Analysis of biological networks in the endothelium with biomimetic microsystem platform.

Kevin Kruse,Mitchell Sun,Mark Maienschein-Cline,Yulia A Komarova,Jeff Klomp,Zhang Chen,Fei Huang,Dianicha Santana,Pinal Kanabar

doi:10.1152/ajplung.00392.2018

Abstract

Here we describe a novel method for studying the protein "interactome" in primary human cells and apply this method to investigate the effect of posttranslational protein modifications (PTMs) on the protein's functions. We created a novel "biomimetic microsystem platform" (Bio-MSP) to isolate the protein complexes in primary cells by covalently attaching purified His-tagged proteins to a solid microscale support. Using this Bio-MSP, we have analyzed the interactomes of unphosphorylated and phosphomimetic end-binding protein-3 (EB3) in endothelial cells. Pathway analysis of these interactomes demonstrated the novel role of EB3 phosphorylation at serine 162 in regulating the protein's function. We showed that phosphorylation "switches" the EB3 biological network to modulate cellular processes such as cell-to-cell adhesion whereas dephosphorylation of this site promotes cell proliferation. This novel technique provides a useful tool to study the role of PTMs or single point mutations in activating distinct signal transduction networks and thereby the biological function of the protein in health and disease.

Highlights

Posttranslational protein modifications (PTMs), such as phosphorylation, can “switch on” and “switch off” the biological function of proteins
While many different methods exist for affinity purification (AP)-MS, including the use of specific antibodies to immunoprecipitate endogenously and exogenously expressed proteins attached to various tags [9], they all suffer from contamination with the bait protein, often making the identification of other proteins difficult [32]
L395 metic end-binding protein-3 (EB3)-S162E were covalently linked to nickel-nitrilotriacetic acid (Ni-NTA) beads using ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC) and NHS (Fig. 2A)

Summary

INTRODUCTION

Posttranslational protein modifications (PTMs), such as phosphorylation, can “switch on” and “switch off” the biological function of proteins. Techniques involving the overexpression of a bait protein fused to a prokaryotic biotin ligase molecule (BirA), such as the recently developed proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS; 39), are not possible when the amount of sample is limited or when using human tissue or primary cells, which are not amenable to transfection (e.g., primary neurons and platelets). To circumvent these problems, we have developed a simple and robust method for isolating protein complexes using the “biomimetic microsystem platform” (Bio-MSP), which we describe in the present studies. To determine how changes in EB3 phosphorylation alter downstream signaling pathways, we isolated proteins bound to EB3 in phosphorylated and dephosphorylated states using our novel technique

MATERIALS AND METHODS

RESULTS

DISCUSSION

DISCLOSURES