Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.     

Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase

3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).     

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

The polytopic endoplasmic reticulum (ER)–localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.     

Amatoxins,Phallotoxins, Phallolysin,and Antamanide: The Biologically Active Components of Poisonous Amanita Mushroom

Multiple mechanisms for feedback control of cholesterol synthesis converge on the rate-limiting enzyme in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase. This complex feedback regulatory system is mediated by sterol and nonsterol metabolites of mevalonate, the immediate product of reductase activity. One mechanism for feedback control of reductase involves rapid degradation of the enzyme from membranes of the endoplasmic reticulum (ER). This degradation results from the accumulation of sterols in ER membranes, which triggers binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the recruitment of a membrane-associated ubiquitin ligase called gp78 that initiates ubiquitination of reductase. Ubiquitinated reductase then becomes extracted from ER membranes and is delivered to cytosolic 26S proteasomes through an unknown mechanism that is mediated by the gp78-associated ATPase Valosin-containing protein/p97 and appears to be augmented by nonsterol isoprenoids. Here, we will highlight several advances that have led to the current view of mechanisms for sterol-accelerated, ER-associated degradation of reductase. In addition, we will discuss potential mechanisms for other aspects of the pathway such as selection of reductase for gp78-mediated ubiquitination, extraction of the ubiquitinated enzyme from ER membranes, and the contribution of Insig-mediated degradation to overall regulation of reductase in whole animals.     

Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase

The endoplasmic reticulum (ER) enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase, catalyzes the production of mevalonate, a rate-controlling step in cholesterol biosynthesis. Excess sterols promote ubiquitination and subsequent degradation of reductase as part of a negative feedback regulatory mechanism. To characterize the process in more detail, we here report the development of a permeabilized cell system that supports reductase ubiquitination stimulated by the addition of sterols in vitro. Sterol-dependent ubiquitination of reductase in permeabilized cells is dependent upon exogenous cytosol, ATP, and either Insig-1 or Insig-2, two membrane-bound ER proteins shown previously to mediate sterol regulation of reductase degradation in intact cells. Oxysterols, but not cholesterol, promote reductase ubiquitination under our conditions. Finally, we show that ubiquitin-activating enzyme (E1) can efficiently replace cytosol to ubiquitinate reductase in response to sterol treatment, suggesting that other molecules required for ubiquitination of reductase, such as the ubiquitin-conjugating and -ligating enzymes (E2 and E3), are localized to ER membranes.     

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.     

Regulation of hepatic Insig-2 by the farnesoid X receptor

Activation of the farnesoid X receptor (FXRalpha) affects genes controlling many pathways, including those involved in bile acid and glucose homeostasis. Here we report that a critical gene involved in cholesterol homeostasis, Insig-2, was induced when mice or cultured cells were treated with FXRalpha agonists or infected with constitutively active FXRalpha. No such induction was observed in agonist-treated FXRalpha-/- mice. Further analysis, which included EMSAs, reporter gene activation, and chromatin immunoprecipitation, identified two functional FXRalpha response elements within intron 2 of the mouse Insig-2 gene. In addition to increasing hepatic Insig-2 protein levels in wild-type mice, FXRalpha activation also reduced lanosterol 14alpha-demethylase mRNA levels and 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein levels. Together, these changes likely account for the decrease in cholesterol synthesis observed after activation of FXR in primary hepatocytes. In conclusion, the current study links hepatic FXRalpha activation to regulation of genes involved in cholesterol synthesis.     

Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol

Feedback control of cholesterol synthesis is mediated in part by sterol-induced binding of HMG CoA reductase to Insig proteins in the endoplasmic reticulum (ER). Binding leads to ubiquitination and proteasomal degradation of reductase, a rate-controlling enzyme in cholesterol synthesis. Using in vitro and in vivo assays, we show that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations. Lanosterol is not effective in mediating the other action of Insigs, namely to promote ER retention of SCAP-SREBP complexes, a reaction that is mediated directly by cholesterol. A pair of methyl groups located in the C4 position of lanosterol confers this differential response. These data indicate that buildup of cholesterol synthesis intermediates represses the pathway selectively at reductase and reveal a previously unappreciated link between feedback inhibition of reductase and carbon flow through the cholesterol synthetic pathway.     

Regulated Endoplasmic Reticulum-associated Degradation of a Polytopic Protein: p97 RECRUITS PROTEASOMES TO Insig-1 BEFORE EXTRACTION FROM MEMBRANES*

Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes.     

Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain

Sterols accelerate degradation of the ER enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), which catalyzes a rate-controlling step in cholesterol biosynthesis. This degradation contributes to feedback inhibition of synthesis of cholesterol and nonsterol isoprenoids. Here, we show that degradation of HMG CoA reductase is accelerated by the sterol-induced binding of its sterol-sensing domain to the ER protein insig-1. Accelerated degradation is inhibited by overexpression of the sterol-sensing domain of SREBP cleavage-activating protein (SCAP), suggesting that both proteins bind to the same site on insig-1. Whereas insig-1 binding to SCAP leads to ER retention, insig-1 binding to HMG CoA reductase leads to accelerated degradation that is blocked by proteasome inhibitors. Insig-1 appears to play an essential role in the sterol-mediated trafficking of two proteins with sterol-sensing domains, HMG CoA reductase and SCAP.     

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

Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.     

Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis

Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol. Neither drug prevented suppression of reductase activity by 25-hydroxylanosterol, 25-hydroxycholesterol, or mevalonolactone added to the medium. Each drug increased the binding, uptake, and degradation of 125I-labeled LDL and inhibited the re-esterification of free cholesterol to cholesteryl oleate and cholesteryl palmitate. The release of free cholesterol from [3H]cholesteryl linoleate LDL could not account for the differential effect of ketoconazole and miconazole on the prevention of low density lipoprotein suppression of reductase activity. The differential effect of the drugs on low density lipoprotein suppression of reductase activity was not unique to IEC-6 cells, but was also observed in several cell lines of different tissue origin such as human skin fibroblast cells (GM-43), human hepatoblastoma cells (HepG2), and Chinese hamster ovary cells (wild type, K-1; 4 alpha-methyl sterol oxidase mutant, 215). These observations suggest that the suppressive action of low density lipoprotein on reductase activity 1) does not require the de novo synthesis of cholesterol, or 24(S), 25-epoxysterols; 2) is not mediated via the same mechanism as that of mevalonolactone; and 3) does not involve cholesteryl reesterification. Ketoconazole blocks a site in the process of LDL suppression of reductase activity that is not affected by miconazole.     

Modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by azole antimycotics requires lanosterol demethylation, but not 24,25-epoxylanosterol formation

The lanosterol 14 alpha-methyl demethylase inhibitors miconazole and ketoconazole have been used to assess their effects upon cholesterol biosynthesis in cultured Chinese hamster ovary cells. In Chinese hamster ovary cells treated with either agent, an initial accumulation of lanosterol and dihydrolanosterol has been observed. At elevated concentrations, however, ketoconazole, but not miconazole, causes the preferential accumulation of 24,25-epoxylanosterol and squalene 2,3:22,23-dioxide. These metabolites accumulate at the expense of lanosterol, thereby demonstrating a second site of inhibition for ketoconazole in the sterol biosynthetic pathway. Both demethylase inhibitors produced a biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. The biphasic modulation is characterized by low levels of the drugs suppressing HMG-CoA reductase activity which is restored to either control or above control values at higher drug concentrations. This modulatory effect of the lanosterol demethylase inhibitors upon HMG-CoA reductase was not observed in the lanosterol 14 alpha-methyl demethylase-deficient mutant AR45. Suppression of HMG-CoA reductase activity is shown to be due to a decrease in the amount of enzyme protein consistent with a steroidal regulatory mechanism. Collectively, the results establish that lanosterol 14 alpha-methyl demethylation, but not 24,25-epoxylanosterol formation, is required to suppress HMG-CoA reductase in the manner described by lanosterol demethylase inhibitors.