Probing the Inflammatory Response Behind Diabetes and Obesity via the Biochemical Characterization of NOD1, an Innate Immune Receptor

Abstract

Innate immunity is an intricate system employed by humans to regulate the trillions of bacteria that live in our body. One protein integral to the innate immune response is called nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1). NOD1 is a cytosolic, membrane‐associated protein that is linked to irritable bowel syndrome, asthma, diabetes, and obesity. These diseases are thought to arise from improper mRNA splicing of NOD1 transcripts leading to a poorly functioning NOD1 protein receptor that can no longer regulate an overstimulated inflammatory response. Activation occurs when NOD1 binds to a bacterial cell wall component called iE‐DAP, which then signals the the NF‐κB cascade to produce pro‐inflammatory cytokines and chemokines.Current research is trying to describe the binding of iE‐DAP to NOD1, however, these studies almost exclusively use expensive and low‐yielding insect cells to obtain their active protein. This project aims to develop a bacterial expression system to express wildtype NOD1 and the LRR domain, which is the region thought to be involved in ligand binding. Currently I have produced both constructs in high yield from an E. coli system to allow their biochemical characterization through peptidoglycan pull‐down experiments and circular dichroism. Future work aims to obtain crystal structures of NOD1 bound to its ligand and perform NMR studies to observe ligand binding events.