Abstract 109: Preliminary Structural Into the Sterol Regulatory Element-Binding Protein (SREBP) Interaction With SREBP Cleavage-Activating Protein (SCAP)

Abstract

Maintenance of cholesterol and fatty acid homeostasis is critical for membrane architecture, protein localization, and protein trafficking. Dysregulation can lead to severe health concerns including obesity, diabetes, heart attack and stroke. Vital to the management of lipid molecules are the sterol regulatory element binding protein (SREBP) family of transcription factors, which transcribe more than 30 genes controlling lipid homeostasis. The activation of SREBP is tightly regulated through association with SREBP cleavage-activating protein (SCAP). When sterol levels are high, SCAP maintains SREBP in the endoplasmic reticulum. As sterol concentrations are reduced, SCAP escorts SREBP to the Golgi apparatus where it is activated. Liver-specific knockout of SCAP results in a 70-80% decrease in lipid biosynthesis and germline knockout is hypothesized to be embryonic lethal. Nonetheless, how SCAP recognizes and restricts the location of SREBP is relatively unknown. Structural insight to this protein-protein interface may yield future drug targets; however, their large molecular mass makes them challenging. We hypothesize both CTDs can be functionally produced in vitro to investigate the complex affinity, stoichiometry, and architecture. So far, we have recombinantly produced milligram quantities of both proteins. Circular dichroism and 1D NMR spectroscopy support in vitro SCAP refolding into a β-propeller fold. In contrast to previously published suggestions of a seven-bladed propeller motif, our sequence analysis and homology modeling support an eight-bladed architecture. Both X-ray crystallography and NMR are currently being pursued to elucidate the SCAP structure. In conclusion, recombinant expression of SCAP and SREBP is feasible and future research will use a combined biochemical and cell-based approach to determine the complex affinity, dynamics, and binding interface.