S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 129

7.4.2 Metallothionein
Metallothionein is a 6-kDa zinc-binding chaperone and superoxide radical scaven-
ger that regulates zinc homeostasis and detoxifies heavy metals. Twenty of the
approximately sixty amino acid residues of mammalian metallothioneins are
cysteines, whose side chains are the only ligands to the seven zinc atoms in two
clusters. Zinc can be released from metallothionein by oxidized thiols including
cysteamine disulfide, coenzyme A disulfide, and glutathione disulfide (GS-SG).
GS-SG is known to participate in a thiol/disulfide interchange with the solvent-
accessible zinc-bound thiolates in metallothionein, which causes the clusters to
collapse and release zinc [455].
Treatment of endothelial cells with Hcy also causes increase in free zinc, which
is detected by preloading the cells with Zinquin AM [146]. In contrast, similar
treatment with cysteine does not induce zinc release. As a direct consequence of
free zinc elevation, the expression of early growth response-1 (Egr-1) protein, a
zinc finger transcription factor that regulates proinflammatory and procoagulant
genes associated with atherothrombosis [456], is transiently elevated in Hcy-treated
cells [146]. In addition, the superoxide radical scavenging ability of metallothionein
is inhibited after treatment with Hcy, thereby leading to superoxide production and
increased oxidative stress.
Experiments with endothelial cells labeled with [ 35 S]Hcy followed by resolution
of cell extracts on nonreducing SDS-PAGE gels and detection by autoradiography
and Western blotting with anti-metallothionein antibodies suggest that
metallothionein is a target for S-homocysteinylation [146]. Autoradiograms show
a[ 35 S]-labeled band of 10 kDa that coincides with the metallothionein band
detected by Western blotting. The radiolabeled band is absent in a sample that
has been reduced with 2-mercaptoethanol. Surprisingly, however, the radiolabeled
band is still present in samples reduced with glutathione. These findings suggest a
mechanism in which Hcy binding to metallothionein disrupts intracellular redox
homeostasis and induces endothelial cell dysfunction. However, the proposed mode
of interaction between Hcy and metallothionein remains to be confirmed in in vitro
studies with purified components.
7.4.3 Dimethylarginine Dimethylaminohydrolase
Endothelial dysfunction due to decreased bioavailability of nitric oxide is observed
in hyperhomocysteinemia [457]. Asymmetrical dimethylarginine (ADMA), a com-
petitive inhibitor of the enzyme nitric oxide synthase, is an important regulator of
vascular function. Increased levels of Hcy are associated with increased levels of
ADMA, which suggests that ADMA may mediate Hcy-induced endothelial dysfunc-
tion due to reduced nitric oxide production [458]. ADMA is known to be hydrolyzed
by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) [459].