Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in the social amoeba Dictyostelium (Vwa1 IP)

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O2-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxW2 and Vwa1, which was found in the FbxWD interactome by co-immunoprecipitation and is classified as a vault protein inter-alpha- trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and their similar transcript expression profiles during multicellular development suggest they function together. FbxWD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally expressed blocked culmination by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. However, vwa1-disruption alone did not affect development. In contrast, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 which in turn imbalances its synergistic function with FbxWD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

与多数真核生物（eukaryotes）类似，后生动物前的社会变形虫盘基网柄菌（Dictyostelium）依赖SCF（Skp1/酷蛋白-1/F-box蛋白）家族的E3泛素连接酶（E3 ubiquitin ligases）调控其蛋白质组（proteome）。在盘基网柄菌中，饥饿胁迫可诱导其从单细胞摄食阶段向多细胞蛞蝓体转变；该多细胞结构可响应外界信号，最终发育形成包含终末分化柄细胞与孢子细胞的子实体。上述发育转变过程受F-box蛋白（F-box proteins）以及Skp1的氧依赖型翻译后修饰（posttranslational modifications）调控。本研究进一步深入探究了FbxW2与Vwa1的关键调控作用：二者通过免疫共沉淀（co-immunoprecipitation）被鉴定为FbxWD互作组（interactome）的成员，且被归类为含穹顶蛋白间-α-胰蛋白酶（VIT）结构域与血管性血友病因子A（vWFA）结构域的蛋白。使用基因标记菌株（gene-tagged strains）开展的反向免疫共沉淀（reciprocal co-IPs）验证了二者的互作关系；二者在多细胞发育过程中的转录本表达谱（transcript expression profiles）高度相似，提示二者可能协同发挥功能。FbxWD过表达与蛋白酶体抑制剂（proteasome inhibitors）处理均未对Vwa1的蛋白水平产生影响，表明二者并非底物与E3泛素连接酶的对应关系。在正常表达FbxW2的蛞蝓顶端细胞（slug tip cells）中强制过表达FbxW2，会通过依赖于其F-box结构域与RING结构域（RING domains）、且依赖于Vwa1表达的机制阻断子实体形成过程。然而，仅敲除Vwa1基因（vwa1-disruption）并不会影响其正常发育过程。与之相反，过表达其三个保守结构域（conserved domains）中的任意一个均会阻断发育进程，且该效应依赖于Vwa1的表达。基于结构预测（structure predictions）结果，我们提出Vwa1结构域的负调控效应是通过人工激活Vwa1实现的，而激活后的Vwa1会打破其与FbxWD的协同功能平衡。自身抑制（autoinhibition）或同源二聚化（homodimerization）可能与人类中进化同源的VWA5A/BCSC-1尚不明确的抑癌功能（tumor suppressor role）相关。