**1.1.2 Proteolytic regulation of HMG-CoA**

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 contains a sterol-sensing domain, SSD. When sterol levels increase in cells there is a concomitant increase in the rate of HMGR degradation. The degradation of HMGR occurs within the proteosome, multiprotein complex dedicated for 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 (Kimura and Tanaka, 2010). 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.

#### **1.2 Disorders of post-sequalene cholesterol biosynthesis**

Seven disorders (Smith-Lemli-Opitz, desmosterolosis, X-linked dominant chondrodysplasis punctata, child syndrome, lathosterolosis and hydrops-ectopic calcification-moth-eaten skeletal dysplasia) of post-squalene cholesterol biosynthesis have been reported, only the most commonly disorder, Smith-Lemli-Opitz syndrome (SLOS) will be discussed in this section of the book as described and reviewed else where (Herman, 2003). SLOS was the first described disorder of post-squalene cholesterol biosynthesis and is by far the most common, with an incidence of approximately 1/40 000 to 1/50 000 in the USA (reviewed in (Kelley and Hennekam, 2000). It was initially described in 1964 as an autosomal recessive major malformation syndrome. In 1993, Irons *et al.(Irons et al., 1993)* detected decreased plasma cholesterol levels and elevated 7DHC in several patients with SLOS, suggesting an enzymatic efficiency of 7-dehydrocholesterol reductase (7DHCR).

#### **1.3 Regulation of cholesterol biosynthesis at transcriptional level**

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 earlier in this book chapter, 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-1 (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/ADD1 (ADD1 is adipocyte differentiation-1) as a consequence of alternative exon usage. SREBP-1a regulates all SREBP-responsive genes in both the cholesterol and fatty acid biosynthetic pathways. SREBP-1c controls the expression of genes involved in fatty acid synthesis and is involved in the differentiation of adipocytes. SREBP-1c is also an essential transcription factor downstream of the actions of insulin at the level of carbohydrate and lipid metabolism. SREBP-2 is the predominant form of this transcription factor in the liver and it exhibits preference at controlling the expression of genes involved in cholesterol homeostasis, including all of the genes encoding the sterol biosynthetic enzymes. In addition SREBP-2 controls expression of the LDL receptor gene.

Regulated expression of the SREBPs is complex in that the effects of sterols are different on the SREBP-1 gene versus the SREBP-2 gene. High sterols activate expression of the SREBP-1 gene but do not exert this effect on the SREBP-2 gene. The sterol-mediated activation of the SREBP-1 gene occurs via the action of the liver X receptors (LXRs). The LXRs are members

covalently attached to proteins targeted for degradation by ubiquitin ligases (Kimura and Tanaka, 2010). 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

Seven disorders (Smith-Lemli-Opitz, desmosterolosis, X-linked dominant chondrodysplasis punctata, child syndrome, lathosterolosis and hydrops-ectopic calcification-moth-eaten skeletal dysplasia) of post-squalene cholesterol biosynthesis have been reported, only the most commonly disorder, Smith-Lemli-Opitz syndrome (SLOS) will be discussed in this section of the book as described and reviewed else where (Herman, 2003). SLOS was the first described disorder of post-squalene cholesterol biosynthesis and is by far the most common, with an incidence of approximately 1/40 000 to 1/50 000 in the USA (reviewed in (Kelley and Hennekam, 2000). It was initially described in 1964 as an autosomal recessive major malformation syndrome. In 1993, Irons *et al.(Irons et al., 1993)* detected decreased plasma cholesterol levels and elevated 7DHC in several patients with SLOS, suggesting an

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 earlier in this book chapter, 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-1 (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/ADD1 (ADD1 is adipocyte differentiation-1) as a consequence of alternative exon usage. SREBP-1a regulates all SREBP-responsive genes in both the cholesterol and fatty acid biosynthetic pathways. SREBP-1c controls the expression of genes involved in fatty acid synthesis and is involved in the differentiation of adipocytes. SREBP-1c is also an essential transcription factor downstream of the actions of insulin at the level of carbohydrate and lipid metabolism. SREBP-2 is the predominant form of this transcription factor in the liver and it exhibits preference at controlling the expression of genes involved in cholesterol homeostasis, including all of the genes encoding the sterol biosynthetic enzymes. In addition SREBP-2 controls expression of the LDL receptor gene.

Regulated expression of the SREBPs is complex in that the effects of sterols are different on the SREBP-1 gene versus the SREBP-2 gene. High sterols activate expression of the SREBP-1 gene but do not exert this effect on the SREBP-2 gene. The sterol-mediated activation of the SREBP-1 gene occurs via the action of the liver X receptors (LXRs). The LXRs are members

cholesterol increase in cells, the rate of HMGR degradation increases.

**1.2 Disorders of post-sequalene cholesterol biosynthesis** 

enzymatic efficiency of 7-dehydrocholesterol reductase (7DHCR).

**1.3 Regulation of cholesterol biosynthesis at transcriptional level** 

of the steroid/ thyroid hormone super family of cytosolic ligand binding receptors that migrate to the nucleus upon ligand binding and regulate gene expression by binding to specific target sequences. There are two forms of the LXRs: LXRα and LXRβ. The LXRs form heterodimers with the retinoid X receptors (RXRs) and as such can regulate gene expression either upon binding oxysterols (e.g. 22*R*-hydroxycholesterol) or 9-*cis*-retinoic acid. All 3 SREBPs are proteolytically activated and the proteolysis is controlled by the level of sterols in the cell. 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. The C-terminal domain (CTD) interacts with a protein called 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. The regulation of SREBP activity is further controlled within the ER by the interaction of SCAP with insulin regulated protein (**Insig**). When cells have sufficient sterol content SREBP and SCAP are retained in the ER via the SCAP-Insig interaction. 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) and as a consequence of this domain SCAP functions as the cholesterol sensor in the protein complex. When cells have sufficient levels of sterols, SCAP will bind cholesterol which promotes the interaction with Insig and the entire complex will be maintained in the ER.

The Insig proteins bind to oxysterols which in turn affects their interactions with SCAP. Insig proteins can cause ER retention of the SREBP/SCAP complex. In addition to their role in regulating sterol-dependent gene regulation, both Insig proteins activate steroldependent degradation of HMGR.

When sterols are scarce, SCAP does not interact with Insig. Under these conditions the SREBP-SCAP complex migrates to the Golgi where SREBP is subjected to proteolysis. The cleavage of SREBP is carried out by 2 distinct enzymes. The regulated cleavage occurs in the lumenal loop between the 2 transmembrane domains. This cleavage is catalyzed by site-1 protease, **S1P**. The function of SCAP is to positively stimulate S1P-mediated cleavage of SREBP. 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. The result of the S2P cleavage is the release of the Nterminal bHLH motif into the cytosol. The bHLH domain then migrates to the nucleus where it will dimerize and form complexes with transcriptional coactivators leading to the activation of genes containing the SRE motif. To control the level of SREBP-mediated transcription, the soluble bHLH domain is itself subject to rapid proteolysis.
