Affinity-based proteomics reveals novel binding partners for Rab46 in endothelial cells

Lucia Pedicini,Katie J Simmons,Ashley Money,Lynn Mckeown,Sabina D Wiktor

doi:10.1038/s41598-021-83560-y

Lucia Pedicini, Katie J Simmons + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-83560-y

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Journal: Scientific Reports	Publication Date: Feb 18, 2021
Citations: 7	License type: open-access

Affiliation: University of Leeds

Abstract

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.

Highlights

Rab[46] is a novel Ca2+-sensing Rab guanosine tri-phosphatases (GTPases) shown to have important functions in endothelial and immune cells
Volcano plot created with MATLAB 2018b using a custom written algorithm. (c) Proteins co-precipitate with GFP-Q604L in reference to GFPN658I, were sorted by fold change and visualized using a bubble plot where the circle area is proportional to p values
To isolate potential Rab[46] effector proteins, we considered the constitutively active GTP-bound mutant (Q604L) compared to the inactive GDP-bound mutant (N658I) for proteomic analysis, as most known Rab effector proteins preferentially interact with the GTP-bound form of the Rab GTPase

Summary

Introduction

Rab[46] is a novel Ca2+-sensing Rab GTPase shown to have important functions in endothelial and immune cells. Using liquid chromatography tandem mass spectrometry (LC– MS/MS) to identify affinity purified proteins that bind to constitutively active GFP-Rab[46] or inactive GFP-Rab[46] expressed in endothelial cells, we have revealed 922 peptides that interact with either the GTP-bound Rab[46] or GDP-bound Rab[46]. Most Rabs lack the inherent ability to efficiently hydrolyse GTP and require guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to regulate their nucleotide binding status[5,6]. In this way, Rabs act as molecular switches that mediate downstream events by interacting with effector molecules when anchored, in their GTP-bound form, to their target membrane compartment. Bubble plot created with MATLAB 2018b (https://www.mathworks.com/matlabcentral/fileexchange/48005-bubbleplot-multidimensional-scatter-plots)

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Conclusion