Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, LS2 9JT, UK.
Sci Rep. 2021 Feb 18;11(1):4054. doi: 10.1038/s41598-021-83560-y.
Rab46 is a novel Ca-sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca-binding, coiled-coil and Rab domains suggest that Rab46 will be important for coupling rapid responses to signalling in many cell types. The molecular mechanisms underlying Rab46 function are currently unknown. Here we provide the first resource for studying Rab46 interacting proteins. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify affinity purified proteins that bind to constitutively active GFP-Rab46 or inactive GFP-Rab46 expressed in endothelial cells, we have revealed 922 peptides that interact with either the GTP-bound Rab46 or GDP-bound Rab46. To identify proteins that could be potential Rab46 effectors we performed further comparative analyses between nucleotide-locked Rab46 proteins and identified 29 candidate effector proteins. Importantly, through biochemical and imaging approaches we have validated two potential effector proteins; dynein and the Na/ K ATPase subunit alpha 1 (ATP1α1). Hence, our use of affinity purification and LC-MS/MS to identify Rab46 neighbouring proteins provides a valuable resource for detecting Rab46 effector proteins and analysing Rab46 functions.
Rab46 是一种新型的 Ca 感应 Rab GTPase，被证明在血管内皮细胞和免疫细胞中具有重要功能。其存在功能的 Ca 结合、卷曲螺旋和 Rab 结构域表明，Rab46 将是许多细胞类型中快速反应与信号传递偶联的重要因素。Rab46 功能的分子机制目前尚不清楚。在这里，我们提供了研究 Rab46 相互作用蛋白的第一个资源。我们使用液相色谱串联质谱（LC-MS/MS）来鉴定与在血管内皮细胞中表达的组成性激活 GFP-Rab46 或非活性 GFP-Rab46 结合的亲和纯化蛋白，我们发现了 922 个与 GTP 结合的 Rab46 或 GDP 结合的 Rab46 相互作用的肽段。为了鉴定可能是 Rab46 效应物的蛋白质，我们对核苷酸锁定的 Rab46 蛋白进行了进一步的比较分析，并鉴定了 29 个候选效应物蛋白。重要的是，通过生化和成像方法，我们验证了两个潜在的效应物蛋白；动力蛋白和 Na+/K+ATP 酶亚基α1（ATP1α1）。因此，我们使用亲和纯化和 LC-MS/MS 来鉴定 Rab46 邻近蛋白，为检测 Rab46 效应物蛋白和分析 Rab46 功能提供了有价值的资源。
