N-Homocysteinylation Induces Different Structural and Functional Consequences on Acidic and Basic Proteins

Abstract

One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation). It has been believed that protein N-homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivity of HTL towards proteins with different physico-chemical properties and hence their structural and functional alterations were studied using different spectroscopic approaches. We found that N-homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N-homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration.

Modification of erythropoietin structure by N‐homocysteinylation affects its antiapoptotic and proliferative functions

“…The electropherograms showed a lower electrophoretic mobility for the modified proteins with an impaired biological function. In line with these results, many acidic proteins have been reported to form oligomers and aggregates upon modification by HTL [10,13,15,17], which may account for their lack of function. An explanation for the lower positive net charge observed in N-homocysteinylated proteins was given by Jakubowski [10], who reported that the e-amino group of lysine (pK = 10.5) is much more basic than the a-amino group of Hcy bound to lysine (eN(Hcy) lysine, pK = 7.1).…”

“…Besides, it has been reported that Nhomocysteinylation may cause protein aggregation, as documented for several proteins [10,[13][14][15][16][17][18]. Several kinds of insults such as oxidative stress, ionizing radiation or drug exposure, as well as the pathophysiological accumulation of reactive metabolites in the body, can induce nonenzymatic post-translational modifications in proteins, thus causing the alteration of their functional properties and denaturation.…”

Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity

“…Our present data show that in insulin-producing cells HC toxicity requires higher concentrations than in other cell types, like endothelial cells [7] or neurons [48,52] as well as prolonged exposure. One of possible explanation for this difference could be the lack of a strong endogenous NO synthase expression in insulinproducing cells [53].…”

“…Other possible mechanisms might involve the inactivation of other components of the glycolytic flux or proteins, which affect the insulin secretory machinery by so-called N-homocysteinylation. The methionylated metabolite of HC, HC thiolactone, can modify proteins resulting in N-homocysteine-linked protein formation and can therefore influence protein structure and function, leading to enzyme inactivation, protein aggregation, and precipitation [38,48,49]. The modification by HC-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors [41].…”

“…The range of concentrations of chemical compounds used in this study was selected based on the preliminary studies of the concentration (0e10,000 mM)-and time (6,12,24,48, 72 h)-dependency experiments (data not shown).…”