Nonsterol Isoprenoids Research Articles

In vivo CRISPR screening identifies geranylgeranyl diphosphate as a pancreatic cancer tumor growth dependency

ObjectiveCancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. In this study, we examined the requirement of SREBP target genes for pancreatic ductal adenocarcinoma (PDAC) tumor growth. MethodsHere we constructed a custom CRISPR knockout library containing known SREBP target genes and performed in vitro 2D culture and in vivo orthotopic xenograft CRISPR screens using a patient-derived PDAC cell line. In vitro, we grew cells in medium supplemented with 10% fetal bovine serum (FBS) or 10% lipoprotein-deficient serum (LPDS) to examine differences in gene essentiality in different lipid environments. In vivo, we injected cells into the pancreata of nude mice and collected tumors after 4 weeks. ResultsWe identified terpenoid backbone biosynthesis genes as essential for PDAC tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that geranylgeranyl diphosphate synthase 1 (GGPS1) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model. ConclusionsThese findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.

Identification of FDA-approved drugs that increase mevalonate kinase in hyper IgD syndrome.

Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder caused by bi-allelic loss-of-function variants in the MVK gene, resulting in decreased activity of the encoded mevalonate kinase (MK). Clinical presentation ranges from the severe early-lethal mevalonic aciduria to the milder hyper-IgD syndrome (MKD-HIDS), and is in the majority of patients associated with recurrent inflammatory episodes with often unclear cause. Previous studies with MKD-HIDS patient cells indicated that increased temperature, as caused by fever during an inflammatory episode, lowers the residual MK activity, which causes a temporary shortage of non-sterol isoprenoids that promotes the further development of inflammation. Because an increase of the residual MK activity is expected to make MKD-HIDS patients less sensitive to developing inflammatory episodes, we established a cell-based screen that can be used to identify compounds and/or therapeutic targets that promote this increase. Using a reporter HeLa cell line that stably expresses the most common MKD-HIDS variant, MK-V377I, C-terminally tagged with bioluminescent NanoLuc luciferase (nLuc), we screened the Prestwick Chemical Library®, which includes 1280 FDA-approved compounds. Multiple compounds increased MK-V377I-nLuc bioluminescence, including steroids (i.e., glucocorticoids, estrogens, and progestogens), statins and antineoplastic drugs. The glucocorticoids increased MK-V377I-nLuc bioluminescence through glucocorticoid receptor signaling. Subsequent studies in MKD-HIDS patient cells showed that the potent glucocorticoid clobetasol propionate increases gene transcription of MVK and other genes regulated by the transcription factor sterol regulatory element-binding protein 2 (SREBP-2). Our results suggest that increasing the flux through the isoprenoid biosynthesis pathway by targeting the glucocorticoid receptor or SREBP-2 could be a potential therapeutic strategy in MKD-HIDS.

P140Cap modulates the mevalonate pathway decreasing cell migration and enhancing drug sensitivity in breast cancer cells

p140Cap is an adaptor protein involved in assembling multi-protein complexes regulating several cellular processes. p140Cap acts as a tumor suppressor in breast cancer (BC) and neuroblastoma patients, where its expression correlates with a better prognosis. The role of p140Cap in tumor metabolism remains largely unknown. Here we study the role of p140Cap in the modulation of the mevalonate (MVA) pathway in BC cells. The MVA pathway is responsible for the biosynthesis of cholesterol and non-sterol isoprenoids and is often deregulated in cancer. We found that both in vitro and in vivo, p140Cap cells and tumors show an increased flux through the MVA pathway by positively regulating the pace-maker enzyme of the MVA pathway, the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), via transcriptional and post-translational mechanisms. The higher cholesterol synthesis is paralleled with enhanced cholesterol efflux. Moreover, p140Cap promotes increased cholesterol localization in the plasma membrane and reduces lipid rafts-associated Rac1 signalling, impairing cell membrane fluidity and cell migration in a cholesterol-dependent manner. Finally, p140Cap BC cells exhibit decreased cell viability upon treatments with statins, alone or in combination with chemotherapeutic at low concentrations in a synergistic manner. Overall, our data highlight a new perspective point on tumor suppression in BC by establishing a previously uncharacterized role of the MVA pathway in p140Cap expressing tumors, thus paving the way to the use of p140Cap as a potent biomarker to stratify patients for better tuning therapeutic options.

Phosphomevalonate kinase deficiency expands the genetic spectrum of systemic autoinflammatory diseases

In the isoprenoid biosynthesis pathway, mevalonate is phosphorylated in two subsequent enzyme steps by mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) to generate mevalonate pyrophosphate that is further metabolized to produce sterol and non-sterol isoprenoids. Biallelic pathogenic variants in the MVK gene result in the autoinflammatory metabolic disorder MVK Deficiency (MKD). So far, however, no patients with proven PMVK deficiency due to biallelic pathogenic variants in the PMVK gene have been reported. This study aims to report the first patient with functionally confirmed PMVK deficiency, including the clinical, biochemical and immunological consequences of a homozygous missense variant in the PMVK gene. We performed whole-exome sequencing and functional studies in cells from a patient who, upon clinical and immunological evaluation, was suspected of an autoinflammatory disease. We identified a homozygous PMVK p.Val131Ala (NM_006556.4: c.392T>C,) missense variant in our patient. Pathogenicity was supported by genetic algorithms and modeling analysis and confirmed in patient cells that revealed a markedly reduced PMVK enzyme activity due to a virtually complete absence of PMVK protein. Clinically, the patient showed various similarities, but also distinct features compared to MKD patients, and responded well to therapeutic IL-1 inhibition. In this study we report the first patient with proven PMVK deficiency due to a homozygous missense variant in PMVK leading to an autoinflammatory disease. PMVK deficiency expands the genetic spectrum of systemic autoinflammatory diseases (SAID), characterized by recurrent fevers, arthritis and cytopenia and thus should be included in the differential diagnosis and genetic testing for SAIDs.

Abstract 232: Searching for Novel Regulators of the Mevalonate Pathway

The mevalonate pathway is an important metabolic pathway that provides cells with vital bioactive molecules. The pathway converts mevalonate into various sterol and nonsterol isoprenoids required for cellular maintenance and health, including, cholesterol, ubiquinione, dolichol, heme A, farnesyl, and gernaylgeranyl protein anchors. However, these molecules are energetically expensive to synthesize and toxic in excess, so tight regulation of the pathway is necessary at multiple steps. In order to identify novel proteins that help regulate the mevalonate pathway, we performed unbiased proteomic screens to detect protein-protein interactions in Huh7 cells, a human hepatocellular carcinoma cell line. Proximity labeling with a promiscuous biotin ligase (BioID), and affinity purification were performed using multiple enzymes from the pathway as bait (MVK, MVD, FDFT1, FDPS, SQLE, and LSS). Several proteins were detected using both techniques at statistically significant levels compared to control. An extensive literature review revealed that two hits, NAB1 (NGFI-A-binding protein 1) and KHDRBS1 (KH RNA Binding Domain Containing, Signal Transduction Associated 1), could have relevant and understudied interactions with mevalonate kinase (MVK). Therefore, to determine if NAB1 or KHDRBS1 regulate MVK, they were independently knocked down using siRNAs in vitro . Knockdown of NAB1 led to decreased mRNA expression and protein levels of MVK under sterol depleted conditions, and decreased expression of high-density lipoprotein (HDL) associated genes ( ABCA1 , ApoA1 , etc). Conversely, when MVK was knocked down under sterol replete conditions, NAB1 protein levels were similarly reduced. Thus, these two proteins undergo a previously unknown interaction which warrants further study. NAB1-MVK interaction might help explain the association between MVK single nucleotide polymorphisms (SNP) and circulating HDL levels in humans, and the increase of circulating HDL in NAB1 knock out (KO) mice.

Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages

Abstract Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.

Decoding the crosstalk between mevalonate metabolism and T cell function.

The mevalonate pathway is an essential metabolic pathway in T cells regulating development, proliferation, survival, differentiation, and effector functions. The mevalonate pathway is a complex, branched pathway composed of many enzymes that ultimately generate cholesterol and nonsterol isoprenoids. T cells must tightly control metabolic flux through the branches of the mevalonate pathway to ensure sufficient isoprenoids and cholesterol are available to meet cellular demands. Unbalanced metabolite flux through the sterol or the nonsterol isoprenoid branch is metabolically inefficient and can have deleterious consequences for T cell fate and function. Accordingly, there is tight regulatory control over metabolic flux through the branches of this essential lipid synthetic pathway. In this review we provide an overview of how the branches of the mevalonate pathway are regulated in T cells and discuss our current understanding of the relationship between mevalonate metabolism, cholesterol homeostasis and T cell function.

Synthesis, function, and regulation of sterol and nonsterol isoprenoids.

Cholesterol, the bulk end-product of the mevalonate pathway, is a key component of cellular membranes and lipoproteins that transport lipids throughout the body. It is also a precursor of steroid hormones, vitamin D, and bile acids. In addition to cholesterol, the mevalonate pathway yields a variety of nonsterol isoprenoids that are essential to cell survival. Flux through the mevalonate pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGCR), the rate limiting enzyme in the mevalonate pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. This review highlights transcriptional and post-translational regulation of HMGCR. Transcriptional regulation of HMGCR is mediated by the Scap-SREBP pathway. Post-translational control is initiated by the intracellular accumulation of sterols, which causes HMGCR to become ubiquitinated and subjected to proteasome-mediated ER-associated degradation (ERAD). Sterols also cause a subfraction of HMGCR molecules to bind the vitamin K2 synthetic enzyme, UbiA prenyltransferase domain-containing protein-1 (UBIAD1). This binding inhibits ERAD of HMGCR, which allows cells to continuously synthesize nonsterol isoprenoids such as geranylgeranyl pyrophosphate (GGPP), even when sterols are abundant. Recent studies reveal that UBIAD1 is a GGPP sensor, dissociating from HMGCR when GGPP thresholds are met to allow maximal ERAD. Animal studies using genetically manipulated mice disclose the physiological significance of the HMGCR regulatory system and we describe how dysregulation of these pathways contributes to disease.

Post-Translational Regulation of HMG CoA Reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an endoplasmic reticulum (ER)-localized integral membrane protein that catalyzes the rate-limiting step in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). HMGCR is subjected to strict feedback control through multiple mechanisms to ensure cells constantly produce essential nonsterol isoprenoids, but do not overaccumulate cholesterol. Here, we focus on the mechanism of feedback control of HMGCR that involves its sterol-induced ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We will also discuss the how GGpp-regulated intracellular trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) inhibits HMGCR ERAD to balance the synthesis of sterol and nonsterol isoprenoids. Finally, we will summarize various mouse models, the characterization of which establish that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMGCR and cholesterol metabolism in vivo.

When All Is Not as It Seems: Recurrent Fever and New-onset Joint Pain in a 17-Month-old Girl.

When All Is Not as It Seems: Recurrent Fever and New-onset Joint Pain in a 17-Month-old Girl.

The Vitamin K2Synthetic Enzyme UBIAD1 Moonlights as a Key Regulator of Cholesterol Synthesis

UbiA prenyltransferase domain‐containing protein‐1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone‐4 (MK‐4). Mutations in UBIAD1 cause Schnyder corneal dystrophy (SCD), which is characterized by corneal opacification owing to over‐accumulation of cholesterol. Our studies disclosed a key role for UBIAD1 in regulating endoplasmic reticulum (ER)‐localized HMG CoA reductase, the rate‐limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Feedback control of reductase involves sterol‐induced ubiquitination, an obligatory reaction for its ER‐associated degradation (ERAD) that is augmented by GGpp. Sterols also cause UBIAD1 to bind reductase, which inhibits ERAD and allows continued synthesis of nonsterol isoprenoids in sterol‐replete cells. GGpp triggers release of reductase from UBIAD1, enhancing ERAD and stimulating translocation of UBIAD1 to Golgi. SCD‐associated UBIAD1 resists GGpp‐induced release from reductase and becomes sequestered in ER to inhibit ERAD.Gene knockout studies in mice were attempted to explore the in vivo function of UBIAD1; however, homozygous germ‐line deletion of Ubiad1 caused embryonic lethality. We generated homozygous deletion of Ubiad1 in knock‐in mice expressing ubiquitination‐resistant HMGCR, implying embryonic lethality results from enhanced ERAD of HMGCR. The study of Ubiad1‐deficient mice offers the opportunity to determine the physiological significance of UBIAD1‐mediated synthesis of MK‐4.

Abstract 425: Genetic Inhibition Of Hepatic Dolichol Biosynthesis Results In Hypolipidemia

Dolichol is a nonsterol isoprenoid derived from the mevalonate pathway. Dehydrodolichyl diphosphate synthase subunit (DHDDS) is a branch point enzyme that - in complex with Nogo-B receptor (NgBR) - catalyzes the first committed step of dolichol biosynthesis. Next, dolichol is phosphorylated, and used as carrier for oligosaccharides, which are transferred to nascent proteins in the N-linked glycosylation pathway. A growing body of evidence highlights the importance of N-linked glycosylation in regulating plasma lipid levels in humans. However, the underlying molecular mechanisms are largely unexplored. Therefore, the goal of this project is to define the role of dolichol biosynthesis in lipid metabolism in the liver, the major organ for the biosynthesis and clearance of lipoproteins. We used Adeno-Associated Virus (AAV)-mediated delivery of CRISPR/Cas9 (AAV-CRISPR) to delete Dhdds in mouse liver, to inhibit the hepatic biosynthesis of dolichol. Mice were injected with AAV-CRISPR or saline as control, and fed a high-fat diet for 4 to 12 weeks. Surprisingly, Dhdds liver-specific knockout (LSKO) mice gained less weight compared to control mice. Plasma total cholesterol and triglyceride levels were also reduced over time in Dhdds LSKO mice. Moreover, we observed less cholesterol content in the high-density lipoprotein (HDL) fractions as well as less triglycerides in very-low-density lipoproteins (VLDL). At the cellular level, Dhdds LSKO mice showed hepatic endoplasmic reticulum (ER) stress and decreased N-linked glycosylation. In addition, we observed significant downregulation of Sterol Regulatory Element-Binding Protein 1c ( Srebp-1c ) - a key regulator of de novo lipogenesis (DNL) - and downstream-regulated genes in the DNL pathway. Conversely, the expression of genes regulating cholesterol biosynthesis was unaffected. Taken together, our data show an important role for hepatic dolichol biosynthesis in regulating plasma lipids, potentially through modulation of Srebp-1c expression and DNL.

Intestinal Deletion of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Promotes Expansion of the Resident Stem Cell Compartment.

The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.

Type 1 polyisoprenoid diphosphate phosphatase modulates geranylgeranyl-mediated control of HMG CoA reductase and UBIAD1.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.

Compromised Protein Prenylation as Pathogenic Mechanism in Mevalonate Kinase Deficiency.

Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder characterized by life-long recurring episodes of fever and inflammation, often without clear cause. MKD is caused by bi-allelic pathogenic variants in the MVK gene, resulting in a decreased activity of the encoded enzyme mevalonate kinase (MK). MK is an essential enzyme in the isoprenoid biosynthesis pathway, which generates both non-sterol and sterol isoprenoids. The inflammatory symptoms of patients with MKD point to a major role for isoprenoids in the regulation of the innate immune system. In particular a temporary shortage of the non-sterol isoprenoid geranylgeranyl pyrophosphate (GGPP) is increasingly linked with inflammation in MKD. The shortage of GGPP compromises protein prenylation, which is thought to be one of the main causes leading to the inflammatory episodes in MKD. In this review, we discuss current views and the state of knowledge of the pathogenetic mechanisms in MKD, with particular focus on the role of compromised protein prenylation.

Abstract 123: Intestinal Deletion Of 3-hydroxy-3-methylglutaryl-coenzyme A Reductase Promotes Expansion Of The Resident Stem Cell Compartment

While the intestine is the critical interface between cholesterol absorption and excretion, surprisingly little is known about the role of de novo cholesterol synthesis in this organ and how it affects whole body cholesterol homeostasis. The mevalonate pathway is most well-known for the production of cholesterol, but it is also required for the production of essential non-sterol isoprenoids. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, is regulated by a three-protein complex made up of INSIG, SCAP, and SREBP2. Intestine specific knockouts of Scap and Srebp2 both result in severe enteropathy and reduced mouse survival. Here, we assessed the hypothesis that Hmgcr is required for enterocyte viability. Mice harboring floxed alleles for Hmgcr were bred with the Villin-Cre transgene to specifically knock out this enzyme in the villus and crypt epithelial cells of the small intestine (i-KO). The i-KO mice are viable through adulthood and fertile. Hmgcr is efficiently deleted based on analysis via drop digital PCR and qPCR. RNA sequencing shows reduction in all SREBP2 target genes throughout the mevalonate pathway in intestinal epithelial cells. Lipidomics confirms substantial reductions in abundance of all sterol and non-sterol isoprenoids, except 7-dehydrocholesterol and cholesterol. Cholesterol is likely maintained through reabsorption of biliary cholesterol or increased uptake from the circulation. Circulating cholesterol levels and cholesterol absorption are not altered in i-KO mice, but triglyceride absorption is increased through compensatory changes in bile acid composition and intestinal growth. At the cellular level, the intestine rapidly compensates for loss of Hmgcr via dramatic expansion of the stem cell compartment within the crypts. In conclusion, the mechanisms by which the intestine compensates for genetic loss of Hmgcr include altered triglyceride absorption, bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment. Together these studies provide insight into the effects of HMGCR knockout in intestinal development and lipid metabolism.

Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol.

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Enhanced ER-associated degradation of HMG CoA reductase causes embryonic lethality associated with Ubiad1 deficiency

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) synthesizes the vitamin K subtype menaquinone-4 (MK-4). Previous studies in cultured cells (Schumacher et al., 2015) revealed that UBIAD1 also inhibits endoplasmic reticulum (ER)-associated degradation (ERAD) of ubiquitinated HMG CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway that produces cholesterol and essential nonsterol isoprenoids. Gene knockout studies were previously attempted to explore the function of UBIAD1 in mice; however, homozygous germ-line elimination of the Ubiad1 gene caused embryonic lethality. We now report that homozygous deletion of Ubiad1 is produced in knockin mice expressing ubiquitination/ERAD-resistant HMGCR. Thus, embryonic lethality of Ubiad1 deficiency results from depletion of mevalonate-derived products owing to enhanced ERAD of HMGCR rather than from reduced synthesis of MK-4. These findings provide genetic evidence for the significance of UBIAD1 in regulation of cholesterol synthesis and offer the opportunity in future studies for the discovery of new physiological roles of MK-4.