Leveraging in vivo protein tracking to identify novel immune markers associated with obesity driven disease - PROJECT SUMMARY. To date, biochemical tools to study secreted proteins’ origins and destinations leading to the discovery of new biomarkers associated with disease are underdeveloped. Deciphering interorgan communication pathways will provide insights into key modulators of obesity driven inflammation. Interorgan dysfunction from overconsumption, hence overstimulation of key metabolic pathways, has a significant impact on the development of metabolic diseases, such as Type II Diabetes, cardiovascular disease, liver disease, and cancer. Specifically, disrupting the interaction between the intestine and adipose tissue plays a crucial role in the development of complications of obesity. Therefore, uncovering intestinal secreted proteins trafficked to adipose tissue can in the future lead to the development of druggable targets that may attenuate the progression of obesity related metabolic diseases. The objective of this proposal is to utilize novel tools to study the interorgan communication pathway between the intestine and adipose tissue immune cells. We have developed a Cre-inducible endogenous biotin-ligase BirA*G3 affinity tracking system expressed in the mouse intestine, with the capability of biotin-tagging proteins sent through the endoplasmic reticulum of intestinal epithelial cells. After Cre-induction, the addition of free-biotin induces rapid labeling of the intestinal epithelium, and of all secreted proteins. Utilizing streptavidin affinity enrichment, we capture the proteins trafficked to our target organ and employ tandem mass- tag (TMT) quantitative mass spectrometry proteomics to identify the low-abundance trafficked proteins, as well as their origins and destinations. Using this approach, we identified PlexinB2 which is secreted from intestine to regulate subcutaneous white adipose tissue lipolysis and systemic metabolism. I hypothesize that the intestines regulate adipose tissue remodeling and inflammation by secreting signals including PlexinB2 to distinct immune cells populations in response to diet induced inflammation. Specifically, in Aim 1, I will elicit novel proteins trafficked from the intestines to adipose tissue immune cells, in mice. I will determine and validate the intestinally secreted proteins targeting adipose tissue immune cells during a high fat, fructose, and cholesterol diet which induces obesity. Then, in Aim 2, I will define the impact of enriched intestinal proteins on immune cell function. I will determine the effect of these identified secreted proteins, including PlexinB2, on macrophages, dendritic cells, T-cells and B-cells in vitro. Finally, I will demonstrate the biological significance of discovered proteins using in vivo models of overexpression as well as the effects our validated Plexin-B2 knock-out mouse has on immune cell function in vivo. Advised by Dr. Ilia Droujinine, co-advised by Dr. Enrique Saez, and in collaboration with Drs. Ben Cravatt, Lindsey Miles, and Howard Hang, my multidisciplinary training will enable me to advance proteomic tools to study organ-to-organ communication, and during its course to become an expert and an independent investigator in the field of metabolic disease.
