Interactome and evolutionary conservation of Dictyostelid small GTPases and their direct regulators

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.被称为小GTP酶（small GTPases）的GTP结合蛋白是最大的调控蛋白家族之一，几乎掌控着活细胞的全部功能活动。其活性分别由鸟苷酸交换因子（guanine nucleotide exchange factors, GEFs）与GTP酶激活蛋白（GTPase activating proteins, GAPs）进行正向与负向调控；上述蛋白与其上游调控因子以及小GTP酶的下游靶标共同构成了精密复杂的信号转导网络。尽管基因组学研究已揭示了GTP酶、GEF以及GAP家族的庞大成员规模，但目前仅对其中极小一部分的相互作用与功能开展了实验探索。盘基网柄菌（Dictyostelid social amoebas）已成为解析Rac-Rho与Ras-Rap家族GTP酶相关蛋白在定向细胞迁移及肌动蛋白细胞骨架调控中功能的经典模式生物。目前已有覆盖该类群从单细胞祖先算起的5亿年演化历程的多个盘基网柄菌物种的基因组、细胞类型特异性转录组以及发育阶段转录组数据。本研究从代表4个主要分类群的代表性基因组中鉴定得到了全部的GTP酶、GEF以及GAP蛋白，并对它们的系统发育关系、演化保守性、功能域结构的变化，以及其在发育阶段和细胞类型特异性的表达模式进行了系统分析。我们对本次分析的约2000个基因的表达谱开展了层级聚类分析，以预测GTP酶、GEF与GAP之间的相互作用模块；该分析不仅验证了经实验证实的已知相互作用模块，还发现了大量全新的组合模式。本研究可为细胞调控所有领域的相关研究提供极具价值的参考资源。