ABSTRACT The prevalence of obesity in the United States continues to rise in both adults and children on an annual basis. Obesity is a risk factor for a multitude of chronic diseases, including type 2 diabetes and cardiovascular disease (CVD), among others. Adipocytes, the primary cells for fat accumulation, contain lipid droplets that expand in response to taking up excess circulating triglycerides (TG) as a protective mechanism to prevent organ lipotoxicity. Adipocytes contain approximately a quarter of the body’s pool of free cholesterol, but have a reduced ability to synthesize its own cholesterol. Therefore, adipocytes rely heavily on circulating lipoproteins to acquire the necessary amount of cholesterol for hypertrophic expansion. This also means that cholesterol transport is an essential determinant for adipocyte function. However, there remains a significant knowledge gap regarding the mechanism and regulation of cholesterol transport in adipocytes. In this application, we build on exciting preliminary data that shows that the bi-directional cholesterol transport functions of the HDL receptor, scavenger receptor class B type I (SR-BI), are compromised in the absence of procollagen peptidase endopeptidase enhancer 2 (PCPE2), an extracellular matrix protein, in mouse adipose tissue and adipocytes differentiated from mouse embryonic fibroblasts (MEFs). Based on these findings, experiments described in this fellowship proposal will test the novel hypothesis that PCPE2 facilitates SR-BI’s cholesterol transport functions to regulate adipocyte cholesterol homeostasis. Aim 1 is designed to determine the mechanism by which PCPE2 may be facilitating SR-BI oligomerization in adipocytes, a process required for cholesterol transport. We will use perfluorooctanoic acid polyacrylamide gel electrophoresis [PFO-PAGE] to test SR-BI oligomerization in the presence or absence of PCPE2. Further, using cutting-edge mass spectrometry technology, we will validate the SR-BI/PCPE2 interaction in adipocytes and determine if this complex contains other protein partners. Aim 2 is designed to investigate the mechanisms by which PCPE2 may impact SR-BI-mediated signaling and membrane localization. First, we will measure activation of signaling networks known to trigger HDL-cholesterol ester uptake (e.g. MAPK pathway). Next, we will perform discontinuous sucrose gradient fractionation methods to isolate adipocyte lipid raft microdomains to visualize SR-BI’s membrane localization in the absence and presence of PCPE2. Together, these studies will identify (1) the mechanism by which SR-BI and PCPE2 may physically interact to facilitate bidirectional cholesterol flux, and (2) how PCPE2 may be impacting SR-BI’s role in mediating bidirectional cholesterol flux in adipocytes. We anticipate that the findings from these studies will help identify novel information that will help us better understand the mechanisms driving obesity, cardiovascular disease, and other complications ...