the key behind human vascular disease?
In an article published in The Federation of American Societies for Experimental Biology (FASEB), Dr. Hieronim Jakubowski offers an interesting scenario involving Homocysteine (Hcy) in possible mechanisms for human vascular disease.
He concludes his article by stating that:
The data presented in this study establish that:
1) proteins can be easily homocysteinylated with Homocysteine thiolactone under physiological conditions;
2) side chain amino groups of lysine residues are the major sites of homocysteinylation in proteins;
3) homocysteinylation leads to protein damage.
These data support a hypothesis that
metabolic conversion of Hcy to the thiolactone, homocysteinylation of proteins,
and the resulting protein damage underlie involvement of Hcy in the pathology of
human vascular disease, as outlined below.
Formation of Homocysteine Thiolactone
Because Hcy forms as a byproduct of methylation reactions in human cells, metabolic conversion of Hcy to the thiolactone is inadvertent and occurs to a lesser or greater extent depending on specific genetic and metabolic conditions. The extent of thiolactone synthesis depends on relative levels of Hcy and Methionine (Met), the levels of expression and activity of MetRS, methionine synthase, and cystathionine ▀-synthase. Because the activity of major Hcy-metabolizing enzymes requires cofactors such as vitamin B12 and folate (methionine synthase) as well as vitamin B6 (cystathionine ▀-synthase), the extent of thiolactone synthesis also depends on the levels of these vitamins. Mutations in genes encoding methionine synthase and cystathionine ▀-synthase, lead to enhanced metabolic conversion of Hcy to the thiolactone. In the extreme cases when the function of Hcy-metabolizing enzymes is abolished, all Hcy is converted to the thiolactone. These effects on Hcy thiolactone synthesis occur in all cell types investigated, from bacterial to human.
Regarding human cells, cystathionine ▀-synthase-deficient fibroblasts produce more Hcy thiolactone than unaffected cells. Inhibition of methionine synthase by an antifolate drug, aminopterin, leads to enhanced synthesis of Hcy thiolactone in normal human fibroblasts as well. Human umbilical vein endothelial cells limited for vitamin B12 and folate synthesize micromolar concentrations of Hcy thiolactone (H. Jakubowski, unpublished data). Concentrations of the thiolactone in tissue cultures are about one-tenth the concentration of Hcy.
Hcy thiolactone easily reacts with proteins under physiological conditions. In tissue cultures of human fibroblasts cellular proteins become homocysteinylated. Recent experiments with cultured human endothelial cells show that both cellular and extracellular proteins become extensively homocysteinylated under conditions of vitamin B12 and folate limitation. The concentration of homocysteinylated proteins can reach up to 40 ÁM within 48 h under these conditions (H. Jakubowski, unpublished data). In in vitro experiments with human serum spiked with [35S]Hcy thiolactone, the thiolactone disappeared with a half-life of ~1h, and each serum protein became homocysteinylated. Protein homocysteinylation in human serum occurs at as low as 10 nM thiolactone and increases directly in proportion to the increase in the thiolactone concentration, up to millimolar range. Thus, regardless of how small or large quantities of Hcy thiolactone are made, the thiolactone modifies proteins. If conditions favoring synthesis of Hcy thiolactone, such as elevated levels of Hcy, are maintained, there will be a concomitant increase in the degree of protein homocysteinylation.
Homocysteinylation of Lysine
Chemical reactivities of homocysteinylated proteins as well as kinetics of protein homocysteinylation reported here suggest that side chain amino groups of lysine residues are the major sites of homocysteinylation in most proteins. For example, Hcy thiolactone reacts with most proteins at rates similar to the rate of the reaction with an equivalent concentration of lysine or -N-acetyl-lysine. This explains why protein homocysteinylation correlates better with protein lysine content than with protein size. Homocysteinylated proteins do not release Hcy on treatments with agents that hydrolyze ester bonds. However, Hcy is released from homocysteinylated proteins by acid hydrolysis or Edman degradation. The possibility that side chains of histidine and arginine could also be modified is unlikely because free arginine or -N-acetyl-histidine do not appreciably react with Hcy thiolactone .
Results of Homocysteinylation
Homocysteinylation can lead to protein damage. As demonstrated here, homocysteinylation of 33% of lysine residues in MetRS and 88% lysine residues in trypsin resulted in complete loss of their enzymatic activities. Homocysteinylated proteins were prone to multimerization and underwent gross structural changes that led to their denaturation. Homocysteine thiolactone may also inactivate enzymes by other mechanisms. For example, lysine oxidase, an important enzyme responsible for posttranslational collagen modification essential for the biogenesis of connective tissue matrices, is inactivated by Hcy thiolactone, which derivatizes the active site tyrosinequinone cofactor.
In addition to a loss of function, protein homocysteinylation can also generate modified proteins that are physiologically detrimental in other ways. For example, homocysteinylated LDL has been recently shown to elicit immune response in rabbits. Rabbit antiserum against an Hcy-LDL adduct was also shown to react with adducts of Hcy and other proteins, such as bovine serum albumin, hemoglobin, and serum proteins. This antigen specificity suggests that the rabbit antiserum reacts with Hcy-Lys-epitopes.
How can protein damage lead to cell injury, a hallmark of atherosclerosis? One plausible scenario is that homocysteinylated proteins on the surface of vascular vessels will be recognized by macrophages either directly or indirectly. Macrophages will attempt to phagocytize damaged proteins on the surface of endothelial cells, which would lead to destruction of endothelial cells and damage to vascular wall. Alternatively, homocysteinylated endothelial cells will attract anti-Hcy-protein antibodies and form antigen-antibody complexes on the surface of vascular vessel. Endothelial cells coated with antibody will be recognized and then bound by macrophages through their Fc receptors. After binding, the endothelial cells will be ingested and destroyed, which will result in injury to the vascular surface. If the agent responsible for the injury (homocysteinylated proteins) is present continuously, attempts to repair the damaged vascular wall will eventually lead to an atherosclerotic plaque.
Protein homocysteinylation is a novel example of protein damage that may explain the involvement of Hcy in the pathology of human vascular diseases. However, other protein modifications by drugs or cellular metabolites have been implicated in other human diseases. For example, acetylation of proteins by aspirin is thought to be an underlying cause of aspirin intolerance. Peniciloylation of proteins by the antibiotic penicillin is involved in penicillin allergy. Modification of proteins by glucose is believed to underlie the pathogenesis of diabetes and Alzheimerĺs disease. Proteins modified by products of lipid oxidation are implicated in the etiology of atherosclerosis. A common aspect of these modification reactions is the involvement of protein lysine residues as sites of modifications.
Another interesting finding discovered by Jakubowski is the fact that the only enzyme able to inactivate homocysteine thiolactone (Hcy-thiolactonase) is identical to the enzyme paraoxonase. In other words, they are one and the same. The significance of this fact is that it has been proposed that HDL is the "good" cholesterol because it transports cholesterol to the liver thereby theoretically lowering cholesterol levels and reducing risk of heart attacks. However, since it has been shown that paraoxonase is found exclusively on the HDL molecule, this may better explain the association between higher HDL levels and reduced risk of MI (and conversely lower HDL levels would increase the risk). If thiolactone induced injury to vascular vessels is the significant initial step in atherosclerotic plaque formation, than HDL levels would be of primary importance in your ability to arrest this process. Another important point is that smoking has been shown to lower paraoxonase levels and therefore theoretically this would lead to higher Hcy thiolactone levels.
Bad Cholesterol and other Myths
This also raises the whole question of whether cholesterol itself can be categorized as either "good" or "bad". The notion that what you eat is going to determine if your going to have a heart attack (the Diet-Heart theory) may have to be re-examined. If Homocysteine Thiolactone does indeed cause vascular inflammation via protein damage as proposed above by Dr. Jakubowski, high serum cholesterol levels and subsequent plaque formation may be an anti-inflammatory response to this condition. Blaming high cholesterol for heart attacks could then be compared to accusing firemen for starting all the fires because wherever there is a fire you always find firemen. This is where there is still much confusion about high cholesterol levels. Cholesterol has never been shown to be the cause of heart attacks, but a risk factor. In other words some studies have shown an association. A good website that looks at this topic in depth is The Cholesterol Myths by Dr. Uffe Ravnskov.
Another idea whose time may have passed into the realm of mythology involves our salt intake.