Regulation of HMGR by covalent modification. HMGR is most active in the dephosphorylated state. Phosphorylation is catalyzed by AMP-activated protein kinase, AMPK, (used to be termed HMGR kinase), an enzyme whose activity is also regulated by phosphorylation. Phosphorylation of AMPK is catalyzed by AMPK kinase (AMPKK). Hormones such as glucagon and epinephrine negatively affect cholesterol biosynthesis by increasing the activity of the inhibitor of phosphoprotein phosphatase inhibitor-1, PPI-1. Conversely, insulin stimulates the removal of phosphates and, thereby, activates HMGR activity. Additional regulation of HMGR occurs through an inhibition of its' activity as well as of its' synthesis by elevation in intracellular cholesterol levels. This latter phenomenon involves the transcription factor SREBP described below.
The activity of HMGR is additionally controlled by the cAMP signaling pathway. Increases in cAMP lead to activation of cAMP-dependent protein kinase, PKA. In the context of HMGR regulation, PKA phosphorylates phosphoprotein phosphatase inhibitor-1 (PPI-1) leading to an increase in its' activity. PPI-1 can inhibit the activity of numerous phosphatases including protein phosphatase 2C and HMG-CoA reductase phosphatase which remove phosphates from AMPK and HMGR, respectively. This maintains AMPK in the phosphorylated and active state, and HMGR in the phosphorylated and inactive state. As the stimulus leading to increased cAMP production is removed, the level of phosphorylations decreases and that of dephosphorylations increases. The net result is a return to a higher level of HMGR activity.
Since the intracellular level of cAMP is regulated by hormonal stimuli, regulation of cholesterol biosynthesis is hormonally controlled. Insulin leads to a decrease in cAMP, which in turn activates cholesterol synthesis. Alternatively, glucagon and epinephrine, which increase the level of cAMP, inhibit cholesterol synthesis.
The ability of insulin to stimulate, and glucagon to inhibit, HMGR activity is consistent with the effects of these hormones on other metabolic pathways. The basic function of these two hormones is to control the availability and delivery of energy to all cells of the body.
Long-term control of HMGR activity is exerted primarily through control over the synthesis and degradation of the enzyme. When levels of cholesterol are high, the level of expression of the HMGR gene is reduced. Conversely, reduced levels of cholesterol activate expression of the gene. Insulin also brings about long-term regulation of cholesterol metabolism by increasing the level of HMGR synthesis.
Proteolytic Regulation of HMG-CoA Reductase
The stability of HMGR is regulated as the rate of flux through the mevalonate synthesis pathway changes. When the flux is high the rate of HMGR degradation is also high. When the flux is low, degradation of HMGR decreases. This phenomenon can easily be observed in the presence of the
statin drugs.
HMGR is localized to the ER and like SREBP (see below) contains a sterol-sensing domain, SSD. When sterol levels increase in cells there is an concomitant increase in the rate of HMGR degradation. The degradation of HMGR occurs within the proteosome, a multiprotein complex dedicated to protein degradation. The primary signal directing proteins to the proteosome is ubiquitination. Ubiquitin is a 7.6kDa protein that is covalently attached to proteins targeted for degradation by ubiquitin ligases. These enzymes attach multiple copies of ubiquitin allowing for recognition by the proteosome. HMGR has been shown to be ubiquitinated prior to its degradation. The primary sterol regulating HMGR degradation is cholesterol itself. As the levels of free cholesterol increase in cells, the rate of HMGR degradation increases.
The Utilization of Cholesterol
Cholesterol is transported in the plasma predominantly as cholesteryl esters associated with
lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDLs. The liver synthesizes VLDLs and these are converted to LDLs through the action of endothelial cell-associated lipoprotein lipase. Cholesterol found in plasma membranes can be extracted by HDLs and esterified by the HDL-associated enzyme LCAT. The cholesterol acquired from peripheral tissues by HDLs can then be transferred to VLDLs and LDLs via the action of cholesteryl ester transfer protein (Apo-D) which is associated with HDLs.
Reverse cholesterol transport allows peripheral cholesterol to be returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile as free cholesterol or as bile salts following conversion to bile acids in the liver.
Regulation of Cellular Sterol Content
The continual alteration of the intracellular sterol content occurs through the regulation of key sterol synthetic enzymes as well as by altering the levels of cell-surface LDL receptors. As cells need more sterol they will induce their synthesis and uptake, conversely when the need declines synthesis and uptake are decreased. Regulation of these events is brought about primarily by sterol-regulated transcription of key rate limiting enzymes and by the regulated degradation of HMGR. Activation of transcriptional control occurs through the regulated cleavage of the membrane-bound transcription factor
sterol regulated element binding protein, SREBP. As discussed above, degradation of HMGR is controlled by the ubiquitin-mediated pathway for proteolysis.
Sterol control of transcription affects more than 30 genes involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids. Transcriptional control requires the presence of an octamer sequence in the gene termed the
sterol regulatory element, SRE-1. It has been shown that SREBP is the transcription factor that binds to SRE-1 elements. It turns out that there are 2 distinct SREBP genes, SREBP-1 and SREBP-2. In addition, the SREBP-1 gene encodes 2 proteins, SREBP-1a and SREBP-1c as a consequence of alternative exon usage. All 3 proteins are proteolytically regulated by sterols. Full-length SREBPs have several domains and are embedded in the membrane of the endoplasmic reticulum (ER). The N-terminal domain contains a
transcription factor motif of the basic helix-loop-helix (bHLH) type that is exposed to the cytoplasmic side of the ER. There are 2 transmembrane spanning domains followed by a large C-terminal domain also exposed to the cytosolic side. When sterols are scarce, cleavage of the full-length SREBP takes place with the result being that the N-terminal bHLH motif is released into the cytosol. The bHLH domain then migrates to the nucleus to direct transcription. Conversely, when sterols are abundant, cleavage of SREBP is inhibited. To control the level of SREBP-mediated transcription, the soluble bHLH domain is itself subject to rapid proteolysis.
The cleavage of SREBP is carried out by 2 distinct enzymes, one of which is regulated by sterols. The regulated cleavage occurs in the lumenal loop between the 2 transmembrane domains. This cleavage is catalyzed by
site-1 protease, S1P. High sterol content blocks the activity of S1P. The second cleavage, catalyzed by
site-2 protease, S2P, occurs in the first transmembrane span, leading to release of active SREBP. In order for S2P to act on SREBP, site-1 must already have been cleaved.
Additional studies on sterol-regulated gene expression demonstrated that cleavage of SREBP by S1P is controlled by the level and action of an additional protein termed,
SREBP cleavage-activating protein, SCAP. SCAP is a large protein also found in the ER membrane and contains at least 8 transmembrane spans. The C-terminal portion, which extends into the cytosol, has been shown to interact with the C-terminal domain of SREBP. This C-terminal region of SCAP contains 4 motifs called
WD40 repeats. The WD40 repeats are required for interaction of SCAP with SREBP. Interestingly, the N-terminus of SCAP, including membrane spans 2-6, resembles HMGR which itself is subject to sterol-stimulated degradation (see above). This shared motif is called the
sterol sensing domain, SSD. Several proteins whose functions involve sterols also contain the SSD. These include patched, an important development regulating receptor whose ligand,
hedgehog, is modified by attachment of cholesterol and the
Neimann Pick C1 (NPC1) protein which is involved in cholesterol transport in the secretory pathway. NPC1 is one of several genes whose activities, when disrupted, lead to severe neurological dysfunction.
The function of SCAP is to positively stimulate S1P-mediated cleavage of SREBP. The function of sterols is to inhibit this positive action of SCAP. The activity of SCAP involves movement from the ER to the Golgi and back. Because the C-terminus of SCAP interacts with SREBP, movement of SCAP takes SREBP along for the ride. When sterols are low, SCAP and SREBP move to the Golgi. This transit is required for SREBP cleavage as S1P is Golgi-localized. When sterols are high, movement of SCAP is halted. Thus, the overall effect of sterols is to regulate the ability of SCAP to present SREBP to S1P.
Diagrammatic representation of the interactions between SREBP and SCAP in the membrane of the ER
CTD = C-terminal domain
Bile Acids Synthesis and Utilization
The end products of cholesterol utilization are the bile acids, synthesized in the liver. Synthesis of bile acids is one of the predominant mechanisms for the excretion of excess cholesterol. However, the excretion of cholesterol in the form of bile acids is insufficient to compensate for an excess dietary intake of cholesterol.
Synthesis of the 2 primary bile acids, cholic acid and chenodeoxycholic acid. The reaction catalyzed by the 7a-hydroxylase is the rate limiting step in bile acid synthesis. Conversion of 7a-hydroxycholesterol to the bile acids requires several steps not shown in detail in this image. Only the relevant co-factors needed for the synthesis steps are shown.
The most abundant bile acids in human bile are
chenodeoxycholic acid (45%) and
cholic acid (31%). These are referred to as the
primary bile acids. Within the intestines the primary bile acids are acted upon by bacteria and converted to the
secondary bile acids, identified as deoxycholate (from cholate) and lithocholate (from chenodeoxycholate). Both primary and secondary bile acids are reabsorbed by the intestines and delivered back to the liver via the portal circulation.
Structure of the conjugated cholic acids
Within the liver the carboxyl group of primary and secondary bile acids is conjugated via an amide bond to either glycine or taurine before their being resecreted into the bile canaliculi. These conjugation reactions yield glycoconjugates and tauroconjugates, respectively. The bile canaliculi join with the bile ductules, which then form the bile ducts. Bile acids are carried from the liver through these ducts to the gallbladder, where they are stored for future use. The ultimate fate of bile acids is secretion into the intestine, where they aid in the emulsification of dietary lipids. In the gut the glycine and taurine residues are removed and the bile acids are either excreted (only a small percentage) or reabsorbed by the gut and returned to the liver. This process of secretion from the liver to the gallbladder, to the intestines and finally reabsorbtion is termed the enterohepatic circulation.
Clinical Significance of Bile Acid Synthesis
Bile acids perform four physiologically significant functions:
· 1. Their synthesis and subsequent excretion in the feces represent the only significant mechanism for the elimination of excess cholesterol.
· 2. Bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the precipitation of cholesterol in the gallbladder.
· 3. They facilitate the digestion of dietary triacylglycerols by acting as emulsifying agents that render fats accessible to pancreatic lipases.
· 4. They facilitate the intestinal absorption of fat-soluble vitamins.