First by regulation of transcription of the reductase gene, which is activated by sterol regulatory element binding protein, a protein that binds to the promoter of the HMGR gene when cholesterol levels fall.
The third level of HMGR regulation involves the degradation of intact reductase. Helices of the HMGR transmembrane domain, called the sterol-sensing domain, sense the increased levels of sterols.
Additionally, as sterol levels increase, the helices can expose Lysine which can be ubiquitinated and subsequently trigger proteolytic degradation. A final level of HMGR regulation is achieved by inhibition via. Elevated cholesterol levels have been identified as a major risk factor for coronary artery disease, the narrowing of arteries of the heart, which affected over 13 million people in the United States alone.
It is a major cause of disability and death, killing over thousand people in the USA in Since HMGR is the first committed enzyme in the cascade that eventually produces cholesterol, use of statins can dramatically reduce blood cholesterol levels. The structure of HMGR with bound statins 1hwk marketed as Lipitor shows that the cis loop forms a number of polar interactions with the statin inhibitor, particularly residues Ser , Asp , Lys , Lys , and hydrogen bond interactions between Glu and Asp with the O5-hydroxyl of the statins.
The catalytic domain is highly conserved in eukaryotes, but the membrane-anchor domain consisting of between two and eight membrane-spanning helices is poorly conserved, and the HMGRs of archaea and of certain eubacteria lack a membrane-anchor domain.
A phylogenetic tree of HMGRs. The tree includes 98 selected organisms that have hmgr genes; for plants, which have multiple isoforms, only isoform 1 of each species is included in the tree. Roman numerals indicate the division of the family into two classes [2,3]. Phylogenetic analysis was performed using aligned amino-acid sequences of HMGR catalytic domains; membrane anchor domains were excluded from analysis.
Full species names and GenBank accession numbers of the sequences used are provided in Table 1. The HMGRs of different organisms are multimers of a species-specific number of identical monomers. As reviewed in detail by Istvan [ 10 ], structural comparisons reveal both similarities and significant differences between the two classes of enzyme.
The core regions containing the catalytic domains of the two enzymes have similar folds. Despite differences in amino-acid sequence and overall architecture, functionally similar residues participate in the binding of coenzyme A by the two enzymes, and the position and orientation of four key catalytic residues glutamate, lysine, aspartate and histidine is conserved in both classes of HMGR.
Unlike the central cores, the amino- and carboxy-terminal regions of the catalytic domains show little similarity between the human and P. In contrast, the P. There is a considerable difference in specific interactions with inhibitor between the two enzymes, however [ 8 , 9 ], accounting for the almost 10 4 -fold higher K i values for inhibition of HMGR P by statin relative to the inhibition of HMGR H K i is the equilibrium constant for an inhibitor binding to an enzyme.
There are significant differences in the regions of the two proteins that bind statins. For HMGR P , this impairs closure over the active site of the 'tail' domain that contains the catalytic histidine. HMGRs of eukaryotes are localized to the endoplasmic reticulum ER , and are directed there by a short portion of the amino-terminal domain prokaryotic HMGRs are soluble and cytoplasmic.
In humans, the reaction catalyzed by HMGR is the rate-limiting step in the synthesis of cholesterol, which maintains membrane fluidity and serves as a precursor for steroid hormones. In plants, a cytosolic HMG-CoA reductase participates in the synthesis of sterols, which are involved in plant development, certain sesquiterpenes, which are important in plant defense mechanisms against herbivores, and ubiquinone, which is critical for cellular protein turnover.
In plastids, however, these compounds are synthesized via a pathway that does not involve mevalonate or HMGR [ 1 ]. Various plant HMGR isozymes function in fruit ripening and in the response to environmental challenges such as attack by pathogens. This three-stage reaction involves two reductive stages and the formation of enzyme-bound mevaldyl-CoA and mevaldehyde as probable intermediates:.
The side groups of the key catalytic residues, Lys, Asp, Glu83, and His, are shown, and the substrate and products are shown with R representing the HMG portion.
The reaction follows three stages see text for details. A highly regulated enzyme, HMGR H is subject to transcriptional, translational, and post-translational control [ 12 ] that can result in changes of over fold in intracellular levels of the enzyme.
Cleavage releases the amino-terminal basic helix-loop-helix bHLH domain, which enters the nucleus, where it functions as a transcription factor that recognizes non-palindromic decanucleotide sequences of DNA called sterol-regulatory elements SREs. Degradation of HMGR H involves its transmembrane regions [ 14 ]: removal of two or more transmembrane regions abolishes the acceleration of HMGR H degradation that occurs under certain conditions [ 12 , 15 ]: degradation is induced by a non-sterol, mevalonate-derived metabolite alone or by a sterol plus a mevalonate-derived non-sterol metabolite, possibly farnesyl pyrophosphate or farnesol.
Four conserved phenylalanines in the sixth membrane span of the transmembrane region are essential for degradation of HMGR H [ 16 ].
The location of this serine - six residues from the catalytic histidine, a spacing conserved in all higher eukaryote HMGRs - suggests that the phosphoserine may interfere with the ability of this histidine to protonate the inhibitory CoAS - thioanion that is released in stage 2 of the reaction mechanism.
Alternatively, it may interfere with closure of the flap domain, a carboxy-terminal region that is thought to close over the active site to facilitate catalysis, a step thought to be essential for formation of the active site [ 7 ]. Subsequent dephosphorylation restores full catalytic activity. Phosphorylation of serine of A. As many plant genes encode a putative target serine surrounded by an apparent AMP kinase recognition motif, it is probable that most plant HMGRs are regulated by phosphorylation.
The phosphorylation state of HMGR does not affect the rate at which the protein is degraded. Several basic unresolved questions concern how phosphorylation controls the catalytic activity of HMGRs; solution of the structures of phosphorylated HMGRs should reveal more of the precise mechanism.
The protein kinases, phosphatases, and signal-transduction pathways that participate in short-term regulation of HMGR activity are yet to be elucidated.
Finally, the physiological roles served by the multiple ways in which HMGR is regulated require clarification. On the medical side, continuing intense competition between drug companies for a share of the lucrative worldwide market for hypercholesterolemic agents should result in new statin drugs with modified pharmacodynamic and metabolic properties that not only lower plasma cholesterol levels more effectively but more importantly minimize undesirable side effects.
A study of the regulation of both mevalonate and mevalonate independent pathways for isoprenoid synthesis in plants. Mol Genet Metab. The authors utilized phylogenetic analysis to analyze a plethora of genomic sequences of various organisms. J Bacteriol. A review article detailing current research and thought concerning Class II forms of the enzyme, including the HMGRs of many pathogenic bacteria. Fractionated S from Hela cells was utilized to determine which component of the reductase ubiquitinating machinery E1, E2 and E3 is provided by rat liver cytosol in the permeabilized cell system These fractions were first described by Hershko and co-workers 43 , 44 and were generated by separating Hela cell S into fractions that bind Fraction II or do not bind Fraction I an anion exchange resin.
Fraction II effectively replaces rat liver cytosol for regulated ubiquitination of reductase in permeabilized cells, but Fraction I does not. Two observations indicate that Fraction II provides a source of ubiquitin activation in the permeabilized cell system.
First, purified E1 replaces rat liver cytosol for sterol-regulated ubiquitination of reductase in permeabilized cells. Second, immunodepletion of E1 eliminates the reductase ubiquitinating activity of rat liver cytosol. These results demonstrate that E1 is the only cytosolic protein required for reductase ubiquitination, which indicates the reductase E2 and E3 are membrane-associated proteins. This notion is consistent with the localization of apparent sites of reductase ubiquitination, lysines 89 and , which are cytosolically exposed and are predicted to lie immediately adjacent to transmembrane helices three and seven Figure 2B.
Results from the analysis of reductase ubiquitination in permeabilized cells indicated that Insig binding results in recruitment of enzymes that ubiquitinate reductase. Coimmunoprecipitation experiments, coupled with tandem mass spectroscopy, were utilized to identify membrane proteins that associate with the sterol-dependent reductase-Insig complex.
These studies revealed that Insig-1 binds to a known membrane-anchored ubiquitin ligase called gp78 The cDNA for gp78 predicts a amino acid protein that can be divided into four domains. The N-terminal domain of amino acids contains five to seven membrane-spanning helices that anchor the protein to ER membranes and mediate association with Insig The membrane attachment region of gp78 is followed by a amino acid region with a RING finger consensus sequence that confers ubiquitin ligase activity Following the RING domain is a amino acid region homologous to Cue1p, an ER membrane protein in yeast that serves as a membrane anchor for Ubc7p, a cytosolic ubiquitin-conjugating enzyme Recently, this region of gp78 has been shown to directly bind to Ufd1, a cytosolic protein that modulates gp78 ubiquitin ligase activity, thereby enhancing ubiquitination and degradation of the enzyme's substrates At least three lines of evidence indicate that gp78, through its binding to Insig-1, initiates sterol-accelerated degradation of reductase.
Importantly, the effect of gp78 knockdown is specific inasmuch as knockdown of a related membrane-bound ubiquitin ligase, Hrd1, does not affect reductase ubiquitination. Another function of gp78, besides its role as a ubiquitin ligase, is to couple ubiquitination of reductase to degradation through its association with VCP. Indeed, coimmunoprecipitation experiments show that gp78 is an intermediary in association of VCP and Insig The identification of gp78 as an E3 ubiquitin ligase that mediates reductase ubiquitination has important implications for yet another mode of sterol regulation.
The regulation of Insig-1 contrasts that of reductase in that Insig-1 becomes ubiquitinated and is rapidly degraded by proteasomes in sterol-depleted cells Ubiquitination of Insig-1 is mediated by gp78 When sterols induce reductase to bind Insig-1, ubiquitination is diverted toward reductase and the enzyme becomes rapidly degraded. However, when sterols cause Scap to bind Insig-1, gp78 is displaced and no longer ubiquitinates Insig-1, thereby stabilizing the protein.
This reaction helps to explain why reductase is degraded when it binds to Insig-1, whereas Scap binding to Insig-1 leads to retention in the ER.
Increased transcription of the Insig-1 gene leads to increased synthesis of Insig-1 protein, but the protein is ubiquitinated and degraded until sterols build up to levels sufficient to trigger Scap binding.
Oxysterols are derivatives of cholesterol that contain hydroxyl groups at various positions in the iso-octyl side chain 53 , These compounds are synthesized in many tissues by specific enzymes called hydroxylases; oxysterols play key roles in cholesterol export and they are also intermediates in the synthesis of bile acids Oxysterols are significantly more soluble than cholesterol in aqueous solution, and thus can readily pass across the plasma membrane and enter cells.
This property renders oxysterols such as , , and hydroxycholesterol extremely potent in inhibiting cholesterol synthesis by stimulating binding of both reductase and Scap to Insigs. Oxysterols are present at very low concentrations 10 4 - to 10 6 -fold less than cholesterol in tissues and blood, which raises questions as to whether they act through a similar mechanism as LDL-derived cholesterol to block cholesterol synthesis.
In the case of Scap, the mode of action of these two classes of sterols is becoming clear. Cholesterol directly binds to the membrane domain of Scap in a specific and saturable fashion The interaction causes a conformational change in Scap that promotes Insig binding The addition of cholesterol in vitro to membranes isolated from sterol-depleted cells causes exposure of a cryptic trypsin cleavage site, thereby altering the tryptic digestion pattern of Scap that can be monitored by immunoblot analysis Co-expression of Insigs lowers the amount of cholesterol required to induce the conformational change in Scap.
Oxysterols neither alter Scap's conformation in vitro nor bind to the protein's membrane domain, leading to the postulation of the existence of a membrane-bound oxysterol binding protein. Remarkably, Insig-2 has been recently defined as a membrane-bound oxysterol binding protein with binding specificity that correlates with the ability of oxysterols to inhibit SREBP processing 32 , Thus, formation of the Scap-Insig complex can be initiated by either binding of cholesterol to the membrane domain of Scap or by binding of oxysterols to Insigs.
By analogy, the likely mechanism by which oxysterols stimulate degradation of reductase is through their binding to Insigs. In striking contrast to results obtained with Scap, the analysis of reductase ubiquitination in permeabilized cells revealed that the reaction was potently stimulated by oxysterols, but not by cholesterol These results led to a search for endogenous sterol regulators of reductase ubiquitination and degradation.
Previous indirect studies implicated that lanosterol, the first sterol produced in the cholesterol biosynthetic pathway Figure 1 , or one of its metabolites participates in feedback inhibition of reductase. These observations led to the evaluation of lanosterol and its metabolite 24,dihydrolanosterol as endogenous regulators of reductase ubiquitination and degradation When added to intact cells, lanosterol and 24,dihydrolanosterol potently stimulate ubiquitination and degradation of reductase through a reaction that requires the presence of Insigs.
This is consistent with the inability of lanosterol to directly bind to Scap and Insig or alter Scap's conformation in vitro The action of lanosterol and 24,dihydrolanosterol is direct and does not require their conversion into an active metabolite as indicated by the reconstitution of reductase ubiquitination by simply incubating isolated membranes with the sterols and purified E1.
Insig-mediated regulation of reductase is controlled by three classes of sterols: oxysterols, cholesterol, and methylated sterols such as lanosterol and 24,dihydrolanosterol.
Oxysterols, which are derived from cholesterol, have dual actions in that they accelerate degradation of reductase and block ER to Golgi transport of Scap-SREBP through their direct binding to Insigs. Notably, the demethylation of lanosterol has been implicated as a rate-limiting step in the post-squalene portion of cholesterol synthesis, suggesting the reaction as a potential focal point in sterol regulation 63 , Considering that lanosterol is the first sterol produced in cholesterol synthesis, it seems reasonable that it controls early steps in the pathway i.
This assures that mRNAs encoding enzymes catalyzing reactions subsequent to lanosterol remain elevated and lanosterol is metabolized to cholesterol. The importance of this conversion is highlighted by the observation that lanosterol cannot support cell growth in the absence of cholesterol and may be toxic This toxicity is likely due to the inability to optimize certain physiologic properties of cell membranes with regard to biological functions.
The physiologic relevance of lanosterol as an endogenous regulator of reductase ubiquitination and degradation was deduced by the recognition that cholesterol synthesis is a highly oxygen-consumptive process.
This led to speculation that oxygen deprivation hypoxia might block demethylation of lanosterol and 24,dihydrolanosterol and thereby stimulate degradation of reductase. Indeed, a recent study shows that hypoxia blunts cholesterol synthesis by inhibiting lanosterol and 24,dihydrolanosterol demethylation, causing both sterols to accumulate in cells Rapid degradation of reductase parallels hypoxia-induced accumulation of lanosterol and 24,dihydrolanosterol.
This finding is consistent with the observation described above that exogenous lanosterol stimulates degradation of reductase without inhibiting SREBP processing. Considered together, these observations establish a connection between cholesterol synthesis and oxygen sensing in animal cells Figure 4. Convergence of these signals triggers rapid degradation of reductase, which ultimately limits synthesis of cholesterol and helps to guard against the wasting of cellular oxygen in the face of hypoxia.
Mechanism for oxygen sensing in the cholesterol synthetic pathway. Convergence of these responses leads to rapid degradation of HMG CoA reductase, thereby limiting synthesis of cholesterol. Despite the recent advances in the understanding of molecular mechanisms underlying sterol-accelerated degradation of reductase, much remains to be determined. For instance, what is the mechanism by which lanosterol and 24,dihydrolanosterol trigger binding of reductase to Insigs?
Do these methylated sterols directly bind the membrane domain of reductase in a reaction analogous to that of cholesterol and Scap? Unfortunately, attempts to demonstrate direct binding of methylated sterols to the membrane domain of reductase have been unsuccessful. Moreover, addition of lanosterol or 24,dihydrolanosterol to reductase-containing membranes in vitro fails to alter the tryptic pattern of the enzyme. Thus, the possibility exists that a distinct ER membrane protein binds to methylated sterols and, in turn, triggers binding of reductase to Insigs, thereby initiating reductase ubiquitination.
Reductase is the target of statins, which are the most widely prescribed cholesterol-lowering drugs in humans. Interest in developing additional strategies that inhibit reductase has led to the discovery of nonsterol compounds, such as vitamin E tocotrienols and the bisphosphonate SR, that mimic sterols in accelerating reductase degradation 76 , The availability of such reagents may prove useful in the ongoing quest to define the molecular mechanisms for the reductase sterol-sensing reaction.
Another unresolved question in reductase degradation is the mechanism for delivery of ubiquitinated forms of the enzyme from the membrane to the cytosol for proteasomal degradation. Unlike model ERAD substrates that are either completely lumenal or contain one transmembrane domain, proteasome inhibition leads to accumulation of ubiquitinated reductase on membranes, rather than in the cytosol This suggests that degradation of reductase is coupled to its ubiquitination and proceeds through a membrane-bound intermediate.
However, reductase must be degraded as a unit without releasing the catalytic domain into the cytosol, which would defeat the purpose of regulated degradation. Efficient degradation of reductase requires nonsterol isoprenoids derived from mevalonate in addition to sterols.
This was borne out of experiments showing that in compactin-treated cells sterols can trigger binding of reductase to Insigs and subsequent ubiquitination of the enzyme. However, the ubiquitinated reductase is not efficiently degraded unless the cells are also treated with mevalonate.
This mevalonate requirement can be bypassed by the addition of geranylgeraniol GG-OH , a carbon isoprenoid, but not by the carbon farnesol GGOH does not appear to trigger reductase ubiquitination, even though it augments sterol-accelerated degradation of the enzyme.
This suggests the action of nonsterol isoprenoids in a post-ubiquitination step of reductase degradation. The current view of sterol-accelerated degradation of reductase is illustrated in the model shown in Figure 5. The reaction is initiated by sensing of membrane-embedded sterols through direct or indirect interactions with the membrane domain of reductase. This interaction causes reductase to bind to a subset of Insigs that are associated with gp78, which mediates transfer of ubiquitin from the E2 Ubc7 to lysines 89 and of reductase.
Ubiquitination targets reductase for recognition by gpassociated VCP, which, together with its cofactors, somehow extract ubiquitinated reductase from membranes and deliver it to proteasomes for degradation. The extraction step appears to be augmented by GG-OH. It seems likely that GG-OH, after its conversion to metabolically active geranylgeranyl-pyrophosphate GG-PP , is incorporated into a protein that enhances the effect of sterols on reductase degradation.
Possible candidates include geranylgeranylated Rab proteins, which are known to play key roles in various aspects of vesicular transport Thus, the possibility exists that a vesicle-mediated transport event delivers ubiquitinated reductase to a specific organelle or subdomain of the ER in which the protein is degraded.
Notably, Ufd1 appears to play a key role in this pathway by enhancing gp78 ubiquitin ligase activity and modulating a post-ubiquitination step in reductase degradation. In addition, Ufd1 seems to bind to gp78 in a sterol-regulated fashion 49 , but the significance of this is presently unknown. Complete elucidation of reductase degradation will likely require the reconstitution of post-ubiquitination steps of reductase degradation in a cell-free system.
Accumulation of hydroxycholesterol, lanosterol, or 24,dihydrolanosterol in ER membranes triggers binding of the reductase to Insigs. A subset of Insigs is associated with the membrane-anchored ubiquitin ligase, gp78, which binds the E2 Ubc7 and VCP, an ATPase that plays a role in extraction of ubiquitinated proteins from ER membranes. Through the action of gp78 and Ubc7, reductase becomes ubiquitinated, which triggers its extraction from the membrane by VCP, and subsequent delivery to proteasomes for degradation.
The post-ubiquitination step is postulated to be enhanced by geranylgeraniol through an undefined mechanism that may involve a geranylgeranylated protein, such as one of the Rab proteins. What is the contribution of reductase degradation to overall cholesterol homeostasis in whole animals?
Insigs appear to play a major role in regulation of reductase in the mouse liver. Genetic deletion of Insigs results in the accumulation of reductase to a level approximately fold higher than that in wild type mice This accumulation is presumably attributable to the combination of both transcriptional and post-transcriptional regulation of reductase, but the extent to which each level of regulation contributed to the massive increase in reductase is unknown.
Thus, studies that directly focus on reductase degradation are required in order to determine the contribution of protein stability to overall regulation of reductase in mice in vivo under various physiologic conditions, such as hypoxia. The significance of Insig-mediated regulation of reductase in maintenance of cholesterol homeostasis is highlighted by the effectiveness of reductase inhibition in lowering plasma LDL-cholesterol in humans However, the inhibition of reductase disrupts normal feedback inhibition of the enzyme, and animals respond by developing a compensatory increase in reductase levels in the liver 81 , Remarkably, a similar response has been observed in livers of statin-treated humans as well Knowledge of the mechanisms for this compensatory increase, particularly the contribution of degradation, may facilitate development of novel drugs that improve the effectiveness of statins, or in some cases provide alternative treatments.
Such a drug would be modeled after lanosterol and 24,dihydrolanosterol, which selectively stimulate reductase degradation without affecting the Scap-SREBP pathway or LDL-receptor activity. In addition, elucidation of the underlying mechanisms for sterol-accelerated, ERAD of reductase may have implications for degradation of other clinically important proteins such as the cystic fibrosis transmembrane conductance regulator CFTR.
Thus, further excitement will undoubtedly ensue once questions posed in this review begin to become clear. Regulation of the mevalonate pathway. Nature ; — Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J Lipid Res ; 21 — Competitive inhibition of 3-hydroxymethylglutaryl coenzyme A reductase by MLA and MLB fungal metabolites, having hypocholesterolemic activity.
FEBS Lett ; 72 — Induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin MLB , a competitive inhibitor of the reductase.
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