Rab46 is a novel Ca2+-sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca2+-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 Na2+/ 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.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021). Severe COVID-19 is characterised in part by vascular thrombosis and cytokine storms due to increased plasma concentrations of factors secreted from endothelial and T-cells. Here, using patient data recorded in the UK Biobank, we demonstrate the importance of variations in Rab46 (CRACR2A) and clinical outcomes. Using logistic regression analysis, we determined that three single nucleotide polymorphisms (SNPs) in the gene EFCAB4B cause missense variations in Rab46, which are associated with COVID-19 fatality independently of risk factors. All three SNPs cause changes in amino acid residues that are highly conserved across species, indicating their importance in protein structure and function. Two SNPs, rs17836273 (A98T) and rs36030417 (H212Q), cause amino acid changes in important functional domains: the EF-hand and coiled-coil domain respectively. By using molecular modelling, we suggest that the substitution of threonine at position 98 causes structural changes in the EF-hand calcium binding domain. Rab46 is a Rab GTPase that plays regulates both regulating endothelial cell secretion and T-cell cytokine signalling and this study supports the hypothesis that genetic variations in Rab46 plays a role in COVID-19 severity.

AbstractRab46 is a novel Ca2+- sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca2+- 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 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 possible interacting proteins. Further comparative analyses between nucleotide-locked Rab46 proteins enriched this dataset to confidently identify 29 effector proteins. Importantly, through biochemical and imaging approaches we have validated two potential effector proteins; dynein and the Na2+/ 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.