**1.1.1 Regulation of cholesterol biosynthetic pathway**

Regulation of the pathway includes sterol-mediated feedback of transcription of several of the genes, including the rate-limiting enzyme HMGR and HMG-CoA synthase, as well as a variety of post-transcriptional mechanisms. Recently, increased attention has been focused on the regulated degradation of HMGR in the ER. The cellular supply of cholesterol is maintained at a steady level by four distinct mechanisms:


The first seven enzymes of cholesterol biosynthesis are soluble proteins with the exception of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which is an integral endoplasmic reticulum (ER) membrane protein (Gaylor, 2002; Goldstein and Brown, 1990; Kovacs et al., 2002). HMG-CoA for cholesterol biosynthesis is generated within the cytosol. It is also synthesized within the mitochondria where its hydrolysis by HMG-CoA lyase generates ketones for energy during fasting (Herman, 2003). Reactions generating mevalonate can also occur within the peroxisome, and the subsequent reactions that result in the production of farnesyl-pyrophosphate are exclusively peroxisomal. The remainder of the biosynthetic reactions occurs in the ER with enzymes and substrates that are membrane-bound. Thus, cholesterol biosynthetic enzymes are compartmentalized in the cytosol, ER and/or peroxisome, adding another level of complexity to the regulation of this metabolic pathway. Particularly, direct regulation of HMGCoA reductase activity means for controlling the level of cholesterol biosynthesis. Recently, increased attention has been focused on the regulated degradation of HMGR in the ER.

Fig. 3. Regulation of HMGCoA reductase

*In vivo* system, the activity of HMGCoA reductase is controlled by four distinct mechanisms: feed-back inhibition, control of gene expression, rate of enzyme degradation and

3. Regulation of excess intracellular free cholesterol through the activity of acyl-CoA:

4. Regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-

The first seven enzymes of cholesterol biosynthesis are soluble proteins with the exception of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which is an integral endoplasmic reticulum (ER) membrane protein (Gaylor, 2002; Goldstein and Brown, 1990; Kovacs et al., 2002). HMG-CoA for cholesterol biosynthesis is generated within the cytosol. It is also synthesized within the mitochondria where its hydrolysis by HMG-CoA lyase generates ketones for energy during fasting (Herman, 2003). Reactions generating mevalonate can also occur within the peroxisome, and the subsequent reactions that result in the production of farnesyl-pyrophosphate are exclusively peroxisomal. The remainder of the biosynthetic reactions occurs in the ER with enzymes and substrates that are membrane-bound. Thus, cholesterol biosynthetic enzymes are compartmentalized in the cytosol, ER and/or peroxisome, adding another level of complexity to the regulation of this metabolic pathway. Particularly, direct regulation of HMGCoA reductase activity means for controlling the level of cholesterol biosynthesis. Recently, increased attention has been focused on the regulated

**HMG-CoA reductase phosphatase**

**PPI-1(a)**

**-ve**

**P**

**PPI-1 (a)**

*In vivo* system, the activity of HMGCoA reductase is controlled by four distinct mechanisms: feed-back inhibition, control of gene expression, rate of enzyme degradation and

**OH**

**OH P**

**P**

**OH**

**Inactive AMP-activated Kinase**

**Active AMP-activated Kinase**

**Pi**

**-ve**

**Protein phosphatase**

**Pi**

**ADP**

**HMG-CoA reductase Active** 

**Protein phosphatase**

**-ve**

cholesterol acyltransferase, ACAT (internalization of excessive cholesterol).

mediated reverse transport.

degradation of HMGR in the ER.

**ATP**

**LKB1+AMP CaMKK +Ca+2**

**ADP**

**Cholesterol**

**HMG-CoA** 

**-ve**

**HMG-CoA reductase Active** 

**Pi**

**cAMP**

Fig. 3. Regulation of HMGCoA reductase

**ADP**

**ATP**

**PKA**

**ATP**

phosphorylation-dephosphorylation. Cholesterol controls the first three mechanisms itself as cholesterol acts as a feed-back inhibitor of pre-existing HMGR as well as inducing rapid degradation of the enzyme. The latter is the result of cholesterol-induced polyubiquitination of HMGR and its degradation in the proteosome (explained in separate section in details). This ability of cholesterol is a consequence of the **sterol sensing domain, SSD** of HMGR. In addition, when cholesterol is in excess the amount of mRNA for HMGR is reduced as a result of decreased expression of the gene. By covalent modification of HMGreductase through the process phosphorylation/ dephosphorylation plays a very important role in regulation of HMGR. The enzyme is most active in its unmodified form. Phosphorylation of the enzyme decreases its activity (Figure 3). HMGR is phosphorylated by adenosine mono phosphate-activated protein kinase, **(AMPK)which is** itself activated via phosphorylation catalyzed by 2 enzymes. LKB1 is the primary kinase sensitive to rising AMP levels which was first identified as a gene in humans carrying an autosomal dominant mutation in Peutz-Jeghers syndrome, PJS and mutated in lung adenocarcinomas. The second AMPK phosphorylating enzyme is calmodulin-dependent protein kinase kinase-beta (CaMKKβ) which induces phosphorylation of AMPK due to increase in the level of intracellular Ca2+ as a result of muscle contraction. The activity of HMGR is additionally controlled by the cAMP signaling pathway (Figure 3). Enhanced level of cAMP lead to activation of cAMPdependent 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 (PP2C) 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. The intracellular level of cAMP itself is regulated by hormonal stimuli, thus regulation of cholesterol biosynthesis is hormonally controlled. One of the most common hormone, insulin leads to a decrease in cAMP, which in turn activates cholesterol biosynthesis. Alternatively, glucagon and epinephrine increase the level of cAMP in turn lead to inhibition of cholesterol biosynthesis. The basic function of epinephrine and glucagon hormones is to control the availability and delivery of energy in all the cells in our body. Degradation of HMGR and inhibition of its biosynthesis, are the two long term regulatory processes for cholesterol biosynthesis as when the levels of cholesterol are high, resulted in reduction in the expression of the HMGR gene. On other hand, low levels of cholesterol activate its expression.
