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Post-translational control of the long and winding road to cholesterol

Open AccessPublished:December 18, 2020DOI:https://doi.org/10.1074/jbc.REV120.010723
      The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.
      Like most animals, humans need cholesterol, but too much can be harmful. Therefore, multiple layers of intricate interwoven mechanisms have evolved to keep cholesterol levels in check. Cholesterol is essential as the precursor for steroid hormones, bile acids, and oxysterols (Fig. 1) and as an important component of cell and organelle membranes. Providing strength and fluidity to the membrane, cholesterol acts as a barrier to permeability and an organizer of specialized membrane domains, notably lipid rafts, which can serve as signaling hubs (
      • Sezgin E.
      • Levental I.
      • Mayor S.
      • Eggeling C.
      The mystery of membrane organization: composition, regulation and roles of lipid rafts.
      ). However, excess cholesterol contributes to disease, notably atherosclerotic cardiovascular disease (
      • Wang N.
      • Fulcher J.
      • Abeysuriya N.
      • Park L.
      • Kumar S.
      • Di Tanna G.L.
      • Wilcox I.
      • Keech A.
      • Rodgers A.
      • Lal S.
      Intensive LDL cholesterol-lowering treatment beyond current recommendations for the prevention of major vascular events: a systematic review and meta-analysis of randomised trials including 327 037 participants.
      ), but it is also increasingly recognized to play a role in some cancers (
      • Huang B.
      • Song B.L.
      • Xu C.
      Cholesterol metabolism in cancer: mechanisms and therapeutic opportunities.
      ) and in neurodegenerative disease (
      • Martín M.G.
      • Pfrieger F.
      • Dotti C.G.
      Cholesterol in brain disease: sometimes determinant and frequently implicated.
      ). Therefore, balancing cholesterol to ensure levels are adequate but not excessive is crucial.
      Figure thumbnail gr1
      Figure 1Branches and alternative end products of the cholesterol synthesis pathway. Cholesterol synthesis proceeds via the early pathway, which converts acetyl-CoA to lanosterol, and the post-lanosterol pathway, which converts lanosterol to cholesterol through the Bloch or modified Kandutsch–Russell pathways. Double-headed arrows indicate multiple enzymatic steps. For the effect of cholesterol on the post-translational regulation of pathway enzymes, refer to . Alternative end products are formed by branches of the cholesterol synthesis pathway. The early pathway intermediate farnesyl diphosphate is the precursor to isoprenoids such as geranylgeranyl pyrophosphate, which augments the sterol-induced degradation of HMGCR. The Kandutsch–Russell pathway intermediate 7-dehydrocholesterol is the precursor to vitamin D, which promotes the degradation of the downstream enzyme DHCR7 in keratinocytes. Cholesterol itself can be converted to other products such as bile acids, steroid hormones, and oxysterols.
      Omnivores ingest some cholesterol, but humans generally synthesize considerably more cholesterol than we consume (
      • Brown A.J.
      • Sharpe L.J.
      Cholesterol synthesis.
      ). Building from acetyl-CoA and harnessing more than 20 enzymes, cholesterol synthesis is an especially energy-intensive process, providing another reason why exquisite regulation is required (
      • Brown A.J.
      • Sharpe L.J.
      Cholesterol synthesis.
      ). Many of these mechanisms are feedback loops, triggered when cholesterol levels accumulate sufficiently that no more needs to be made. Thus, almost all genes in the cholesterol synthesis pathway are induced by the sterol-regulatory element binding protein-2 transcription factor when cholesterol levels are low and down-regulated when cholesterol levels are high (
      • Brown M.S.
      • Radhakrishnan A.
      • Goldstein J.L.
      Retrospective on cholesterol homeostasis: the central role of Scap.
      ). This relatively well-studied transcriptional program allows gradual oscillations of control. Layered on top is the more acute post-translational regulation, including inactivation and demolition of individual enzymes, for instance by phosphorylation and the ubiquitin–proteasome system, respectively.
      Cholesterol is not the only significant sterol in humans, and like oxysterols (
      • Brown A.J.
      • Sharpe L.J.
      • Rogers M.J.
      Oxysterols: from physiological tuners to pharmacological opportunities.
      ), intermediates in cholesterol synthesis often exhibit biological activities very different from cholesterol itself (
      • Brown A.J.
      • Ikonen E.
      • Olkkonen V.M.
      Cholesterol precursors: more than mere markers of biosynthesis.
      ). Indeed, in recent years intermediates in the cholesterol synthesis pathway have been implicated in a range of biological and pathological contexts, including immunity (
      • Bekkering S.
      • Arts R.J.W.
      • Novakovic B.
      • Kourtzelis I.
      • van der Heijden C.
      • Li Y.
      • Popa C.D.
      • Ter Horst R.
      • van Tuijl J.
      • Netea-Maier R.T.
      • van de Veerdonk F.L.
      • Chavakis T.
      • Joosten L.A.B.
      • van der Meer J.W.M.
      • Stunnenberg H.
      • et al.
      Metabolic induction of trained immunity through the mevalonate pathway.
      ), neurological conditions (
      • Segatto M.
      • Tonini C.
      • Pfrieger F.W.
      • Trezza V.
      • Pallottini V.
      Loss of mevalonate/cholesterol homeostasis in the brain: a focus on autism spectrum disorder and Rett syndrome.
      ), and cancers (
      • Gabitova L.
      • Restifo D.
      • Gorin A.
      • Manocha K.
      • Handorf E.
      • Yang D.-H.
      • Cai K.Q.
      • Klein-Szanto A.J.
      • Cunningham D.
      • Kratz L.E.
      • Herman G.E.
      • Golemis E.A.
      • Astsaturov I.
      Endogenous sterol metabolites regulate growth of EGFR/KRAS-dependent tumors via LXR.
      ). This provides yet more impetus to better understand how flux through the pathway is controlled.

      Cholesterol synthesis pathway

      The cholesterol synthesis pathway (Table 1) encompasses 22 enzymes and can be divided almost equally into the early sterol synthesis pathway up to lanosterol (the first true sterol) and the post-lanosterol pathway, which has two parallel branches leading to cholesterol (Fig. 1). The early pathway contains HMGCR, the classic control point and well-studied target of the statins, first-line therapy for cardiovascular disease. Also in the early pathway is a second rate-limiting enzyme, SM, which is exquisitely regulated, but in different ways than HMGCR (Fig. 2). The post-lanosterol enzymes also have several interesting modes of regulation that provide yet more control mechanisms regulating this highly intricate pathway.
      Table 1Cholesterol synthesis enzymes
      EnzymeProtein name (UniProt)UniProt identifierECGene symbol
      Early pathwayACAT2Acetyl-CoA acetyltransferase, cytosolicQ9BWD12.3.1.9ACAT2
      HMGCSHydroxymethylglutaryl-CoA synthase, cytoplasmicQ015812.3.3.10HMGCS1
      HMGCR3-Hydroxy-3-methylglutaryl-coenzyme A reductaseP040351.1.1.34HMGCR
      MVKMevalonate kinaseQ034262.7.1.36MVK
      PMVKPhosphomevalonate kinaseQ151262.7.4.2PMVK
      MVDDiphosphomevalonate decarboxylaseP536024.1.1.33MVD
      IDI1/IDI2Isopentenyl-diphosphate Δ-isomerase 1/2Q13907/Q9BXS15.3.3.2IDI1/2
      FDPSFarnesyl pyrophosphate synthaseP143242.5.1.10, 2.5.1.1FDPS
      GGPPSGeranylgeranyl pyrophosphate synthaseO957492.5.1.1, 2.5.1.10, 2.5.1.29GGPS1
      SQSSqualene synthaseP372682.5.1.21FDFT1
      SMSqualene monooxygenaseQ145341.14.13.132SQLE
      LSSLanosterol synthaseP484495.4.99.7LSS
      Post-lanosterol pathwayLDMLanosterol 14-α-demethylaseQ168501.14.13.70CYP51A1
      DHCR14Δ14-Sterol reductase TM7SF2O760621.3.1.70TM7SF2
      LBRΔ14-Sterol reductase LBRQ147391.3.1.70LBR
      SC4MOLMethylsterol monooxygenase 1Q158001.14.13.72SC4MOL
      NSDHLSterol-4-α-carboxylate 3-dehydrogenase, decarboxylatingQ157381.1.1.170NSDHL
      HSD17B73-Keto-steroid reductaseP569371.1.1.270HSD17B7
      EBP3-β-Hydroxysteroid-Δ87-isomeraseQ151255.3.3.5EBP
      SC5DLathosterol oxidaseO758451.14.21.6SC5D
      DHCR77-Dehydrocholesterol reductaseQ9UBM71.3.1.21DHCR7
      DHCR24Δ24-Sterol reductaseQ153921.3.1.72DHCR24
      Figure thumbnail gr2
      Figure 2Post-translational regulation of the long and winding cholesterol synthesis pathway. Cholesterol synthesis can be divided into the early pathway and post-lanosterol pathway, which in turn proceeds via the Bloch pathway or the Kandutsch–Russell pathway (containing C24-saturated derivatives of Bloch pathway intermediates). Depicted is the more widely utilized modified Kandutsch–Russell pathway, which bypasses upstream intermediates and downstream entry points. For simplicity, chemical structures are shown only for the sterol intermediates at the start and end of the Bloch and modified Kandutsch–Russell pathways. Red broken lines and inhibitory arrows indicate intermediates and E3 ligases that promote the degradation of HMGCR, whereas the red solid lines indicate intermediates that promote the degradation of other cholesterol synthesis enzymes (inhibitory arrows) or stabilize MARCHF6 (arrowheads). Note that for clarity, we have indicated only the major contributors to degradation of each enzyme. It is also likely that additional intermediates promote degradation of some enzymes, but these have yet to be explored. The E3 ubiquitin ligase MARCHF6 targets the enzymes indicated in purple. For more details, please refer to the text.
      From lanosterol, the pathway can take one of two intertwined routes, creating a long and winding road to cholesterol through various branch points and side tracks (Fig. 1). Lanosterol can be acted upon by LDM to enter the Bloch pathway, or DHCR24 to enter the Kandutsch–Russell pathway, both of which use the same enzymes to ultimately produce cholesterol, via distinct intermediates (Fig. 2). DHCR24 can theoretically act on any intermediate from lanosterol through to desmosterol to transfer intermediates from the Bloch to the Kandutsch–Russell pathway. A modified Kandutsch–Russell pathway (
      • Mitsche M.A.
      • McDonald J.G.
      • Hobbs H.H.
      • Cohen J.C.
      Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways.
      ) is believed to be more widely utilized, in which DHCR24 converts zymosterol to zymostenol as the major step between the pathways (Fig. 2). Cholesterol synthesis occurs largely in the endoplasmic reticulum (ER), and this is where most enzymes are localized. In fact, there is some evidence that they interact with each other to optimize efficiency of cholesterol synthesis.
      In 2013, we reviewed the control of cholesterol synthesis by enzymes beyond the classic control point, HMGCR (
      • Sharpe L.J.
      • Brown A.J.
      Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR).
      ). At the time, only degradation of HMGCR and SM was known, but we now know that at least four additional enzymes are subject to controlled degradation. In 2013, degradation by E3 ligases was only partially explored for HMGCR, but now we know that five E3 ligases may be involved in its degradation, including MARCHF6, which also degrades at least three more cholesterol synthesis enzymes. This expanding field of research is uncovering many complexities that involve intricate regulation of a large number of enzymes along the pathway. We anticipate that each enzyme will have its own story to tell in due course. Here, we delve into the latest developments on the post-translational control of cholesterol synthesis, notably the degradation of its enzymes. We begin by introducing some post-translational modifications and then explore in depth the regulatory mechanisms of the enzymes in the pathway.

      Post-translational modifications

      Post-translational regulation of enzymes often occurs by post-translational modifications (PTMs), such as ubiquitination (also called ubiquitylation) and phosphorylation, which typically lead to degradation or altered activity, respectively. To survey the PTM landscape of the cholesterol synthesis pathway, we utilized several databases (notably, PhosphoSite Plus (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      )), alongside targeted literature searches.
      Of the >450 known PTMs (
      The UniProt Consortium
      UniProt: the universal protein knowledgebase.
      ), the most commonly reported on cholesterol synthesis enzymes are ubiquitination, phosphorylation, and acetylation, in that order. There are a handful of enzymes that are SUMOylated, monomethylated, and/or succinylated on specific lysine residues. Only one enzyme is known to be glycosylated (N-linked glycosylation on HMGCR) (
      • Zielinska D.F.
      • Gnad F.
      • Wiśniewski J.R.
      • Mann M.
      Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.
      ), and one is both O-GalNAc and O-GlcNAc modified (EBP (
      • Myers S.A.
      • Panning B.
      • Burlingame A.L.
      Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells.
      ,
      • Steentoft C.
      • Vakhrushev S.Y.
      • Joshi H.J.
      • Kong Y.
      • Vester-Christensen M.B.
      • Schjoldager K.T.
      • Lavrsen K.
      • Dabelsteen S.
      • Pedersen N.B.
      • Marcos-Silva L.
      • Gupta R.
      • Bennett E.P.
      • Mandel U.
      • Brunak S.
      • Wandall H.H.
      • et al.
      Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.
      )). Although one might expect modification with lipid anchors such as prenylation or long-chain fatty acylation given the membranous location of most of the enzymes, no such modifications are reported.
      When examining the extent of PTMs in the context of the total human proteome (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ,
      The UniProt Consortium
      UniProt: a worldwide hub of protein knowledge.
      ), cholesterol synthesis enzymes tended to be more heavily ubiquitinated but less phosphorylated, particularly at serine and threonine residues (Fig. 3; for details of modified sites, please refer to Table S1). PTMs are continually being added to the PhosphoSite database, and so there are likely many more modifications on these enzymes that are yet to be discovered. This is particularly true for enzymes that are likely to be under-represented in large-scale proteomic studies, such as IDI2, which has low expression except in muscle (
      • Clizbe D.B.
      • Owens M.L.
      • Masuda K.R.
      • Shackelford J.E.
      • Krisans S.K.
      IDI2, a second isopentenyl diphosphate isomerase in mammals.
      ), and DHCR14, which is lowly abundant in many tissues (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ), as well as being particularly hydrophobic (Fig. 3), which may hinder mass spectrometric analysis. Another important note is that each enzyme is very likely to have a large number of “proteoforms,” a term adopted to refer to the complexity of proteins taking into account each possible variation, including its modifications (
      • Smith L.M.
      • Kelleher N.L.
      Consortium for Top Down Proteomics
      Proteoform: a single term describing protein complexity.
      ).
      Figure thumbnail gr3
      Figure 3Post-translational modifications on cholesterol synthesis enzymes. For each enzyme, length (aa) and sequence were obtained from UniProt (
      The UniProt Consortium
      UniProt: a worldwide hub of protein knowledge.
      ) (the identifiers are listed in ). Hydrophobicity was determined by the percentage of hydrophobic residues, grand average of hydropathy (gravy) score (where a positive score indicates hydrophobic and a negative score indicates hydrophilic), and predicted number of transmembrane domains (mem, determined by TOPCONS (
      • Tsirigos K.D.
      • Peters C.
      • Shu N.
      • Käll L.
      • Elofsson A.
      The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides.
      ), as a measure of the likelihood of membrane association). The post-translational modifications for each enzyme were obtained from PhosphoSite (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ) and presented as the total percentage of modified residues per enzyme (PTM), and the percentage of each specific residue that has a particular modification (e.g. %S indicates the percentage of serine residues in that enzyme that are known to be phosphorylated). Median values are for the cholesterol synthesis enzymes. Proteome values are the median values based on a median length of ∼400 amino acids (
      • Brocchieri L.
      • Karlin S.
      Protein length in eukaryotic and prokaryotic proteomes.
      ) with amino acid distribution as per UniProt (
      The UniProt Consortium
      UniProt: a worldwide hub of protein knowledge.
      ). For the percentage of modified residues, all modifications from PhosphoSite (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ) were compared with the composition of the proteome. The intensity of shading reflects increasing values in the mem column and collectively for the PTM columns. For more details, please see . Ub, ubiquitination; Ac, acetylation.

      Ubiquitin–proteasome system (UPS)

      The UPS involves the conjugation of ubiquitin to substrates through an E1 activating enzyme, E2 conjugating enzyme, and an E3 ubiquitin ligase. The mammalian genome encodes two E1s and ∼40 E2s, but many hundreds of E3s (
      • Zheng N.
      • Shabek N.
      Ubiquitin ligases: structure, function, and regulation.
      ). Therefore, the E3 ligases provide specificity toward particular target proteins and are classified into one of three families: the majority comprise the RING ligases, and the remainder are either HECT or U-box ligases. Ubiquitin is typically conjugated to lysine residues of the target protein but can less commonly be attached to noncanonical residues (serine, threonine, and cysteine), as well as to the N-terminal amine. To add another level of sophistication, there are ∼100 deubiquitinating enzymes, with the ability to remove, or at least trim, the polyubiquitin chains (
      • Clague M.J.
      • Urbé S.
      • Komander D.
      Breaking the chains: deubiquitylating enzyme specificity begets function.
      ).
      There are several E3 ligases implicated in the control of HMGCR (Gp78, TRC8, HRD1, MARCHF6, and RNF145) (
      • Jo Y.
      • Lee P.C.
      • Sguigna P.V.
      • DeBose-Boyd R.A.
      Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.
      ,
      • Menzies S.A.
      • Volkmar N.
      • van den Boomen D.J.
      • Timms R.T.
      • Dickson A.S.
      • Nathan J.A.
      • Lehner P.J.
      The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.
      ,
      • Tsai Y.C.
      • Leichner G.S.
      • Pearce M.M.
      • Wilson G.L.
      • Wojcikiewicz R.J.
      • Roitelman J.
      • Weissman A.M.
      Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.
      ,
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ), and one E3 ligase, MARCHF6, targets four enzymes in the cholesterol synthesis pathway (SM and HMGCR (
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ), LDM and DHCR24 (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      )). These all lead to degradation of the target enzymes. Deubiquitinases are involved in the regulation of SM, although their identities remain unknown (
      • Chua N.K.
      • Scott N.A.
      • Brown A.J.
      Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron.
      ). The deubiquitinase USP19 has been implicated in the regulation of both HRD1 (
      • Harada K.
      • Kato M.
      • Nakamura N.
      USP19-mediated deubiquitination facilitates the stabilization of HRD1 ubiquitin ligase.
      ) and MARCHF6 (
      • Nakamura N.
      • Harada K.
      • Kato M.
      • Hirose S.
      Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6.
      ), suggesting an indirect role in controlling cholesterol synthesis.
      Many ubiquitination sites on cholesterol synthesis enzymes (Table S1) are yet to be explored for their functional consequences. Several enzymes are rapidly degraded by the UPS, typically in response to changing sterol conditions, although the sterol intermediates promoting degradation can be different for each enzyme. In general, the earlier intermediates feedback more effectively on the earlier enzymes, and the later intermediates affect the later enzymes (Fig. 2). Thus, the first sterol lanosterol can feed back to accelerate degradation of the first rate-limiting enzyme, HMGCR, whereas cholesterol itself feeds back to signal destruction of the second rate-limiting enzyme, SM. This segmental control would enable HMGCR to continue making essential isoprenoids when cholesterol levels are high (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ).

      Phosphorylation

      Phosphorylation adds a phosphate group to a serine, threonine, or tyrosine residue by a kinase. The best-understood and most commonly known post-translational modification, phosphorylation is typically associated with signaling pathways and rapid control of downstream targets. In the context of cholesterol synthesis, there are at least three enzymes that are controlled by their phosphorylated residues: HMGCR (
      • Gillespie J.G.
      • Hardie D.G.
      Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes.
      ), DHCR7 (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Phosphorylation regulates activity of 7-dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis.
      ), and DHCR24 (
      • Luu W.
      • Zerenturk E.J.
      • Kristiana I.
      • Bucknall M.P.
      • Sharpe L.J.
      • Brown A.J.
      Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.
      ). These modifications lead to a change in the activity of the enzyme with consequences for cholesterol synthesis.

      Acetylation

      Acetylation is the conjugation of acetate to lysine residues or the N-terminal amine. Protein acetylation, long known for regulating transcription in the nucleus, is increasingly being recognized to play important regulatory roles in metabolism. Virtually every enzyme in several metabolic processes (e.g. glycolysis, the urea cycle, fatty acid metabolism, and glycogen metabolism) is acetylated in human liver tissue (
      • Zhao S.
      • Xu W.
      • Jiang W.
      • Yu W.
      • Lin Y.
      • Zhang T.
      • Yao J.
      • Zhou L.
      • Zeng Y.
      • Li H.
      • Li Y.
      • Shi J.
      • An W.
      • Hancock S.M.
      • He F.
      • et al.
      Regulation of cellular metabolism by protein lysine acetylation.
      ). Furthermore, the effects of acetylation appear to be coordinated to simultaneously shunt metabolic flux down specific pathways and away from others. Interestingly, acetylation is over-represented on the initial two enzymes of cholesterol synthesis (Fig. 3) immediately downstream of acetyl-CoA (Fig. 2). Considering that acetyl-CoA is the starting material for cholesterol synthesis, reversible acetylation may well contribute to flux control through the pathway.

      Interplay between PTMs

      The various PTMs no doubt interact to varying degrees, helping integrate diverse signals and multiplying their regulatory potential (
      • Narita T.
      • Weinert B.T.
      • Choudhary C.
      Functions and mechanisms of non-histone protein acetylation.
      ). For instance, of the 48 acetylated residues recorded to date across all cholesterol synthesis enzymes, 41 (85%) are also reported to be ubiquitinated, which may indicate competitive cross-talk (
      • Yang X.J.
      • Seto E.
      Lysine acetylation: codified crosstalk with other posttranslational modifications.
      ). Conceivably, direct competition between acetylation and ubiquitination for modification of the same lysine residues (
      • Narita T.
      • Weinert B.T.
      • Choudhary C.
      Functions and mechanisms of non-histone protein acetylation.
      ) could reduce degradation and hence preserve enzyme levels. This may favor the increased cholesterol synthesis found in the fed state (
      • Jones P.J.
      Use of deuterated water for measurement of short-term cholesterol synthesis in humans.
      ) in which the starting material acetyl-CoA accumulates in the cytosol (
      • Narita T.
      • Weinert B.T.
      • Choudhary C.
      Functions and mechanisms of non-histone protein acetylation.
      ). SUMOylation, methylation, and succinylation may additionally compete on the same lysine residue, further expanding the repertoire of regulation. Phosphorylation and ubiquitination may also functionally interact (
      • Swaney D.L.
      • Beltrao P.
      • Starita L.
      • Guo A.
      • Rush J.
      • Fields S.
      • Krogan N.J.
      • Villén J.
      Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation.
      ), and indeed we observed 44% of phosphorylated residues and 27% of ubiquitinated residues within 3 amino acids of another modification among the cholesterol synthesis enzymes (Table S1). Proximity to other modified residues is considered a good predictor of functional sites (
      • Beltrao P.
      • Albanèse V.
      • Kenner L.R.
      • Swaney D.L.
      • Burlingame A.
      • Villén J.
      • Lim W.A.
      • Fraser J.S.
      • Frydman J.
      • Krogan N.J.
      Systematic functional prioritization of protein posttranslational modifications.
      ), although what proportion of modified residues overall can be considered functional is unclear. Further work is needed to decode the clamorous cross-talk between PTMs across the cholesterol synthesis pathway. However, rather than the high-throughput methods that first identified the PTMs, low-throughput methods are required to pinpoint effects on individual enzymes. This has presented a serious bottleneck for phosphorylation studies, which will be eased by innovations like machine-based learning approaches that help to prioritize likely functional sites (
      • Ochoa D.
      • Jarnuczak A.F.
      • Viéitez C.
      • Gehre M.
      • Soucheray M.
      • Mateus A.
      • Kleefeldt A.A.
      • Hill A.
      • Garcia-Alonso L.
      • Stein F.
      • Krogan N.J.
      • Savitski M.M.
      • Swaney D.L.
      • Vizcaíno J.A.
      • Noh K.-M.
      • et al.
      The functional landscape of the human phosphoproteome.
      ). PTMs are major inputs in the regulation of cholesterol synthesis, and below we will discuss how these modifications affect individual enzymes, as well as other regulatory mechanisms.

      Early pathway enzymes

      We will focus on the enzymes that have had the most detailed research into their regulation. For the early enzymes, only HMGCR and SM have significant work behind them, with only limited studies into the other enzymes, perhaps because they are not known to be rate-limiting and therefore have not been considered worthy of investigation. Interestingly, phosphomevalonate kinase is the smallest enzyme and has all of its lysines ubiquitinated (Fig. 3), although it is currently unknown whether these have any functional consequences. MVK is best known for its role in mevalonate kinase deficiency (
      • Favier L.A.
      • Schulert G.S.
      Mevalonate kinase deficiency: current perspectives.
      ), although this is linked to the decreased production of isoprenoids in a branch pathway (Fig. 1) rather than the production of cholesterol. Some pathogenic mutations in MVK cause rapid proteasomal degradation of the enzyme (
      • Zhu T.
      • Tian D.
      • Zhang L.
      • Xu X.
      • Xia K.
      • Hu Z.
      • Xiong Z.
      • Tan J.
      Novel mutations in mevalonate kinase cause disseminated superficial actinic porokeratosis.
      ). MVK has also been identified as the luteinizing hormone receptor mRNA-binding protein, for which its role is regulated by SUMOylation, with no known consequence for cholesterol synthesis (
      • Wang L.
      • Gulappa T.
      • Menon K.M.J.
      Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.
      ). Building on studies in Caenorhabditis elegans (
      • Sapir A.
      • Tsur A.
      • Koorman T.
      • Ching K.
      • Mishra P.
      • Bardenheier A.
      • Podolsky L.
      • Bening-Abu-Shach U.
      • Boxem M.
      • Chou T.F.
      • Broday L.
      • Sternberg P.W.
      Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging.
      ), it has been proposed that regulated SUMOylation of HMGCS, along with its ubiquitin–proteasomal degradation, controls the activity of this enzyme with age. Further research into the early enzymes is warranted to uncover regulatory mechanisms that for example control flux entering the sterol versus isoprenoid branches of the pathway. Considering the large number of PTMs on these enzymes (Fig. 3), we believe that many worthwhile findings will eventuate.

      The classic control enzyme: HMGCR

      The largest of the enzymes (Fig. 3), HMGCR catalyzes the first rate-limiting step of the mevalonate pathway, converting HMG-CoA to mevalonate. In a quintessential example of metabolic feedback regulation, its proteasomal degradation is accelerated by the accumulation of pathway intermediates and cholesterol derivatives (Fig. 2). These include C4-dimethylated sterols, in particular lanosterol and its C24-saturated derivative 24,25-dihydrolanosterol (
      • Chen L.
      • Ma M.Y.
      • Sun M.
      • Jiang L.Y.
      • Zhao X.T.
      • Fang X.X.
      • Man Lam S.
      • Shui G.H.
      • Luo J.
      • Shi X.J.
      • Song B.L.
      Endogenous sterol intermediates of the mevalonate pathway regulate HMGCR degradation and SREBP-2 processing.
      ,
      • Song B.L.
      • Javitt N.B.
      • DeBose-Boyd R.A.
      Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol.
      ,
      • Coates H.W.
      • Brown A.J.
      A wolf in sheep's clothing: unmasking the lanosterol-induced degradation of HMG-CoA reductase.
      ), as well as side-chain oxysterols such as 25- and 27-hydroxycholesterol (
      • Song B.L.
      • Javitt N.B.
      • DeBose-Boyd R.A.
      Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol.
      ,
      • Song B.L.
      • DeBose-Boyd R.A.
      Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase.
      ). However, cholesterol itself does not promote HMGCR turnover (
      • Song B.L.
      • DeBose-Boyd R.A.
      Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase.
      ).
      The sterol-induced degradation of HMGCR has been intensely studied and is initiated through a tripartite mechanism. First, HMGCR contains a sterol-sensing transmembrane region (
      • Skalnik D.G.
      • Narita H.
      • Kent C.
      • Simoni R.D.
      The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.
      ) that includes ubiquitination sites required for its regulated degradation (Lys-89 and Lys-248) (
      • Sever N.
      • Song B.L.
      • Yabe D.
      • Goldstein J.L.
      • Brown M.S.
      • DeBose-Boyd R.A.
      Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
      ). A homologous sterol-sensing domain exists in Scap (sterol-regulatory element binding protein-cleavage activating protein), which directly binds to its regulator cholesterol (
      • Radhakrishnan A.
      • Sun L.P.
      • Kwon H.J.
      • Brown M.S.
      • Goldstein J.L.
      Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain.
      ), but interaction with lanosterol or other sterols is yet to be confirmed for HMGCR. Second, ER-resident Insig (insulin-induced gene) proteins are recruited to the HMGCR sterol-sensing domain (
      • Sever N.
      • Yang T.
      • Brown M.S.
      • Goldstein J.L.
      • DeBose-Boyd R.A.
      Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.
      ). This provides a scaffold for the binding and activity of cognate E3 ligases, the third component of the cascade. A suite of E3 ligases are implicated in sterol-mediated degradation of HMGCR, and each E3 ligase may help fine-tune the metabolic regulation of this critical enzyme. Most recently, CRISPR/Cas9 screens identified RNF145 and gp78 as the major regulators of HMGCR, with Hrd1 playing a minor role only when the former two proteins are absent (
      • Menzies S.A.
      • Volkmar N.
      • van den Boomen D.J.
      • Timms R.T.
      • Dickson A.S.
      • Nathan J.A.
      • Lehner P.J.
      The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.
      ). RNF145 transcription is sterol-regulated (
      • Menzies S.A.
      • Volkmar N.
      • van den Boomen D.J.
      • Timms R.T.
      • Dickson A.S.
      • Nathan J.A.
      • Lehner P.J.
      The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.
      ,
      • Cook E.C.
      • Nelson J.K.
      • Sorrentino V.
      • Koenis D.
      • Moeton M.
      • Scheij S.
      • Ottenhoff R.
      • Bleijlevens B.
      • Loregger A.
      • Zelcer N.
      Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene.
      ), whereas gp78 targets Insigs for degradation in the absence of sterols (
      • Lee J.N.
      • Song B.
      • DeBose-Boyd R.A.
      • Ye J.
      Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78.
      ,
      • Liu T.F.
      • Tang J.J.
      • Li P.S.
      • Shen Y.
      • Li J.G.
      • Miao H.H.
      • Li B.L.
      • Song B.L.
      Ablation of gp78 in liver improves hyperlipidemia and insulin resistance by inhibiting SREBP to decrease lipid biosynthesis.
      ), adding further intricacy to the regulatory circuit controlling HMGCR abundance. It is also proposed that the E3 ligases Trc8 (
      • Jo Y.
      • Lee P.C.
      • Sguigna P.V.
      • DeBose-Boyd R.A.
      Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.
      ) and MARCHF6 (
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ) affect HMGCR turnover, although the former result is contested by some researchers (
      • Tsai Y.C.
      • Leichner G.S.
      • Pearce M.M.
      • Wilson G.L.
      • Wojcikiewicz R.J.
      • Roitelman J.
      • Weissman A.M.
      Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.
      ), and the latter may occur through indirect mechanisms (
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ). It seems likely that unique cellular or tissue-specific contexts will dictate which E3 ligase is the major regulator of HMGCR levels at any one time.
      Importantly, the sterol-regulatory network that governs HMGCR levels has been confirmed to be functional in vivo in mouse models. Knockout of gp78 stabilizes HMGCR and up-regulates its activity in mouse liver (
      • Liu T.F.
      • Tang J.J.
      • Li P.S.
      • Shen Y.
      • Li J.G.
      • Miao H.H.
      • Li B.L.
      • Song B.L.
      Ablation of gp78 in liver improves hyperlipidemia and insulin resistance by inhibiting SREBP to decrease lipid biosynthesis.
      ), whereas intake of dimethylated sterol analogues leads to depletion of hepatic HMGCR and reduced serum cholesterol (
      • Jiang S.-Y.
      • Li H.
      • Tang J.-J.
      • Wang J.
      • Luo J.
      • Liu B.
      • Wang J.-K.
      • Shi X.-J.
      • Cui H.-W.
      • Tang J.
      • Yang F.
      • Qi W.
      • Qiu W.-W.
      • Song B.-L.
      Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol.
      ). Furthermore, levels of a transgenic protein comprising the sterol-sensing domain of HMGCR are increased or decreased by statin or cholesterol feeding, respectively (
      • Hwang S.
      • Hartman I.Z.
      • Calhoun L.N.
      • Garland K.
      • Young G.A.
      • Mitsche M.A.
      • McDonald J.
      • Xu F.
      • Engelking L.
      • DeBose-Boyd R.A.
      Contribution of accelerated degradation to feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol metabolism in the liver.
      ), in agreement with previous cell culture experiments (
      • Skalnik D.G.
      • Narita H.
      • Kent C.
      • Simoni R.D.
      The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.
      ). The same study also showed that knock-in of a sterol-resistant HMGCR K89R/K248R mutant leads to its accumulation in the liver and impedes its down-regulation during cholesterol feeding (
      • Hwang S.
      • Hartman I.Z.
      • Calhoun L.N.
      • Garland K.
      • Young G.A.
      • Mitsche M.A.
      • McDonald J.
      • Xu F.
      • Engelking L.
      • DeBose-Boyd R.A.
      Contribution of accelerated degradation to feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol metabolism in the liver.
      ). This collective understanding of sterol-induced HMGCR degradation will undoubtedly be of importance in the future development of cholesterol-lowering therapeutics. Such treatments may be useful in augmenting or substituting statins, which can lose efficacy over time or lead to severe withdrawal effects (
      • Pineda A.
      • Cubeddu L.X.
      Statin rebound or withdrawal syndrome: does it exist?.
      ) because of compensatory up-regulation of HMGCR gene expression and a decline in degradation-promoting sterol intermediates (
      • Jiang S.-Y.
      • Li H.
      • Tang J.-J.
      • Wang J.
      • Luo J.
      • Liu B.
      • Wang J.-K.
      • Shi X.-J.
      • Cui H.-W.
      • Tang J.
      • Yang F.
      • Qi W.
      • Qiu W.-W.
      • Song B.-L.
      Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol.
      ).
      Following its ubiquitination, HMGCR is extracted from the ER membrane by the sequential actions of the AAA+ ATPase VCP and the 19S regulatory particle of the proteasome (
      • Morris L.L.
      • Hartman I.Z.
      • Jun D.J.
      • Seemann J.
      • DeBose-Boyd R.A.
      Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)–associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
      ), enabling its degradation. Interestingly, this process is augmented by geranylgeraniol, a derivative of the endogenous isoprenoid geranylgeranyl pyrophosphate (Fig. 1) (
      • Sever N.
      • Song B.L.
      • Yabe D.
      • Goldstein J.L.
      • Brown M.S.
      • DeBose-Boyd R.A.
      Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
      ,
      • Morris L.L.
      • Hartman I.Z.
      • Jun D.J.
      • Seemann J.
      • DeBose-Boyd R.A.
      Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)–associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
      ,
      • Leichner G.S.
      • Avner R.
      • Harats D.
      • Roitelman J.
      Metabolically regulated endoplasmic reticulum-associated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase: evidence for requirement of a geranylgeranylated protein.
      ). The effects of this molecule are mediated at least in part by the prenyltransferase UBIAD1, which binds HMGCR and impedes its membrane extraction and subsequent degradation (
      • Schumacher M.M.
      • Elsabrouty R.
      • Seemann J.
      • Jo Y.
      • DeBose-Boyd R.A.
      The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.
      ). In the presence of its substrate geranylgeranyl pyrophosphate, UBIAD1 is sequestered to the Golgi and no longer able to inhibit HMGCR degradation (
      • Schumacher M.M.
      • Elsabrouty R.
      • Seemann J.
      • Jo Y.
      • DeBose-Boyd R.A.
      The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.
      ). Therefore, nonsterol mevalonate pathway products also control HMGCR levels, consistent with its key position upstream of the isoprenoid branch of the pathway. The importance of this accessory mode of regulation in whole-body metabolism is demonstrated by the embryonic lethality of UBIAD1 deficiency in mice, which can be rescued by the knock-in of degradation-resistant HMGCR (
      • Jo Y.
      • Kim S.S.
      • Garland K.
      • Fuentes I.
      • DiCarlo L.M.
      • Ellis J.L.
      • Fu X.
      • Booth S.L.
      • Evers B.M.
      • DeBose-Boyd R.A.
      Enhanced ER-associated degradation of HMG CoA reductase causes embryonic lethality associated with Ubiad1 deficiency.
      ).
      Beyond ubiquitination, HMGCR is also subject to regulation by phosphorylation. Here, the energy sensor AMPK phosphorylates Ser-872 within the HMGCR catalytic domain (
      • Clarke P.R.
      • Hardie D.G.
      Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.
      ), inactivating the enzyme and curtailing flux through the energy-intensive mevalonate pathway (
      • Sato R.
      • Goldstein J.L.
      • Brown M.S.
      Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion.
      ). Ser-872 phosphorylation does not affect the sterol-induced degradation of HMGCR (
      • Sato R.
      • Goldstein J.L.
      • Brown M.S.
      Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion.
      ), presumably allowing for HMGCR activity to be rapidly halted when ATP levels fall, irrespective of the current sterol status of the cell.

      A second control enzyme: SM

      SM controls flux through the cholesterol synthesis pathway downstream of HMGCR as recently reviewed (
      • Chua N.K.
      • Coates H.W.
      • Brown A.J.
      Squalene monooxygenase: a journey to the heart of cholesterol synthesis.
      ). The regulatory domain of SM, the first 100 amino acids (dubbed N100) is highly responsive to sterols, and increased cholesterol or desmosterol levels lead to rapid destruction of the protein, in a feedback loop from cholesterol (
      • Gill S.
      • Stevenson J.
      • Kristiana I.
      • Brown A.J.
      Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
      ). In brief, excess cholesterol ejects a spring-loaded amphipathic helix in the N100 domain from the ER membrane, initiating its proteasomal degradation (
      • Chua N.K.
      • Howe V.
      • Jatana N.
      • Thukral L.
      • Brown A.J.
      A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.
      ) by ubiquitination of atypical residues (serines rather than lysines) (
      • Chua N.K.
      • Hart-Smith G.
      • Brown A.J.
      Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.
      ). MARCHF6 is the E3 ubiquitin ligase that targets SM for proteasomal destruction (
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ) and, in a first for an E3 ligase, is itself stabilized by cholesterol and some intermediates, e.g. desmosterol and lanosterol (
      • Sharpe L.J.
      • Howe V.
      • Scott N.A.
      • Luu W.
      • Phan L.
      • Berk J.M.
      • Hochstrasser M.
      • Brown A.J.
      Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.
      ). SM also senses abundance of its substrate squalene via N100 to increase the metabolic capacity at this step (
      • Yoshioka H.
      • Coates H.W.
      • Chua N.K.
      • Hashimoto Y.
      • Brown A.J.
      • Ohgane K.
      A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
      ). This is the first reported example of stabilization of an enzyme by its substrate binding to a site other than its active site. Deubiquitinases also play a role in SM regulation, although the precise DUBs involved are not yet known (
      • Chua N.K.
      • Scott N.A.
      • Brown A.J.
      Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron.
      ). Intriguingly, deubiquitinase inhibitors had opposing effects on full-length SM and its regulatory domain N100, decreasing the former but increasing the latter (
      • Chua N.K.
      • Scott N.A.
      • Brown A.J.
      Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron.
      ). The details of this complex interplay require further investigation.

      Post-lanosterol enzymes

      Lanosterol can be acted upon by LDM or DHCR24 to enter the Bloch or Kandutsch–Russell pathway, respectively. The step beyond LDM is the only step in the late pathway that can be performed by either of two enzymes, DHCR14 or LBR (Fig. 2). Interestingly, of these four enzymes around this critical point of the pathway, only DHCR14 protein levels are subjected to feedback inhibition by sterols (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ,
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ,
      • Zerenturk E.J.
      • Kristiana I.
      • Gill S.
      • Brown A.J.
      The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
      ). At the end of the pathway, either DHCR7 or DHCR24 acts as the final enzyme that produces cholesterol. DHCR7 protein levels (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ), but not DHCR24 (
      • Zerenturk E.J.
      • Kristiana I.
      • Gill S.
      • Brown A.J.
      The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
      ), are decreased in the presence of sterols. Most work on these later enzymes has related to the genes, including transcriptional regulation and genetic mutations, for example, the relatively common Smith–Lemli–Opitz syndrome results from mutations in the DHCR7 gene. By comparison, the post-translational regulation of the remaining enzymes has been relatively neglected and is deserving of further research to explore the regulatory mechanisms, both to fully understand the control of the pathway and as potential future therapeutic targets.

      Gateway enzymes: LDM and DHCR24

      LDM (also commonly known as CYP51A1) and DHCR24 can each act on lanosterol to control entry into either the Bloch pathway or the classic Kandutsch–Russell pathway. Although the action of LDM is restricted to this point, DHCR24 can transfer any intermediate from the Bloch to the Kandutsch–Russell pathway, although it predominantly does this at the point of zymosterol (
      • Mitsche M.A.
      • McDonald J.G.
      • Hobbs H.H.
      • Cohen J.C.
      Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways.
      ) (Fig. 1). DHCR24 also performs the final step to produce cholesterol from desmosterol.
      LDM and DHCR24 protein levels are both unaffected by cholesterol (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ,
      • Zerenturk E.J.
      • Kristiana I.
      • Gill S.
      • Brown A.J.
      The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
      ) but interestingly are both regulated by the E3 ligase MARCHF6 (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ). LDM is additionally targeted by the newly uncovered RNF185–membralin complex (
      • van de Weijer M.L.
      • Krshnan L.
      • Liberatori S.
      • Guerrero E.N.
      • Robson-Tull J.
      • Hahn L.
      • Lebbink R.J.
      • Wiertz E.
      • Fischer R.
      • Ebner D.
      • Carvalho P.
      Quality control of ER membrane proteins by the RNF185/membralin ubiquitin ligase complex.
      ), again highlighting that multiple E3 ligases can target the same substrate. LDM is turned over in the presence of nitric oxide (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ,
      • Park J.W.
      • Byrd A.
      • Lee C-M.
      • Morgan E.T.
      Nitric oxide stimulates cellular degradation of human CYP51A1, the highly conserved lanosterol 14α-demethylase.
      ), although the physiological relevance of this is unclear. Although logical candidates, sterols and hypoxia (which would make sense in light of the high oxygen requirements of LDM) do not promote degradation of LDM (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ). Further research is warranted to uncover a clear physiological signal.
      DHCR24 protein levels are similarly unaffected by sterols like cholesterol and 24,25-epoxycholesterol (
      • Zerenturk E.J.
      • Kristiana I.
      • Gill S.
      • Brown A.J.
      The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
      ) but are decreased by pregnenolone and some tyrosine-kinase inhibitors (
      • Wages P.A.
      • Kim H.-Y.H.
      • Korade Z.
      • Porter N.A.
      Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.
      ). However, these effects on protein levels were observed following 24 h of treatment and may be attributable to transcriptional changes, which tend to occur over longer timeframes. Interestingly, 24,25-epoxycholesterol (
      • Zerenturk E.J.
      • Kristiana I.
      • Gill S.
      • Brown A.J.
      The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
      ) and progesterone (
      • Jansen M.
      • Wang W.
      • Greco D.
      • Bellenchi G.C.
      • di Porzio U.
      • Brown A.J.
      • Ikonen E.
      What dictates the accumulation of desmosterol in the developing brain?.
      ) inhibit the activity of DHCR24 without affecting its protein levels. Similarly, phosphorylation and signaling play important roles in regulating DHCR24 activity, again without affecting protein levels (
      • Luu W.
      • Zerenturk E.J.
      • Kristiana I.
      • Bucknall M.P.
      • Sharpe L.J.
      • Brown A.J.
      Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.
      ). The anti-arrhythmic drug amiodarone inhibits the activity of DHCR24 (
      • Allen L.B.
      • Genaro-Mattos T.C.
      • Anderson A.
      • Porter N.A.
      • Mirnics K.
      • Korade Z.
      Amiodarone alters cholesterol biosynthesis through tissue-dependent inhibition of emopamil binding protein and dehydrocholesterol reductase 24.
      ,
      • Simonen P.
      • Li S.
      • Chua N.K.
      • Lampi A.M.
      • Piironen V.
      • Lommi J.
      • Sinisalo J.
      • Brown A.J.
      • Ikonen E.
      • Gylling H.
      Amiodarone disrupts cholesterol biosynthesis pathway and causes accumulation of circulating desmosterol by inhibiting 24-dehydrocholesterol reductase.
      ), perhaps via a similar mechanism as triparanol, the archetypal DHCR24 inhibitor, which has some structural features in common (
      • Simonen P.
      • Li S.
      • Chua N.K.
      • Lampi A.M.
      • Piironen V.
      • Lommi J.
      • Sinisalo J.
      • Brown A.J.
      • Ikonen E.
      • Gylling H.
      Amiodarone disrupts cholesterol biosynthesis pathway and causes accumulation of circulating desmosterol by inhibiting 24-dehydrocholesterol reductase.
      ). This indicates that there are multiple ways to inhibit the activity of DHCR24, without needing to adjust the levels of the enzyme. This may be somewhat specific to DHCR24, because we are currently unaware of any other cholesterol synthesis enzymes that are comparatively quite stable (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ), yet their activity is very highly regulated. In a fascinating mode of regulation, a protease encoded by hepatitis C virus cleaves DHCR24 to prevent its activity, thereby promoting desmosterol accumulation and its own replication (
      • Tallorin L.
      • Villareal V.A.
      • Hsia C.Y.
      • Rodgers M.A.
      • Burri D.J.
      • Pfeil M.P.
      • Llopis P.M.
      • Lindenbach B.D.
      • Yang P.L.
      Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.
      ).

      Twin enzymes: DHCR14 and LBR

      The Δ14 double bond of FF-MAS can be reduced to form T-MAS by either DHCR14 or LBR. These twin enzymes have a shared function and appear to have complementary levels of expression in different tissues (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ) but vastly different modes of regulation. Like most cholesterol synthesis enzymes, DHCR14 is located in the ER, whereas LBR is found in the inner nuclear membrane, which is contiguous with the ER. Cholesterol and early sterol intermediates lead to rapid degradation of DHCR14 (21). Like HMGCR and SM, this occurred through the proteasome and ubiquitination, but the ubiquitination site and major E3 ligase remain elusive (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ). In contrast, LBR was not rapidly turned over nor affected by sterol levels. This is similar to the transcriptional regulation of the two enzymes; LBR is unchanged by sterol conditions, whereas DHCR14 is highly regulated by sterols (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ). LBR has several phosphorylated serine residues (
      • Sellis D.
      • Drosou V.
      • Vlachakis D.
      • Voukkalis N.
      • Giannakouros T.
      • Vlassi M.
      Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1-insights from molecular dynamics simulations.
      ,
      • Voukkalis N.
      • Koutroumani M.
      • Zarkadas C.
      • Nikolakaki E.
      • Vlassi M.
      • Giannakouros T.
      SRPK1 and Akt protein kinases phosphorylate the RS domain of lamin B receptor with distinct specificity: a combined biochemical and in silico approach.
      ) that affect its chromatin-binding function (
      • Takano M.
      • Koyama Y.
      • Ito H.
      • Hoshino S.
      • Onogi H.
      • Hagiwara M.
      • Furukawa K.
      • Horigome T.
      Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts.
      ), but there is no information on whether this has any effect on its Δ14-reductase activity. LBR is described as a chimeric protein in which its N-terminal domain is needed for chromatin binding, but its C-terminal domain consisting of transmembrane regions is essential for its role in cholesterol synthesis (
      • Kasbekar D.P.
      A cross-eyed geneticist's view: I. Making sense of the lamin B receptor, a chimeric protein.
      ). It remains unclear why LBR has retained its cholesterol synthesis function in the presence of the dedicated DHCR14 enzyme.

      Terminal enzyme: DHCR7

      DHCR7 is the final enzyme in the Kandutsch–Russell pathway, converting 7-dehydrocholesterol to cholesterol. Like SM and DHCR14, DHCR7 is subject to rapid proteasomal degradation in the presence of cholesterol or desmosterol (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ). Like DHCR14, the E3 ligase and ubiquitination sites remain unknown. 7-Dehydrocholesterol (7DHC) is also the precursor to vitamin D (Fig. 1), and therefore there are numerous studies examining links between DHCR7 and vitamin D. Interestingly, vitamin D promotes degradation of DHCR7 in keratinocytes (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ), where vitamin D is synthesized. Reduction in DHCR7 protein levels and activity leads to an accumulation of 7DHC, from which more vitamin D can be produced (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ).
      DHCR7 is well-known as the mutated gene in Smith–Lemli–Opitz syndrome, and the most common mutations destabilize DHCR7 protein, which can be rescued by statin treatment (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ), a promising therapy in Smith–Lemli–Opitz syndrome treatment (
      • Wassif C.A.
      • Kratz L.
      • Sparks S.E.
      • Wheeler C.
      • Bianconi S.
      • Gropman A.
      • Calis K.A.
      • Kelley R.I.
      • Tierney E.
      • Porter F.D.
      A placebo-controlled trial of simvastatin therapy in Smith–Lemli–Opitz syndrome.
      ). Somewhat similar to DHCR24, DHCR7 activity is also regulated by phosphorylation and signaling (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Phosphorylation regulates activity of 7-dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis.
      ). Notably, DHCR7 appears to be activated by the energy sensor AMPK, which as mentioned deactivates the earlier key enzyme HMGCR. Possibly these opposing actions of AMPK may serve to save energy by turning off the pathway early but allowing its completion to prevent accumulation of potentially harmful intermediates (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Phosphorylation regulates activity of 7-dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis.
      ).

      A mediator of multiple steps: MARCHF6

      The E3 ligase MARCHF6 controls protein levels of at least four enzymes in cholesterol synthesis: the rate-limiting enzymes HMGCR and SM (
      • Zelcer N.
      • Sharpe L.J.
      • Loregger A.
      • Kristiana I.
      • Cook E.C.
      • Phan L.
      • Stevenson J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
      ) and the gateway enzymes LDM and DHCR24 (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ). Together with the finding that cholesterol stabilizes MARCHF6, this indicates that MARCHF6 is intricately involved in cholesterol metabolism (
      • Scott N.A.
      • Sharpe L.J.
      • Brown A.J.
      The E3 ubiquitin ligase MARCHF6 as a metabolic integrator in cholesterol synthesis and beyond.
      ). MARCHF6 may play a role in the degradation of other enzymes in the pathway as well. Other tested enzymes, DHCR7 (
      • Prabhu A.V.
      • Luu W.
      • Sharpe L.J.
      • Brown A.J.
      Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
      ), DHCR14 (
      • Capell-Hattam I.M.
      • Sharpe L.J.
      • Qian L.
      • Hart-Smith G.
      • Prabhu A.V.
      • Brown A.J.
      Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
      ), EBP, and lanosterol synthase (
      • Scott N.A.
      • Sharpe L.J.
      • Capell-Hattam I.M.
      • Gullo S.J.
      • Luu W.
      • Brown A.J.
      The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
      ), are unlikely to be MARCHF6 targets, suggesting that MARCHF6 regulates the pathway in a very specific and controlled manner. Future work should determine which of the remaining cholesterol synthesis enzymes are similarly degraded by MARCHF6 to fully understand its intricate control over the pathway. It will also be interesting to determine why and how MARCHF6 targets some enzymes but not others. The current four targets are all either rate-limiting or control entry into the separate branches of the pathway, suggesting that these may be particularly critical enzymes to control.

      Localization and interactions between cholesterol synthesis enzymes

      Cholesterol synthesis occurs largely in the membrane of the ER, likely because of the highly hydrophobic nature of the substrates and products starting from around squalene (
      • Gaylor J.L.
      Membrane-bound enzymes of cholesterol synthesis from lanosterol.
      ). As such, these hydrophobic enzymes (Fig. 3) are largely localized to the ER, where they are likely to interact with each other and indeed may form something of a metabolon or “cholestesome,” a coordinated chain of enzymes to harness substrate channeling benefits. The two terminal enzymes DHCR7 and DHCR24 are known to interact in mammals (
      • Luu W.
      • Hart-Smith G.
      • Sharpe L.J.
      • Brown A.J.
      The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally.
      ), DHCR7 and EBP interact (
      • Kedjouar B.
      • de Médina P.
      • Oulad-Abdelghani M.
      • Payré B.
      • Silvente-Poirot S.
      • Favre G.
      • Faye J.-C.
      • Poirot M.
      Molecular characterization of the microsomal tamoxifen binding site.
      ), and in yeast, there is an “ergosome” in which a number of ergosterol-synthesizing enzymes interact (
      • Mo C.
      • Bard M.
      A systematic study of yeast sterol biosynthetic protein–protein interactions using the split-ubiquitin system.
      ). Determining which enzymes interact with which others in cholesterol synthesis would help confirm or disprove the existence of a cholestesome.
      Some cholesterol synthesis enzymes can be found on lipid droplets, such as SM, lanosterol synthase, and sterol-4-α-carboxylate 3-dehydrogenase (
      • Bersuker K.
      • Peterson C.W.H.
      • To M.
      • Sahl S.J.
      • Savikhin V.
      • Grossman E.A.
      • Nomura D.K.
      • Olzmann J.A.
      A proximity labeling strategy provides insights into the composition and dynamics of lipid droplet proteomes.
      ). There is some suggestion from work in both yeast (
      • Leber R.
      • Landl K.
      • Zinser E.
      • Ahorn H.
      • Spök A.
      • Kohlwein S.D.
      • Turnowsky F.
      • Daum G.
      Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.
      ) and mammalian cells (
      • Ohashi M.
      • Mizushima N.
      • Kabeya Y.
      • Yoshimori T.
      Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets.
      ) that lipid droplet formation could sequester these enzymes and shut down cholesterol synthesis, maybe as a feedback response opposing excess lipid synthesis and storage. Interestingly, DHCR7 has also been observed in the Golgi, whereas DHCR24 and EBP were not (
      • Koczok K.
      • Gurumurthy C.B.
      • Balogh I.
      • Korade Z.
      • Mirnics K.
      Subcellular localization of sterol biosynthesis enzymes.
      ). All three enzymes were colocated in the ER along with the earlier enzyme SC5D, which was additionally localized to the nucleus, which may suggest that it has additional functions beyond sterol synthesis (
      • Koczok K.
      • Gurumurthy C.B.
      • Balogh I.
      • Korade Z.
      • Mirnics K.
      Subcellular localization of sterol biosynthesis enzymes.
      ). Under what physiological conditions DHCR7 and SC5D may be relocated needs to be established. Although yet to be tested, it is entirely possible that the vast array of PTMs on cholesterol synthesis enzymes may influence their cellular localization.

      Outstanding questions and concluding remarks

      Our understanding of the post-translational control of cholesterol has deepened considerably over the past decade, but many questions remain to be answered. Does MARCHF6 target other enzymes in the cholesterol synthesis pathway? Which other cholesterol synthesis enzymes are controlled by changing sterol levels? What inputs control the other enzymes? How do the multitude of PTMs interact? Does regulation of the enzymes inform the effects of disease mutations and vice versa? How are the enzymes differently regulated in different cellular and physiological contexts? How do these various regulatory inputs affect flux through the pathway?
      Although studies of cultured cells are indispensable for the discovery and characterization of enzyme regulatory mechanisms, it is vital that the physiological relevance of these mechanisms is verified at the organismal level, as has been done for HMGCR. Such advances would inform the development of alternative strategies to therapeutically target cholesterol synthesis. In addition to HMGCR, SM is perhaps the next best-understood pathway enzyme and thus an ideal candidate for in vivo studies of its sterol regulation. However, further fundamental insights into less well-characterized enzymes will open a multitude of avenues for modulating pathway activity and broader cholesterol homeostasis.
      Cholesterol synthesis enzymes display a myriad of PTMs (Fig. 3). The vast majority of these have been gleaned from high-throughput screens. Further studies will no doubt add to the number, variety, and certainty of these PTMs. In particular, low-throughput studies are crucial to determine the intricate details of these PTMs and the consequent regulation of the enzymes. Nevertheless, a picture emerges of an ornate overlay of PTMs, suggestive of a complex control panel of signals, exquisitely responsive to changing metabolic conditions, especially with respect to energy demands. Phosphates are added or removed from a multitude of sites on successive enzymes, like lights blinking on and off. Acetylation lights up the beginning of the pathway before flickering at discrete points downstream, and sterol-mediated ubiquitination, as some sort of master switch, shuts down the entire energy-intensive pathway via proteasomal degradation at multiple steps. We look forward to further efforts to decode the complex signals that control traffic along this long and winding road to cholesterol.

      References

        • Sezgin E.
        • Levental I.
        • Mayor S.
        • Eggeling C.
        The mystery of membrane organization: composition, regulation and roles of lipid rafts.
        Nat. Rev. Mol. Cell Biol. 2017; 18 (28356571): 361-374
        • Wang N.
        • Fulcher J.
        • Abeysuriya N.
        • Park L.
        • Kumar S.
        • Di Tanna G.L.
        • Wilcox I.
        • Keech A.
        • Rodgers A.
        • Lal S.
        Intensive LDL cholesterol-lowering treatment beyond current recommendations for the prevention of major vascular events: a systematic review and meta-analysis of randomised trials including 327 037 participants.
        Lancet Diabetes Endocrinol. 2020; 8 (31862150): 36-49
        • Huang B.
        • Song B.L.
        • Xu C.
        Cholesterol metabolism in cancer: mechanisms and therapeutic opportunities.
        Nat. Metab. 2020; 2 (32694690): 132-141
        • Martín M.G.
        • Pfrieger F.
        • Dotti C.G.
        Cholesterol in brain disease: sometimes determinant and frequently implicated.
        EMBO Rep. 2014; 15 (25223281): 1036-1052
        • Brown A.J.
        • Sharpe L.J.
        Cholesterol synthesis.
        in: Biochemistry of Lipids, Lipoproteins, and Membranes. 6th Ed. Elsevier Science Publishing Co., Inc., New York2016: 327-358
        • Brown M.S.
        • Radhakrishnan A.
        • Goldstein J.L.
        Retrospective on cholesterol homeostasis: the central role of Scap.
        Annu. Rev. Biochem. 2018; 87 (28841344): 783-807
        • Brown A.J.
        • Sharpe L.J.
        • Rogers M.J.
        Oxysterols: from physiological tuners to pharmacological opportunities.
        Br. J. Pharmacol. 2020; (32335907)
        • Brown A.J.
        • Ikonen E.
        • Olkkonen V.M.
        Cholesterol precursors: more than mere markers of biosynthesis.
        Curr. Opin. Lipidol. 2014; 25 (24378747): 133-139
        • Bekkering S.
        • Arts R.J.W.
        • Novakovic B.
        • Kourtzelis I.
        • van der Heijden C.
        • Li Y.
        • Popa C.D.
        • Ter Horst R.
        • van Tuijl J.
        • Netea-Maier R.T.
        • van de Veerdonk F.L.
        • Chavakis T.
        • Joosten L.A.B.
        • van der Meer J.W.M.
        • Stunnenberg H.
        • et al.
        Metabolic induction of trained immunity through the mevalonate pathway.
        Cell. 2018; 172 (29328908): 135-146.e9
        • Segatto M.
        • Tonini C.
        • Pfrieger F.W.
        • Trezza V.
        • Pallottini V.
        Loss of mevalonate/cholesterol homeostasis in the brain: a focus on autism spectrum disorder and Rett syndrome.
        Int. J. Mol. Sci. 2019; 20 (31284522)3317
        • Gabitova L.
        • Restifo D.
        • Gorin A.
        • Manocha K.
        • Handorf E.
        • Yang D.-H.
        • Cai K.Q.
        • Klein-Szanto A.J.
        • Cunningham D.
        • Kratz L.E.
        • Herman G.E.
        • Golemis E.A.
        • Astsaturov I.
        Endogenous sterol metabolites regulate growth of EGFR/KRAS-dependent tumors via LXR.
        Cell Rep. 2015; 12 (26344763): 1927-1938
        • Sharpe L.J.
        • Brown A.J.
        Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR).
        J. Biol. Chem. 2013; 288 (23696639): 18707-18715
        • Mitsche M.A.
        • McDonald J.G.
        • Hobbs H.H.
        • Cohen J.C.
        Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways.
        eLife. 2015; 4 (26114596)e07999
        • Hornbeck P.V.
        • Zhang B.
        • Murray B.
        • Kornhauser J.M.
        • Latham V.
        • Skrzypek E.
        PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
        Nucleic Acids Res. 2015; 43 (25514926): D512-D520
        • The UniProt Consortium
        UniProt: the universal protein knowledgebase.
        Nucleic Acids Res. 2018; 46 (29425356)2699
        • Zielinska D.F.
        • Gnad F.
        • Wiśniewski J.R.
        • Mann M.
        Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.
        Cell. 2010; 141 (20510933): 897-907
        • Myers S.A.
        • Panning B.
        • Burlingame A.L.
        Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21606357): 9490-9495
        • Steentoft C.
        • Vakhrushev S.Y.
        • Joshi H.J.
        • Kong Y.
        • Vester-Christensen M.B.
        • Schjoldager K.T.
        • Lavrsen K.
        • Dabelsteen S.
        • Pedersen N.B.
        • Marcos-Silva L.
        • Gupta R.
        • Bennett E.P.
        • Mandel U.
        • Brunak S.
        • Wandall H.H.
        • et al.
        Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.
        EMBO J. 2013; 32 (23584533): 1478-1488
        • The UniProt Consortium
        UniProt: a worldwide hub of protein knowledge.
        Nucleic Acids Res. 2018; 47 (29425356): D506-D515
        • Clizbe D.B.
        • Owens M.L.
        • Masuda K.R.
        • Shackelford J.E.
        • Krisans S.K.
        IDI2, a second isopentenyl diphosphate isomerase in mammals.
        J. Biol. Chem. 2007; 282 (17202134): 6668-6676
        • Capell-Hattam I.M.
        • Sharpe L.J.
        • Qian L.
        • Hart-Smith G.
        • Prabhu A.V.
        • Brown A.J.
        Twin enzymes, divergent control: the cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.
        J. Biol. Chem. 2020; 295 (31911440): 2850-2865
        • Smith L.M.
        • Kelleher N.L.
        • Consortium for Top Down Proteomics
        Proteoform: a single term describing protein complexity.
        Nat. Methods. 2013; 10 (23443629): 186-187
        • Zheng N.
        • Shabek N.
        Ubiquitin ligases: structure, function, and regulation.
        Annu. Rev. Biochem. 2017; 86 (28375744): 129-157
        • Clague M.J.
        • Urbé S.
        • Komander D.
        Breaking the chains: deubiquitylating enzyme specificity begets function.
        Nat. Rev. Mol. Cell Biol. 2019; 20 (30733604): 338-352
        • Jo Y.
        • Lee P.C.
        • Sguigna P.V.
        • DeBose-Boyd R.A.
        Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (22143767): 20503-20508
        • Menzies S.A.
        • Volkmar N.
        • van den Boomen D.J.
        • Timms R.T.
        • Dickson A.S.
        • Nathan J.A.
        • Lehner P.J.
        The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.
        eLife. 2018; 7 (30543180)e40009
        • Tsai Y.C.
        • Leichner G.S.
        • Pearce M.M.
        • Wilson G.L.
        • Wojcikiewicz R.J.
        • Roitelman J.
        • Weissman A.M.
        Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.
        Mol. Biol. Cell. 2012; 23 (23087214): 4484-4494
        • Zelcer N.
        • Sharpe L.J.
        • Loregger A.
        • Kristiana I.
        • Cook E.C.
        • Phan L.
        • Stevenson J.
        • Brown A.J.
        The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.
        Mol. Cell Biol. 2014; 34 (24449766): 1262-1270
        • Scott N.A.
        • Sharpe L.J.
        • Capell-Hattam I.M.
        • Gullo S.J.
        • Luu W.
        • Brown A.J.
        The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.
        Biochem. J. 2020; 477 (31904814): 541-555
        • Chua N.K.
        • Scott N.A.
        • Brown A.J.
        Valosin-containing protein mediates the ERAD of squalene monooxygenase and its cholesterol-responsive degron.
        Biochem. J. 2019; 476 (31471528): 2545-2560
        • Harada K.
        • Kato M.
        • Nakamura N.
        USP19-mediated deubiquitination facilitates the stabilization of HRD1 ubiquitin ligase.
        Int. J. Mol. Sci. 2016; 17 (27827840)1829
        • Nakamura N.
        • Harada K.
        • Kato M.
        • Hirose S.
        Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6.
        Exp. Cell Res. 2014; 328 (25088257): 207-216
        • Gill S.
        • Stevenson J.
        • Kristiana I.
        • Brown A.J.
        Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.
        Cell Metab. 2011; 13 (21356516): 260-273
        • Gillespie J.G.
        • Hardie D.G.
        Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes.
        FEBS Lett. 1992; 306 (1628744): 59-62
        • Prabhu A.V.
        • Luu W.
        • Sharpe L.J.
        • Brown A.J.
        Phosphorylation regulates activity of 7-dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis.
        J. Steroid Biochem. Mol. Biol. 2017; 165 (27520299): 363-368
        • Luu W.
        • Zerenturk E.J.
        • Kristiana I.
        • Bucknall M.P.
        • Sharpe L.J.
        • Brown A.J.
        Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.
        J. Lipid Res. 2014; 55 (24363437): 410-420
        • Zhao S.
        • Xu W.
        • Jiang W.
        • Yu W.
        • Lin Y.
        • Zhang T.
        • Yao J.
        • Zhou L.
        • Zeng Y.
        • Li H.
        • Li Y.
        • Shi J.
        • An W.
        • Hancock S.M.
        • He F.
        • et al.
        Regulation of cellular metabolism by protein lysine acetylation.
        Science. 2010; 327 (20167786): 1000-1004
        • Narita T.
        • Weinert B.T.
        • Choudhary C.
        Functions and mechanisms of non-histone protein acetylation.
        Nat. Rev. Mol. Cell Biol. 2019; 20 (30467427): 156-174
        • Yang X.J.
        • Seto E.
        Lysine acetylation: codified crosstalk with other posttranslational modifications.
        Mol. Cell. 2008; 31 (18722172): 449-461
        • Jones P.J.
        Use of deuterated water for measurement of short-term cholesterol synthesis in humans.
        Can. J. Physiol. Pharmacol. 1990; 68 (2200588): 955-959
        • Swaney D.L.
        • Beltrao P.
        • Starita L.
        • Guo A.
        • Rush J.
        • Fields S.
        • Krogan N.J.
        • Villén J.
        Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation.
        Nat. Methods. 2013; 10 (23749301): 676-682
        • Beltrao P.
        • Albanèse V.
        • Kenner L.R.
        • Swaney D.L.
        • Burlingame A.
        • Villén J.
        • Lim W.A.
        • Fraser J.S.
        • Frydman J.
        • Krogan N.J.
        Systematic functional prioritization of protein posttranslational modifications.
        Cell. 2012; 150 (22817900): 413-425
        • Ochoa D.
        • Jarnuczak A.F.
        • Viéitez C.
        • Gehre M.
        • Soucheray M.
        • Mateus A.
        • Kleefeldt A.A.
        • Hill A.
        • Garcia-Alonso L.
        • Stein F.
        • Krogan N.J.
        • Savitski M.M.
        • Swaney D.L.
        • Vizcaíno J.A.
        • Noh K.-M.
        • et al.
        The functional landscape of the human phosphoproteome.
        Nat. Biotechnol. 2020; 38 (31819260): 365-373
        • Favier L.A.
        • Schulert G.S.
        Mevalonate kinase deficiency: current perspectives.
        Appl. Clin. Genet. 2016; 9 (27499643): 101-110
        • Zhu T.
        • Tian D.
        • Zhang L.
        • Xu X.
        • Xia K.
        • Hu Z.
        • Xiong Z.
        • Tan J.
        Novel mutations in mevalonate kinase cause disseminated superficial actinic porokeratosis.
        Br. J. Dermatol. 2019; 181 (30597534): 304-313
        • Wang L.
        • Gulappa T.
        • Menon K.M.J.
        Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.
        Biochim. Biophys. Acta. 2010; 1803 (20167237): 591-597
        • Sapir A.
        • Tsur A.
        • Koorman T.
        • Ching K.
        • Mishra P.
        • Bardenheier A.
        • Podolsky L.
        • Bening-Abu-Shach U.
        • Boxem M.
        • Chou T.F.
        • Broday L.
        • Sternberg P.W.
        Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25187565): E3880-E3889
        • Chen L.
        • Ma M.Y.
        • Sun M.
        • Jiang L.Y.
        • Zhao X.T.
        • Fang X.X.
        • Man Lam S.
        • Shui G.H.
        • Luo J.
        • Shi X.J.
        • Song B.L.
        Endogenous sterol intermediates of the mevalonate pathway regulate HMGCR degradation and SREBP-2 processing.
        J. Lipid Res. 2019; 60 (31455613): 1765-1775
        • Song B.L.
        • Javitt N.B.
        • DeBose-Boyd R.A.
        Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol.
        Cell Metab. 2005; 1 (16054061): 179-189
        • Coates H.W.
        • Brown A.J.
        A wolf in sheep's clothing: unmasking the lanosterol-induced degradation of HMG-CoA reductase.
        J. Lipid Res. 2019; 60 (31462514): 1643-1645
        • Song B.L.
        • DeBose-Boyd R.A.
        Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase.
        J. Biol. Chem. 2004; 279 (15090540): 28798-28806
        • Skalnik D.G.
        • Narita H.
        • Kent C.
        • Simoni R.D.
        The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.
        J. Biol. Chem. 1988; 263 (2834394): 6836-6841
        • Sever N.
        • Song B.L.
        • Yabe D.
        • Goldstein J.L.
        • Brown M.S.
        • DeBose-Boyd R.A.
        Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
        J. Biol. Chem. 2003; 278 (14563840): 52479-52490
        • Radhakrishnan A.
        • Sun L.P.
        • Kwon H.J.
        • Brown M.S.
        • Goldstein J.L.
        Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain.
        Mol. Cell. 2004; 15 (15260976): 259-268
        • Sever N.
        • Yang T.
        • Brown M.S.
        • Goldstein J.L.
        • DeBose-Boyd R.A.
        Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.
        Mol. Cell. 2003; 11 (12535518): 25-33
        • Cook E.C.
        • Nelson J.K.
        • Sorrentino V.
        • Koenis D.
        • Moeton M.
        • Scheij S.
        • Ottenhoff R.
        • Bleijlevens B.
        • Loregger A.
        • Zelcer N.
        Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene.
        PLoS One. 2017; 12 (28231341)e0172721
        • Lee J.N.
        • Song B.
        • DeBose-Boyd R.A.
        • Ye J.
        Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78.
        J. Biol. Chem. 2006; 281 (17043353): 39308-39315
        • Liu T.F.
        • Tang J.J.
        • Li P.S.
        • Shen Y.
        • Li J.G.
        • Miao H.H.
        • Li B.L.
        • Song B.L.
        Ablation of gp78 in liver improves hyperlipidemia and insulin resistance by inhibiting SREBP to decrease lipid biosynthesis.
        Cell Metab. 2012; 16 (22863805): 213-225
        • Jiang S.-Y.
        • Li H.
        • Tang J.-J.
        • Wang J.
        • Luo J.
        • Liu B.
        • Wang J.-K.
        • Shi X.-J.
        • Cui H.-W.
        • Tang J.
        • Yang F.
        • Qi W.
        • Qiu W.-W.
        • Song B.-L.
        Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol.
        Nat. Commun. 2018; 9 (30510211)5138
        • Hwang S.
        • Hartman I.Z.
        • Calhoun L.N.
        • Garland K.
        • Young G.A.
        • Mitsche M.A.
        • McDonald J.
        • Xu F.
        • Engelking L.
        • DeBose-Boyd R.A.
        Contribution of accelerated degradation to feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol metabolism in the liver.
        J. Biol. Chem. 2016; 291 (27129778): 13479-13494
        • Pineda A.
        • Cubeddu L.X.
        Statin rebound or withdrawal syndrome: does it exist?.
        Curr. Atheroscler. Rep. 2011; 13 (21104165): 23-30
        • Morris L.L.
        • Hartman I.Z.
        • Jun D.J.
        • Seemann J.
        • DeBose-Boyd R.A.
        Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)–associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
        J. Biol. Chem. 2014; 289 (24860107): 19053-19066
        • Leichner G.S.
        • Avner R.
        • Harats D.
        • Roitelman J.
        Metabolically regulated endoplasmic reticulum-associated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase: evidence for requirement of a geranylgeranylated protein.
        J. Biol. Chem. 2011; 286 (21778231): 32150-32161
        • Schumacher M.M.
        • Elsabrouty R.
        • Seemann J.
        • Jo Y.
        • DeBose-Boyd R.A.
        The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.
        eLife. 2015; 4 (25742604)e05560
        • Jo Y.
        • Kim S.S.
        • Garland K.
        • Fuentes I.
        • DiCarlo L.M.
        • Ellis J.L.
        • Fu X.
        • Booth S.L.
        • Evers B.M.
        • DeBose-Boyd R.A.
        Enhanced ER-associated degradation of HMG CoA reductase causes embryonic lethality associated with Ubiad1 deficiency.
        eLife. 2020; 9 (32118581)e54841
        • Clarke P.R.
        • Hardie D.G.
        Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.
        EMBO J. 1990; 9 (2369897): 2439-2446
        • Sato R.
        • Goldstein J.L.
        • Brown M.S.
        Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion.
        Proc. Natl. Acad. Sci. U.S.A. 1993; 90 (8415689): 9261-9265
        • Chua N.K.
        • Coates H.W.
        • Brown A.J.
        Squalene monooxygenase: a journey to the heart of cholesterol synthesis.
        Prog. Lipid Res. 2020; 79 (32360125)101033
        • Chua N.K.
        • Howe V.
        • Jatana N.
        • Thukral L.
        • Brown A.J.
        A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.
        J. Biol. Chem. 2017; 292 (28972164): 19959-19973
        • Chua N.K.
        • Hart-Smith G.
        • Brown A.J.
        Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.
        J. Biol. Chem. 2019; 294 (30940729): 8134-8147
        • Sharpe L.J.
        • Howe V.
        • Scott N.A.
        • Luu W.
        • Phan L.
        • Berk J.M.
        • Hochstrasser M.
        • Brown A.J.
        Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.
        J. Biol. Chem. 2019; 294 (30545937): 2436-2448
        • Yoshioka H.
        • Coates H.W.
        • Chua N.K.
        • Hashimoto Y.
        • Brown A.J.
        • Ohgane K.
        A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.
        Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (32170014): 7150-7158
        • Zerenturk E.J.
        • Kristiana I.
        • Gill S.
        • Brown A.J.
        The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1).
        Biochim. Biophys. Acta. 2012; 1821 (22178193): 1269-1277
        • Prabhu A.V.
        • Luu W.
        • Sharpe L.J.
        • Brown A.J.
        Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis.
        J. Biol. Chem. 2016; 291 (26887953): 8363-8373
        • van de Weijer M.L.
        • Krshnan L.
        • Liberatori S.
        • Guerrero E.N.
        • Robson-Tull J.
        • Hahn L.
        • Lebbink R.J.
        • Wiertz E.
        • Fischer R.
        • Ebner D.
        • Carvalho P.
        Quality control of ER membrane proteins by the RNF185/membralin ubiquitin ligase complex.
        Mol. Cell. 2020; 79 (32738194): 768-781.e7
        • Park J.W.
        • Byrd A.
        • Lee C-M.
        • Morgan E.T.
        Nitric oxide stimulates cellular degradation of human CYP51A1, the highly conserved lanosterol 14α-demethylase.
        Biochem. J. 2017; 474 (28830911): 3241-3252
        • Wages P.A.
        • Kim H.-Y.H.
        • Korade Z.
        • Porter N.A.
        Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.
        J. Lipid Res. 2018; 59 (30087204): 1916-1926
        • Jansen M.
        • Wang W.
        • Greco D.
        • Bellenchi G.C.
        • di Porzio U.
        • Brown A.J.
        • Ikonen E.
        What dictates the accumulation of desmosterol in the developing brain?.
        FASEB J. 2013; 27 (23230282): 865-870
        • Allen L.B.
        • Genaro-Mattos T.C.
        • Anderson A.
        • Porter N.A.
        • Mirnics K.
        • Korade Z.
        Amiodarone alters cholesterol biosynthesis through tissue-dependent inhibition of emopamil binding protein and dehydrocholesterol reductase 24.
        ACS Chem. Neurosci. 2020; 11 (32286791): 1413-1423
        • Simonen P.
        • Li S.
        • Chua N.K.
        • Lampi A.M.
        • Piironen V.
        • Lommi J.
        • Sinisalo J.
        • Brown A.J.
        • Ikonen E.
        • Gylling H.
        Amiodarone disrupts cholesterol biosynthesis pathway and causes accumulation of circulating desmosterol by inhibiting 24-dehydrocholesterol reductase.
        J. Intern. Med. 2020; 288 (32415867): 560-569
        • Tallorin L.
        • Villareal V.A.
        • Hsia C.Y.
        • Rodgers M.A.
        • Burri D.J.
        • Pfeil M.P.
        • Llopis P.M.
        • Lindenbach B.D.
        • Yang P.L.
        Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.
        J. Biol. Chem. 2020; 295 (32641492): 12426-12436
        • Sellis D.
        • Drosou V.
        • Vlachakis D.
        • Voukkalis N.
        • Giannakouros T.
        • Vlassi M.
        Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1-insights from molecular dynamics simulations.
        Biochim. Biophys. Acta. 2012; 1820 (22056509): 44-55
        • Voukkalis N.
        • Koutroumani M.
        • Zarkadas C.
        • Nikolakaki E.
        • Vlassi M.
        • Giannakouros T.
        SRPK1 and Akt protein kinases phosphorylate the RS domain of lamin B receptor with distinct specificity: a combined biochemical and in silico approach.
        PLoS One. 2016; 11 (27105349)e0154198
        • Takano M.
        • Koyama Y.
        • Ito H.
        • Hoshino S.
        • Onogi H.
        • Hagiwara M.
        • Furukawa K.
        • Horigome T.
        Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts.
        J. Biol. Chem. 2004; 279 (14718546): 13265-13271
        • Kasbekar D.P.
        A cross-eyed geneticist's view: I. Making sense of the lamin B receptor, a chimeric protein.
        J. Biosci. 2018; 43 (29872011): 235-237
        • Wassif C.A.
        • Kratz L.
        • Sparks S.E.
        • Wheeler C.
        • Bianconi S.
        • Gropman A.
        • Calis K.A.
        • Kelley R.I.
        • Tierney E.
        • Porter F.D.
        A placebo-controlled trial of simvastatin therapy in Smith–Lemli–Opitz syndrome.
        Genet. Med. 2017; 19 (27513191): 297-305
        • Scott N.A.
        • Sharpe L.J.
        • Brown A.J.
        The E3 ubiquitin ligase MARCHF6 as a metabolic integrator in cholesterol synthesis and beyond.
        Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2020; 1866 (33049405)158837
        • Gaylor J.L.
        Membrane-bound enzymes of cholesterol synthesis from lanosterol.
        Biochem. Biophys. Res. Commun. 2002; 292 (11969204): 1139-1146
        • Luu W.
        • Hart-Smith G.
        • Sharpe L.J.
        • Brown A.J.
        The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally.
        J. Lipid Res. 2015; 56 (25637936): 888-897
        • Kedjouar B.
        • de Médina P.
        • Oulad-Abdelghani M.
        • Payré B.
        • Silvente-Poirot S.
        • Favre G.
        • Faye J.-C.
        • Poirot M.
        Molecular characterization of the microsomal tamoxifen binding site.
        J. Biol. Chem. 2004; 279 (15175332): 34048-34061
        • Mo C.
        • Bard M.
        A systematic study of yeast sterol biosynthetic protein–protein interactions using the split-ubiquitin system.
        Biochim. Biophys. Acta. 2005; 1737 (16300994): 152-160
        • Bersuker K.
        • Peterson C.W.H.
        • To M.
        • Sahl S.J.
        • Savikhin V.
        • Grossman E.A.
        • Nomura D.K.
        • Olzmann J.A.
        A proximity labeling strategy provides insights into the composition and dynamics of lipid droplet proteomes.
        Dev. Cell. 2018; 44 (29275994): 97-112.e7
        • Leber R.
        • Landl K.
        • Zinser E.
        • Ahorn H.
        • Spök A.
        • Kohlwein S.D.
        • Turnowsky F.
        • Daum G.
        Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.
        Mol. Biol. Cell. 1998; 9 (9450962): 375-386
        • Ohashi M.
        • Mizushima N.
        • Kabeya Y.
        • Yoshimori T.
        Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets.
        J. Biol. Chem. 2003; 278 (12837764): 36819-36829
        • Koczok K.
        • Gurumurthy C.B.
        • Balogh I.
        • Korade Z.
        • Mirnics K.
        Subcellular localization of sterol biosynthesis enzymes.
        J. Mol. Histol. 2019; 50 (30535733): 63-73
        • Tsirigos K.D.
        • Peters C.
        • Shu N.
        • Käll L.
        • Elofsson A.
        The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides.
        Nucleic Acids Res. 2015; 43 (25969446): W401-W407
        • Brocchieri L.
        • Karlin S.
        Protein length in eukaryotic and prokaryotic proteomes.
        Nucleic Acids Res. 2005; 33 (15951512): 3390-3400