Haploid Mammalian Genetic Screen Identifies UBXD8 as a Key Determinant of HMGCR Degradation and Cholesterol Biosynthesis

• Published in: Arteriosclerosis, thrombosis, and vascular biology Vol. 37; no. 11; pp. 2064 - 2074
• Main Authors: Loregger, Anke, Raaben, Matthijs, Tan, Josephine, Scheij, Saskia, Moeton, Martina, van den Berg, Marlene, Gelberg-Etel, Hila, Stickel, Elmer, Roitelman, Joseph, Brummelkamp, Thijn, Zelcer, Noam
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 01.11.2017; Lippincott Williams & Wilkins
• Subjects: Animals; Basic Sciences; Blood Proteins - genetics; Blood Proteins - metabolism; Cholesterol - biosynthesis; CRISPR-Cas Systems; Endoplasmic Reticulum - enzymology; Enzyme Stability; Feedback, Physiological; Gene Expression Regulation, Enzymologic; Haploidy; Hep G2 Cells; Hepatocytes - enzymology; Humans; Hydroxymethylglutaryl CoA Reductases - genetics; Hydroxymethylglutaryl CoA Reductases - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mevalonic Acid - metabolism; Microscopy, Confocal; Proteasome Endopeptidase Complex - metabolism; Protein Transport; Proteolysis; Rats; Recombinant Fusion Proteins - metabolism; Transfection; Ubiquitination; cholesterol; sterols; hydroxymethylglutaryl CoA reductases; mammalian haploid genetics; oxysterols
• ISSN: 1079-5642; 1524-4636; 1524-4636
• DOI: 10.1161/ATVBAHA.117.310002

OBJECTIVE—The cellular demand for cholesterol requires control of its biosynthesis by the mevalonate pathway. Regulation of HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), a rate-limiting enzyme in this pathway and the target of statins, is a key control point herein. Accordingly, HMGCR is subject to negative and positive regulations. In particular, the ability of oxysterols and intermediates of the mevalonate pathway to stimulate its proteasomal degradation is an exquisite example of metabolically controlled feedback regulation. To define the genetic determinants that govern this process, we conducted an unbiased haploid mammalian genetic screen. APPROACH AND RESULTS—We generated human haploid cells with mNeon fused to endogenous HMGCR using CRISPR/Cas9 and used these cells to interrogate regulation of HMGCR abundance in live cells. This resulted in identification of known and new regulators of HMGCR, and among the latter, UBXD8 (ubiquitin regulatory X domain-containing protein 8), a gene that has not been previously implicated in this process. We demonstrate that UBXD8 is an essential determinant of metabolically stimulated degradation of HMGCR and of cholesterol biosynthesis in multiple cell types. Accordingly, UBXD8 ablation leads to aberrant cholesterol synthesis because of loss of feedback control. Mechanistically, we show that UBXD8 is necessary for sterol-stimulated dislocation of ubiquitylated HMGCR from the endoplasmic reticulum membrane en route to proteasomal degradation, a function dependent on its UBX domain. CONCLUSIONS—We establish UBXD8 as a previously unrecognized determinant that couples flux across the mevalonate pathway to control of cholesterol synthesis and demonstrate the feasibility of applying mammalian haploid genetics to study metabolic traits.Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.