S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 127

wild-type recombinant annexin A2 into the hyperhomocysteinemic mice. Taken
together, these results indicate that the inhibition of annexin A2-dependent fibrino-
lysis by S-homocysteinylation in vivo generates a prothrombotic phenotype.
If S-homocysteinylation of annexin A2 generates a prothrombotic phenotype, it
should be observed in any model of hyperhomocysteinemia, regardless of whether
the model is dietary or genetic. Surprisingly, however, a prothrombotic phenotype
is not observed in genetic hyperhomocysteinemia. Specifically, severely
hyperhomocysteinemic Tg-I278T Cbs / mice do not display increased suscepti-
bility to arterial or venous thrombosis, measured by several methods, including
photochemical injury of the carotid artery, chemical (FeCl 3 ) injury in the carotid
artery and mesenteric arterioles, and ligation of the inferior vena cava [448]. There
are no significant differences in hemostatic and hemodynamic parameters between
Tg-I278T Cbs / and control mice. Although the authors [448] do not discuss this,
these data suggest that annexin A2 S-homocysteinylation for an unknown reason
does not occur in the Cbs / mice. Another possibility is that other mechanisms of
fibrinolysis are enhanced in severely hyperhomocysteinemic mice.

7.4 Intracellular Proteins

7.4.1 Heterogenous Nuclear Ribonucleoprotein E1
(hnRNP-E1)

Folates participate in one carbon metabolism and are essential for the synthesis of
DNA and the remethylation of Hcy to methionine. Inadequate folate supply leads to
the elevation of Hcy levels. High-affinity folate receptors (FR), discovered in 1981
[449], are critical for the cellular uptake of 5-methylenetetrahydrofolate [450]. In
addition to hyperhomocysteinemia, folate deficiency also induces a marked eleva-
tion of the cell-surface FR protein, but not FR mRNA, in HeLa cells. The FR
expression is regulated posttranslationally by heterologous nuclear ribonucleopro-
tein E1 (hnRNP-E1), which binds to an 18 base cis-element in the 5 0 -UTR of FR-

α

mRNA [451].
The accumulation of intracellular Hcy resulting from folate deficiency has been
shown to trigger the interaction of FR-

mRNA cis-element with hnRNP-E1, which
then stimulates the biosynthesis and upregulation of FR [452]. Studies with purified
components demonstrate that Hcy induces a concentration-dependent increase in
the affinity of hnRNP-E1 to FR-

α

mRNA, which correlates with increase in
translation of the FR in vitro and in cultured human cells [453]. Inhibition of
hnRNP-E1 synthesis by siRNA reduces both constitutive and Hcy-induced FR
biosynthesis.
Mass spectroscopic studies show that Hcy binds to hnRNP-E1 via multiple
disulfide bonds within the K-homology domains that are known to interact with
FR-

α

α mRNA. To identify the sites of S-homocysteinylation, S-homocysteinylated