Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 43

S-NO-Hcy [100]. NO is generated by endothelial nitric oxide synthase and regulates
diverse functions in the endothelium, including ion channel activity, anti-oxidative,
anti-apoptotic, and anti-inflammatory responses. NO diffuses from the endothelial
cells to the vascular smooth muscle cells, where it mediates vasorelaxation. These
functions are mediated by protein S-nitrosylation [249]. Overexpression of the
inducible nitric oxide synthase by transfection of endothelial cells with an adenovi-
rus vector carrying the iNOS gene increases nitric oxide generation and results in a
significant increase in the formation of S-NO-Hcy [101]. While the biological
activities of NO are well established, the role of S-NO-Hcy is unclear, but, by
becoming a constituent of proteins, it can cause protein damage and thus contribute
to the pathophysiology of hyperhomocysteinemia.
Incubation of human umbilical vein endothelial cells (HUVEC) with [ 35 S]Hcy
results in incorporation of the radiolabel into cellular proteins [76]. Compositional
analysis shows that

M[ 35 S]Hcy contains 75 %
[ 35 S]Hcy and 25 % [ 35 S]Met (third row, Table 3.9 ). Because the transsulfuration
pathway is absent in endothelial cells, no radioactivity is found in cysteine. When
the endothelial cell 35 S-protein is subjected to one cycle of Edman degradation,
38 % of 35 S is released as a phenyl-thiohydantoin (PTH) derivative of Hcy. Edman
degradation of N-[ 35 S]Hcy-albumin (positive control; prepared by the modification
of human serum albumin with [ 35 S]Hcy-thiolactone—see Sects. 3.2.4 . and 5.1 .)
shows that

35 S-protein from HUVEC + 10

μ

96 % of posttranslationally incorporated Hcy is released as a PTH-Hcy
(last row, Table 3.9 ). These results suggest that posttranslationally incorporated
Hcy (N-[ 35 S]Hcy-protein) represents about a half (38 %) of total protein N-linked
[ 35 S]Hcy (75 %, third row). Of the 62 % of 35 S-protein resistant to Edman
degradation, translationally incorporated Hcy ([ 35 S]Hcy-protein) represents 37 %
(25 % is [ 35 S]Met-protein). The fraction of [ 35 S]Hcy-protein increases (second
column in Table 3.9 ), and the fraction of [ 35 S]Met-protein decreases (third column)
with increasing Hcy in culture media (row 1 and 2). This suggests that the
translational incorporation is more important at higher Hcy concentrations.
In cultures supplemented with folic acid, all [ 35 S] recovered by acid hydrolysis
of protein is associated only with Met (forth row in Table 3.9 ). This is due to
facilitation by folic acid of the remethylation of Hcy to Met catalyzed by Met
synthase, which uses methyltetrahydrofolate as a cofactor (Fig. 1.1 ). Supplementa-
tion with Met, which competes with Hcy and S-NO-Hcy for the active site of
MetRS and thus prevents both the formation of S-NO-Hcy-tRNA and the conver-
sion of Hcy to Hcy-thiolactone, decreases the incorporation of Hcy into protein (5th
row). However, the supplementation with HDL does not inhibit translational
incorporation of Hcy into protein but inhibits the posttranslational incorporation
because of Hcy-thiolactone-hydrolyzing activity of the PON1 protein carried on
HDL (see Sect. 3.5.1 .).
Taken together, these findings indicate that Hcy can gain an access to the genetic
code by S-nitrosylation-mediated invasion of the methionine-coding pathway.
However, whether S-nitroso-Hcy is generated in mammalian organisms and is a
component of human or animal proteins remains to be investigated.

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