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  • Introduction Cholesterol plays a pivotal role as


    Introduction Cholesterol plays a pivotal role as a constituent of biological membranes and as a precursor for vitamins, hormones and bile acids. Accordingly, its production, distribution, and elimination must be tightly regulated at the cellular and organismal level. Accordingly, dysregulated cholesterol metabolism is extensively linked to a wide variety of human diseases, including atherosclerosis and the metabolic syndrome [1,2]. Cells can acquire cholesterol by taking up low-density lipoprotein (LDL) via the LDL-receptor (LDLR) pathway, or alternatively via de novo cholesterol biosynthesis through the mevalonate pathway. The synthesis of cholesterol by the mevalonate pathway requires the coordinated activity of >20 enzymes and is transcriptionally regulated by the sterol-regulatory Naproxen Sodium binding protein (SREBP) family of transcription factors [3]. However, next to transcriptional regulation, post-transcriptional control exquisitely governs abundance of the rate limiting enzymes Squalene Monooxygenase (SQLE) and 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGCR) [4]. Both enzymes are subject to robust degradation by the ubiquitin-proteasome system in response to metabolic cues [[5], [6], [7]]. Intriguingly, these cues differ for the two enzymes. Whereas proteasomal degradation of HMGCR is stimulated by lanosterol, intermediate metabolites of the biosynthetic pathway, and oxysterols [[8], [9], [10]], that of SQLE is primarily triggered by cholesterol, the end product of the pathway itself [5]. Another distinguishing feature of these processes is that whereas sterol-stimulated degradation of HMGCR requires INSIG proteins, that of SQLE does not [5,8,11]. The ability to differentially control the regulated degradation of these two rate-limiting enzymes may allow fine tuning of synthesis of important products of the isoprenoid branch of the mevalonate pathway, such as ubiquinone, dolichol and isoprenoids, independently from that of cholesterol itself [6,12]. Finally, the enzymes required for HMGCR and SQLE ubiquitylation and proteasomal degradation are different. Ubiquitylation follows the mandatory sequence of enzymatic reactions consisting of activating ubiquitin by an E1 enzyme, binding of ubiquitin to an E2 ubiquitin conjugating enzyme, and finally the ligation of ubiquitin onto the target protein that is supported by the E3 ubiquitin ligase [13]. Extensive research into the ubiquitylation cascade of HMGCR has implicated the E2 enzyme UBE2G2, as well as the E3 ubiquitin ligases gp78, TRC8 and RNF145 in its degradation [4,[14], [15], [16], [17], [18]], while the RING-type ubiquitin ligase MARCH6 has been shown to promote the degradation of SQLE [19]. However, the E2 enzyme(s) governing this process remains elusive. In yeast, the SQLE homologue Erg1 is degraded by the MARCH6 homologue Doa10, in cooperation with the E2 conjugating enzymes Ubc6 and Ubc7 [20]. However, a marked difference in complexity can be observed between ERAD in yeast versus mammalian cells [13,[21], [22], [23]]. We therefore used a CRISPR/Cas9-based screening approach to delineate the ERAD machinery responsible for MARCH6-dependent degradation of SQLE in mammalian cells and report here the identification of the E2 UBE2J2 as a critical determinant of this process.
    Materials and methods
    Results To identify the E2 conjugating enzyme responsible for MARCH6-dependent degradation of SQLE, we used a CRISPR/Cas9-based candidate-gene screening approach. We generated HEK293T cells lacking the E2 ubiquitin conjugating enzymes implicated in ERAD: UBE2J1, UBE2J2, and UBE2G2. We confirmed reduced transcript levels of the three E2s (Fig. 1A) and observed expression of FLAG-tagged Cas9 in the edited cells (Fig. 1B and C). In wild-type (WT) cells, HMGCR and SQLE are subject to rapid sterol-stimulated degradation (Supplementary Fig. 1). However, in UBE2J2- cells, sterol-stimulated degradation of SQLE, but not that of HMGCR, was largely attenuated (Fig. 1B). Of note, cells lacking UBE2G2 exhibited moderate attenuation of SQLE degradation, indicating that although UBE2J2 is the primary E2 necessary for MARCH6-dependent SQLE degradation in HEK293T cells, some redundancy between E2 enzymes in SQLE degradation may exist.