SREBP2 and transcripts LDLR and HMGR

The American biochemists and 1985 Nobel Prize winners, Mike Brown and Joseph Goldstein, showed that cellular cholesterol content is regulated by two parallel mechanisms. When the content of unesterified cholesterol in cells increases the expression of the LDL-receptor (LDLR) protein decreases. In addition, the key enzymes of cholesterol biosynthesis (hydroxymethylglutaryl (HMG)-CoA synthase, HMG-CoA reductase, squalene synthase, farnesyl diphosphate synthase; are repressed. Thus, any further increase in cellular cholesterol is minimised. Alternatively, if intracellular levels of unesterified cholesterol are depleted, these pathways are activated (figure 1.06). The genes encoding for these proteins contain an upstream sequence known as the sterol regulatory element (SRE)-1 (Kim et al. 1995). The transcription factor responsible for binding to SRE-1 DNA sequence TCACNCCAC to activate expression of each gene, is part of a larger protein complex known as sterol regulatory binding protein (SREBP) (Eberlé et al. 2004). SREBP belong to the basic-helix-loop-helix leucine zipper class of membrane bound transcription

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factors which is normally localised in the endoplasmic reticulum (ER) (Sakai 1995). SREBP precursors are maintained in the ER membrane and closely associated with (SREBP)-cleavage activating protein (SCAP) which contains a sterol sensing domain (SSD) to actively monitor sterol levels in the cytosol. SCAP itself interacts with insulin induced gene (Insig) which sequesters the SREBP/SCAP complex in the ER when SCAP senses that sterol levels are high. Sterols directly interact with the SSD of SCAP and modulate SCAP conformation.

In cholesterol-depleted cells, SREBP binds to SCAP which senses the diminished cholesterol levels and disrupts its interaction with Insig allowing SCAP to be sorted into COPII-coated transport vesicles (Sun et al. 2005). SCAP then transports SREBP in the COPII vesicle from the ER to the Golgi apparatus where the N-terminal transcriptionally active domain, nuclear SREBP (nSREBP), is proteolytically cleaved from the SREBP/SCAP complex by site 1 (S1P) and site 2 (S2P) proteases, thus enabling its translocation through the nuclear envelope (Duncan 1997; Espenshade et al. 1999). In the nucleus nSREBP binds with the necessary SRE sequence in the promoter of the target gene (Horton et al. 2002).

Three members of the SREBP family have been described in several mammalian species: SREBP1a and 1c produced from a single gene (SREBP1) located on human chromosome 17p11.2 (Hua et al. 1995) and SREBP2 from a separate gene (SREBP2) located on human chromosome 22q13 (Miserez et al. 1997). SREBP2 is the isoform responsible for regulating cholesterol homeostatic genes transcription HMG-CoA reductase (HMGR) and LDLR, which mediate de novo cholesterol biosynthesis and also uptake of native low density lipoprotein, respectively.

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Figure 1.06 : Regulation of SREBP transcription factor for the up-regulation of cholesterogenic genes. When sterol levels are depleted the SCAP Insig interaction is disrupted enabling SCAP mediated transport of SREBP/SCAP complex into a COPII vesicle (A). The protein complex translocates from the ER to the Golgi apparatus in the COPII vesicle where the transcriptionally active portion of SREBP (nSREBP) is cleaved from the protein complex by S1P and S2P proteases (A). nSREBP then translocates to the nucleus where it binds with the necessary SRE sequence in the promoter of the target gene for transcription (A). When intracellulat cytosolic sterol/cholesterol levels are high SCAP interacts with Insig which sequesters the SREBP/SCAP protein complex deeper within the ER membrane (B)

1.6.2 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)

HMGR is a transmembrane glycoprotein enzyme located on the ER. When activated HMGR catalyses the four-electron reduction of 3-hydroxy-3-methylglutaryl coenzyme A

(HMG-CoA) to mevalonate, which undergoes many subsequent transformations to form

sterol precursors and ultimately cholesterol (Burg & Espenshade 2011). As intracellular sterol levels fluctuate HMGR is tightly regulated at the transcriptional (SREBP/SCAP) and post-translational (phosphorylation and ubiquitination) levels (Burg & Espenshade 2011).

All cells require cholesterol, and lipoproteins normally function to package this insoluble molecule in a form readily transported in the blood. However, as previously discussed, factors such as unhealthy diet and genetic predisposition overload this essential lipid transport pathway and contribute to the dyslipidaemia that promotes atherosclerotic disease. The management of cholesterol begins in the liver where high LDL levels can result from excessive production of triglyceride-rich very low density lipoprotein (VLDL) as well as inadequate uptake of LDL by liver and peripheral cells due to low number of or genetic defects in LDLR. De novo synthesis of cholesterol takes place in the cytosol, where

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three molecules of acetyl-coenzyme A interact to form hydroxylmethylglutaryl-coenzyme A (HMG-CoA). HMG-CoA then reacts with HMG-CoA reductase (HMGR), an enzyme that resides in the membrane of the smooth ER (Burg & Espenshade 2011). HMGR is a tetrameric macromolecule with binding pockets for HMG-CoA deep within each monomer, with neighbouring monomers contributing additional binding interactions. In total, the tetramer has four pockets, one within each monomer. HMGR uses one molecule of NADPH to catalyses the reduction of HMG-CoA to mevalonate (Burg & Espenshade 2011). After leaving the enzyme, mevalonate undergoes many subsequent transformations to form other sterol precursors in the pathway to cholesterol. Statins competitively bind to the active site of HMGR blocking the binding site for HMG-CoA to inhibit its reduction to mevalonate and therefore the pathway to cholesterol.

1.5.2.3.1 HMGR in atherosclerosis

Lee et al. (2011), demonstrated significant localised expression of HMGR in macrophage- rich areas of unstable plaque compared to plaques from patients with stable angina when using anti-HMGR and anti-CD31. These findings suggest that local HMGR is functionally active within the atherosclerotic plaque tissue, and lesion macrophages in praque from patients with unstable angina more actively produce HMGR than in patients with stable angina. Tuomisto et al. (2003) specifically isolated macrophage-rich shoulder regions of AP tissue using laser microdisection and reported a high up-regulation of HMGR compared to disease-free tissue of the same artery. It was proposed the relatively high expression of HMGR may in fact be due to proliferation and/or differentiation of macrophages because similar expression was observed with THP-1 cells when cultured with phorbol 12-myristate 13-acetate (PMA); a chemical used to differentiate THP-1 monocytes to macrophages in vitro. Conversely when THP-1 cells were incubated with ox-LDL, which closer resembles atherogenic conditions, down-regulation of HMGR was observed, which suggests that lipid-loaded THP-1 macrophages may not be an accurate model of macrophages present in shoulder. Thus, HMGR may be expressed as a requirement of macrophage proliferation and/or differentiation rather than dysregulated by inflammatory/lipid stimuli.

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