Monday, April 29, 2024
4/29/2024
PROJECT SUMMARY Flux through the mevalonate (MVA) pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)- localized HMG CoA reductase (HMGCR), the rate limiting enzyme in MVA pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. One mechanism for this control involves sterol-induced ER-associated degradation (ERAD). Previous studies revealed that ERAD of HMGCR is accelerated by two classes of sterols 1) methylated sterols such as the cholesterol synthesis intermediate 24, 25-dihydrolanosterol (DHL) and 2) oxysterols including 25-hydroxycholesterol (25HC). 25HC, but not DHL, blocks proteolytic activation of sterol regulatory element-binding proteins (SREBPs), another Insig-mediated reaction. HMGCR is the molecular target of statin drugs, long prescribed to lower LDL cholesterol; however, statins block ERAD of HMGCR, causing its accumulation that blunts the drugs’ efficacy. This proposal aims to gain mechanistic understanding for how sterols initiate HMGCR ERAD and provide insights on how targeting the reaction can be harnessed to prevent statin resistance. Studies show the 1,1-bisphosphonate ester SRP3042 mimics sterols in accelerating HMGCR ERAD. Photoactivatable srpDHY, a derivative of SRP3042, specifically crosslinks HMGCR, indicating sterols directly bind the enzyme to initiate ERAD. During the K99 phase of my training, I will use cryo-EM to determine how SRP3042 accelerates HMGCR ERAD. In the R00 phase, I will determine whether SRP3042 administered to mice can be used to block the statin-induced accumulation of hepatic HMGCR by accelerating the protein’s ERAD as a viable strategy to augment statin therapy. Lipid homeostasis is maintained by the SREBP family of transcription factors which modulate expression of enzymes required for lipid synthesis. Preliminary studies show the sterol-like molecule, LY295427 acts through a unique unknown mechanism to block 25HC-mediated events. These include inhibition of transcriptional regulation of LXR, regulation of acyl-coenzyme A cholesterol acyltransferase (ACAT) activity, and SREBP cleavage. However, LY295427 does not reverse 25HC-medated HMGCR ERAD. My R00 studies will identify the molecular target of LY295427 utilizing a variety of techniques including a CRISPR/Cas9 knockout screen, genomic siRNA screen, and a biochemical approach coupling click chemistry and mass spectrometry. My mentor, Dr. DeBose-Boyd (Professor of Molecular Genetics), employs a multi-disciplinary approach to explore regulatory mechanisms, and fully supports collaborative opportunities that will ensure my transition away from his interests. I have assembled a strong, scientifically diverse mentoring committee to aid my transition to independence. Completion of these studies will reveal novel lipid sensing pathways that can be harnessed for development of regulatory molecules for diseases associated with lipid dysregulation. Moreover, they will provide strong preliminary data for a future R01 application that will enhance my transition into an independent scientist.
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