异性。因此，K5可能具有与K3不同的底物识别模式；然而，底物识别的分子基础仍不清楚。在这里，我们研究了人类CD8α不被K3和K5识别的原因，以获得对底物特异性的分子基础的理解。CD8α在实验条件下形成二硫化物连接的同型二聚体，以评估病毒连接酶介导的下调。已知在每个CD8α单体（Cys164-Cys164和Cys181-Cys181）之间的柄区有两个链间二硫键介导同二聚化。当Cys181-Cys181的链间二硫键被消除时，CD8α通过细胞质尾部的多泛素化作用，被具有功能环变体（RINGv）结构域的K5下调。天冬氨酸位于CD8α的茎/跨膜界面，是k5介导的CD8α突变体无Cys181-Cys181连锁下调所必需的。这些结果表明，靠近茎/跨膜界面的二硫键严重抑制了K5对底物的靶向作用。膜蛋白胞外近膜柄区域的可及性可能是病毒泛素连接酶K5识别底物的重要因素。
Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164–Cys164 and Cys181–Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181–Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181–Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.