CLICK-chemoproteomics and molecular dynamics simulation reveals pregnenolone targets and their binding conformations in Th2 cells

Abstract

Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. CD4+ and CD8+ immune cells synthesize P5 de novo, P5 in turn play important role in immune homeostasis and regulation. However, P5s biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5-target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live Th2 cells. Subsequently, using high-throughput quantitative proteomics we identified the P5-interactome in CD4+ Th2 cells. Our study revealed P5s conserved mode of action in CD4+ and CD8+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5s biochemistry in CD4+ and CD8+ immune cells. Applying advanced computational algorithms, we were able to generate near-native maps of P5-protein key molecular interactions that can lead to successful designing of novel molecular therapeutics strategies.