Genetic or nutritional disorders in homocysteine or folate metabolism increase proteinN‐homocysteinylation in mice

Abstract

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine beta-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse.

Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

“…Nevertheless, some conditions such as a diet particularly rich in methionine; mutations or inactivation of cystathionine β-synthase, methionine synthase, and 5,10-methylene tetrahydrofolate reductase; and nutritional deficiencies of vitamins (folic acid or vitamin B12) induce a dramatic increase of cellular homocysteine concentration, with the resulting increase in the rate of HTL synthesis 17 and increased accumulation of N-Hcyprotein in humans and mice. 18,19 Although different hypotheses have been proposed to describe the pathological consequences of hyperhomocysteinemia in humans, so far the cause of homocysteine toxicity has not been understood. It has been suggested that increased HTL synthesis and NHcy-protein accumulation is an important risk factor for cardiovascular and neurodegenerative diseases.…”

“…It has been reported that the N-homocysteinylation of four lysine residues of cytochrome c causes structural changes in the protein affecting the redox state of the heme group. 25,26 In addition, N-homocysteinylation causes protein aggregation, [13][14][15][16][17][18][19][20][21] which has been documented for several proteins, including human serum albumin (HSA). 13,27 However, the mechanism of N-Hcy-protein aggregation has not been studied.…”

Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice

“…HHcy leads to the accumulation of Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein also in mouse models [17,18]. For example, accumulation of S-Hcy-annexin A2 inhibits endothelial cell-dependent fibrinolysis in HHcy mice [19].…”

Modulation of paraoxonase 1 and protein N-homocysteinylation by leptin and the synthetic liver X receptor agonist T0901317 in the rat

“…N-Hcy-proteins induce an auto-immune response (Jakubowski 2005) and accumulate in atherosclerotic lesions (Perla-Kajan et al 2008). The levels of free Hcy-thiolactone and N-Hcy-proteins are increased in hyperhomocysteinemia secondary to genetic or nutritional deficiencies (Jakubowski et al , 2009 or renal insufficiency (Perna et al 2006), as well as in patients with ischemic heart disease (Yang et al 2006). The level of Hcy-thiolactone and N-Hcy-protein is also affected by PON1, which hydrolyzes thiolactone to free Hcy.…”