Biosynthesis of Membrane Lipids Notes
Outline HERE

Glycerophospholipids (Phosphoglycerides)

The most abundant phospholipids are made from glycerol. Phospholipids are important components of membranes, and help transport triacylglycerols and cholesterol in the body. Typically, they have saturated fatty acids at carbon #1 (more than 90%) and unsaturated fatty acids at carbon #2 (more than 90%).

There are a few differences in the lipids (and metabolic routes to making them) of eukaryotes and prokaryotes. We will not be concerned about distinguishing the routes of synthesis between these two organisms, but it is important to know that there are differences between the two.

Pathways of Glycerophospholipids

Phosphatidic acid (phosphatidate) is an important intermediate in the synthesis of both glycerophospholipids and fat. Phosphatidate is a branch point between the synthesis of triacyglycerols (fats/oils) and the synthesis of phosphoglycerides. Note that this simple pathway involves the hydrolysis of phosphate from position #3 of the glycerol followed by addition of an acyl group from acyl-CoA.

To synthesize glycerophospholipids from phosphatidate is only slightly more complicated. The first step involves activation of the phosphatidate by CTP to form CDP-diacylglycerol. This activated form of phosphatidate is equivalent to the activation of glucose you saw last term by UTP to form UDP-glucose. In both cases, the nucleotide activation form has energy necessary to drive subsequent reactions forward.

Phosphatidyl Derivatives

Note that CDP-diacylglycerol will react with serine, releasing a CMP and producing phosphatidyl-serine. A related reaction in which serine is replaced by inositol yields phosphatidyl inositol. You may recall from last term that this molecule, when phosphorylated at positions 4 and 5 on inositol acts as a second messenger to activate phospholipase C. One other interesting note about phosphatidyl inositol - the fatty acids at positions 1 and 2 on the glycerol are almost always fixed - stearic acid at position #1 and arachidonic acid at position #2.

Phosphatidylethanolamine is derived from phosphatidylserine by dephosphorylation. Phosphatidylcholine is derived from phosphatidylethanolamine by a series of three methylations. The methyl groups in these reactions are donated by a common methyl-donating molecule known as S-Adenosylmethionine or SAM. After losing its methyl group, SAM is converted to S-Adenosylhomocysteine (SAH).

Synthesis of phosphatidylethanolamine can also occur in two other ways in mammals. One simple way is by swap of the serine on phosphatidylserine for free ethanolamine. Here, free ethanolamine is converted to phosphorylethanolamine, followed by activation to form CDP-ethanolamine and transfer of the ethanolamine to diacylglycerol to form phosphatidylethanolamine. Note that CMP is a byproduct, just as with synthesis through the phosphatidate pathway above.

Metabolism of Sphingolipids

Spingolipids are another class of membrane lipids found in the membranes of all eukaryotic cells, but is found preferentially in of the central nervous system. As seen in the top figure on page 720, synthesis starts with two common cellular molecules - palmitoyl-CoA and the amino acid serine. Reduction of the dehydrosphingosine followed by desaturation of dihydrosphingosine yields sphingosine, the base compound of the sphingolipids.

Addition of an acyl group from acyl-CoA to the amide creates a ceramide. The ceramide is a branch between synthesis of sphingomyelin (important component of the myelin sheath of nerve cells) and cerebrosides (brain membrane sphingolipids. Conversion of a ceramide to sphingomyelin involves addition of a phosphocholine from phosphatidyl choline (forming diacyglycerol in the process). To convert a ceramide to a cerebroside requires addition of a glucose from UDP-glucose. Gangliosides are made from cerebrosides by addition of complicated, activated sugards. Gangliosides are complex sphingolipids containing at least one acidic sugar - either N-acetylneuraminate or N-glycolylneuraminate. These acids sugars are commonly called sialic acids. Sugars are added to cerebrosides in an activated form (UDP-glucose, UDP-galactose, etc.). One unusual activated form that is used is that of CMP-N-acetylneuraminate.

Sphingolipids are sources of second messengers. Ceramide can initiate programmed cell death in some cell types.

Health Considerations of Membrane Lipids

Besides the problems associated with PAF above, other membrane lipids related to health problems include the phosphoglyceride dipalmitoyl phosphatidylcholine (respiratory distress syndrome) and ganglioside GM2 (Tay-Sachs disease). In respiratory stress syndrome, the immature lungs of premature infants do not synthesize enough dipalmitoyl phosphatidylcholine. This molecule is important (along with some proteins) in the extracellular fluid of the alveoli of the lung where they act to prevent lung collapse upon exhalation. Degradation of sphingolipids occurs in the lysosomes of cells. Deficiency of the enzymes that degrade sphingolipids lead to a variety of inherited diseases called sphingolipidoses. In Tay-Sachs disease, the enzyme N-acetylhexosaminidase is deficient. In the absence of this enzyme, the ganglioside GM2 accumulates, primarily in the brain. The disease causes severe problems in the nervous system, and usually is fatal by age four. The disease can be diagnosed during fetal development by assaying for the relevant enzyme in amniocentesis

There are many other ganglioside-metabolism-related diseases not covered by your book. Other important involvement of gangliosides include 1) cholera toxin attachment to a specific ganglioside on the cell surface and 2) influenza virus attachment to a ganglioside.

Cholesterol

Cholesterol is an important biological compound. It helps modulate membrane fluidity and is the precursor of other important biological compounds, such as the steroid hormones and the bile acids. Complete synthesis of cholesterol can be brought about starting only with acetyl-CoA. The synthetic mechanism can be thought of in three stages:

Many compounds can be made from five carbon precursors used to make cholesterol. These compounds (including cholesterol) are known as isoprenoids because the five carbon intermediates (isopentenyl pyrophosphate and dimethylallyl pyrophosphate) are collectively called isoprenes.

Isoprenoids are made by putting together one or more five-carbon isoprene units. Categories of isoprenoids include

Synthesis of Mevalonate and Isopentenyl Pyrophosphate

Mevalonate is a six carbon intermediate that is a precursor of the five carbon molecule known as isopentenyl pyrophosphate. Synthesis of mevalonate from acetoacetyl-CoA (a ketone body - discussed later in the term) and acetyl-CoA is shown HERE. Note that the precursor of mevalonate is a six carbon intermediate linked to CoA, called HMG-CoA. The enzyme that converts HMG-CoA to mevalonate is known as HMG-CoA reductase. This enzyme is allosterically inhibited by cholesterol (feedback inhibition) and is also the target of a drug called lovastatin for reducing cholesterol levels in the body.

Conversion of mevalonate to isopentenyl pyrophosphate requires 3 ATPs and results in a decarboxylation and addition of two phosphates in pyrophosphate form. The five carbon molecule which results, isopentenyl pyrophosphate, is one of the five carbon isoprene units used to assemble the isoprenoids. The other molecule, dimethyallylpyrophosphate, can be produced by isomerization of isopentenyl pyrophosphate.

Synthesis of Higher Isoprenoids

Addition of five carbon units to make bigger structures occurs by the mechanism of a condensation reaction. Addition of dimethylallyl pyrophosphate to isopentenyl pyrophosphate by the condensation reaction yields a 10 carbon isoprenoid, known as geranyl pyrophosphate. Note that a pyrophosphate is lost in the reaction. Addition of another unit of isopentenyl pyrophosphate yields the 15 carbon isoprenoid, farnesyl pyrophosphate. Joining of two farnesyl pyrophosphates (tail to tail) yields the thirty carbon isoprenoid squalene. Note that two pyrophosphate are split in this reaction and that NADPH is required, meaning a reduction is occurring.

Squalene is the last of the "linear" precursors of cholesterol. Synthesis of other isoprenoids that are NOT derived from cholesterol can proceed through modification of squalene. Some of these include vitamins A, E, and K. Cyclization of squalene occurs by a complicated involving formation of an epoxide. The ultimate product of this reaction is the "first" cyclized product, known as lanosterol. Conversion of lanosterol to cholesterol involves release of formic acid, rearrangement/reduction of double bonds, and two decarboxylations. The process requires 19 enzymatic reactions.

Regulation of Cholesterol Biosynthesis

As you are probably aware, the levels of cholesterol in the body are of concern due to the association between high levels of it in the blood and heart disease.

The cholesterol supply in the body is controlled by:

HMG-CoA reductase is bound in the membrane of the endoplasmic reticulum. The many controls on the amount of the enzyme are interesting. HMG-CoA reductase is a prime example of an enzyme exhibiting feedback inhibition. The process refers to inhibition brought about by the end product of a pathway (cholesterol) on an enzyme acting early in the same pathway (HMG-CoA reductase). Since the many steps going from HMG-CoA to cholesterol require much energy (ATP, NADPH, protein synthesis), stopping the process early saves a tremendous amount of energy.

Phosphorylation of the enzyme occurs by an AMP-activated (Note this is NOT cAMP-activated) protein kinase. Remember that AMP rises when ATP decreases, so cholesterol biosynthesis is turned OFF when ATP is low.

Transcription of HMG-CoA reductase is regulated by a transcription factor called the sterol regulatory binding protein (SREBP). This protein, when active, binds near the HMG-CoA reductase gene in the DNA. When SREBP is inactive, it is anchored to the endoplasmic reticulum or nuclear membrane. Lowered cholesterol levels causes the SREBP to be released and it travels to the HMG-CoA-reductase gene, binds it (as well as other cholesterol metabolism genes), and activates transcription. When cholesterol levels rise, free SREBP is degraded.

Translation of HMG-CoA reductase is inhibited by mevalonate derivatives and also by dietary cholesterol.

Degradation of the HMG-CoA reductase is modulated through the portion of it located in the membrane. As cholesterol (and other sterols) increase in concentration, the enzyme forms oligomers that are susceptible to degradation.

Movement of Cholesterol and Other Lipids in the Body

Triacylglycerols (fats, triglycerides) constitute 90% of dietary lipid, and are the major form of energy storage in humans. Oxidative metabolism of fats yields more than twice the energy as an equal weight of dry carbohydrate or protein. Remember that fats and other lipids (including cholesterol) are insoluble in water. Movement of these materials in the body occurs via the bloodstream in lipoprotein complexes described below.

Lipoprotein complexes carry lipids in the bloodstream. Very little free lipid can be detected in the blood. The protein components of the lipoproteins are synthesized in the liver and intestinal mucosa cells. Lipoproteins form micelles with lipids as a mechanism of transporting them in the aqueous environment of the blood.

Triglycerides arising from the digestive system are packaged into lipoproteins in the bloodstream called chylomicrons. They travel into the blood stream via the lymph system. Triacylglycerols are also synthesized by the liver where they are packaged as very low density lipoproteins (VLDLs) and released into the blood. Upon arrival in adipose tissue and muscle cells, lipoprotein lipase cleaves fats in them to free fatty acids and glycerol. In adipose tissue, hydrolysis of fats to fatty acids and glycerol is accomplished by hormone-sensitive triacylglycerol lipase. Free fatty acids are released there into the blood stream where they bind to albumin.

The six categories of lipoproteins are summarized below:

Liver synthesis of cholesterol in the body is important, because tissues outside of the liver and intestine do not synthesize it themselves and rely on delivery of it in the plasma via the LDLs. As noted above, uptake of LDLs by target cells occurs by recognition of apoprotein B-100 in the LDL. Steps in the process include:

1. Binding of B-100 in the LDL in coated pits of the target cell. Clathrin is a protein abundant in the coated pits.

2. Endocytosis of the LDL and formation of an endocytic vesicle.

3. Fusion of the endocytic vesicle with the cellular lysosomes. LDL proteins here are hydrolyzed to free amino acids. Cholesterol esters are hydrolyzed to free cholesterol. The LDL receptor components return to the cell surface.

4. The released cholesterol can be used for membrane biosynthesis or reesterified and stored in the cell. Esterification is a critical way of controlling the level of cholesterol in cells. Too much free cholesterol disrupts membranes. Esterification in the cell joins the cholesterol to an oleic acid or palmitoleic acid (monounsaturated fatty acids), in contrast to the LDL cholesterol esters, which are to linoleic acid (polyunsaturated). Note that when cholesterol is abundant in cells, synthesis of LDL receptors will be inhibited, preventing the uptake of more cholesterol. The LDL receptor gene is ALSO regulated by SREBP (see above).

LDL Receptor

The LDL receptor gene contains 18 exons with widely different functions (HERE) and is a good example of exon shuffling (discussed later in the term). The receptor is a transmembrane protein with six different types of domain. The LDL receptor is implicated in a serious genetic disease of cholesterol metabolism, known as familial hypercholesterolemia. In this disease, LDLs and IDLs are unable to reenter the liver cell and accumulate to very high levels in the blood. Since theses lipoprotein complexes carry cholesterol, cholesterol levels go up astronomically (3-5 times higher than normal), resulting in nodules of cholesterol forming in skin and tendons. Worse, high levels of LDL result in oxidation of the LDL, forming oxidized LDL, which is a target for macrophage cells of the immune system. These complexes that form are known as foam cells. Foam cells become trapped in the walls of blood vessels and contribute to the formation of atherosclerotic plaques, causing arterial narrowing and heart attacks.

Reducing Cholesterol

Reduction of cholesterol levels is attempted by 1) inhibition of the reabsorption of bile acids in the intestinal system (produced from cholesterol - non absorption requires more cholesterol being converted to bile acids) and 2) inhibition of new cholesterol synthesis by a class of compound known as statins. Statins (for example, lovastatin) are inhibitors of HMG-CoA reductase.

Bile Acids

Bile acids, as noted above, are synthesized from cholesterol. Bile acids resemble cholesterol, but have more polar constituents. Like the fatty acids, which have polar and non-polar portions that allow them to act like detergents, the bile acids also act like detergents. They are stored in the bile in the gall bladder and act to solubilize dietary lipids during digestion. Glycocholate is a major bile salt. Bile salts provide one way to break down cholesterol in the body and, as noted in the previous paragraph, blocking their normal reabsorption in the intestine requires the body to break more cholesterol down for their synthesis.

Steroid Hormones

Another group of molecules synthesized from cholesterol is the steroid hormones. There are five major classes of these compounds - progestagen, glucocorticoids, mineralocorticoids, androgens, and estrogens. Progesterone (a progestagen) is essential for maintenance of pregnancy. Androgens are responsible for development of male secondary sex characteristics. Estrogens are required for female secondary sex characteristics and also participate (along with progesterone) in the ovarian cycle. Glucocorticoids enhance the degradation of fat and proteins and also inhibit the inflammatory response. Mineralocorticoids act on the kidney to increase reabsorption of Na+ and excretion of K+ and H+, leading to increases in blood volume and pressure. Steroid hormones are interesting in being able to cross cellular membranes and directly activate transcription of classes of genes.

Hydroxylation and P450

Hydroxylation reactions are important in the synthesis of cholesterol from squalene, of steroid hormones from cholesterol and also in detoxification of toxic compounds by the liver. All of these hydroxylation reactions are the product of a group of enzymes known as monooxygenases (also called mixed function oxygenases). These enzymes use one atom of molecular oxygen (O2) to perform the hydroxylation. They also use the molecule NADPH in the mechanism. These reactions require oxygen to be activated and the molecules that perform the activation are known as cytochrome P450. Cytochrome P450 is actually a family of proteins containing a heme prosthetic group. The reaction mechanism involves adrenodoxin, a nonheme iron protein, as well. Adrenodoxin transfers one electron to reduce the Fe+3 form of P450 to the Fe+2 form, followed by oxygen binding, addition of a second electron by adrenodoxin, splitting of the oxygen - one going to form water and the other abstracting a hydrogen from the substrate to form a hydroxyl on it.

Hydroxylation serves to solubilize substances and permits them to be passed into the urine. It is for this reason that the urine of athletes is tested for many compounds, including steroids, because this is where they will usually be passed. Caffeine also can be eliminated from the body by this mechanism, as can many drugs. In addition, hyddroxylation by the P450 system can actually generate carcinogens from harmless compounds. The P450 system, is obviously a mixed bag.

Steroid Hormone Synthesis

Cholesterol is a precursor of the steroid hormones. An intermediate in this process is pregnenolone, which differs from cholesterol by 6 carbons. The six carbons are removed in an interesting reaction stimulated by the hormone ACTH. In the synthesis of pregnenolone, three molecules of oxygen and three molecules of NADPH are used. Pregnenolone can then serve as the steroid hormone precursor. Note that the female hormone estradiol is synthesized from the male hormone testosterone (HERE). Estradiol is an estrogen hormone and has an aromatic ring, wherease testosterone does not. The enzyme responsible for this conversion is known as an aromatase. Some tumors bind estrogens, so one way to prevent these tumors from growing is to treat the person with an aromatase inhibitor. Testosterone is, of course, known to increase muscle mass (in males and females). It is referred to as an anabolic steroid.

Vitamin D Synthesis

Vitamin D is derived from a cholesterol derivative, 7-dehydrocholesterol, in a reaction that is initiated by ultraviolet light and which splits one of the rings. The active form, calcitrol, is formed from Vitamin D3 by hydroxylation reactions in the liver and kidney. Vitamin D deficiency causes a syndrome known as rickets, which is characterized by inadequate calcification of cartilage and bone. Vitamin D deficiency in children was signficant in 17th century England. Today, many foods, including milk are fortified with vitamin D.