S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 128

hnRNP-E1 is prepared in vitro by incubation of recombinant GST-hnRNP-E1 with
50- M Hcy. Digestion with trypsin followed by LC-MS/MS analysis shows that S-

linked Hcy is present at several cysteine residues of hnRNP-E1 (Cys54, Cys109,
Cys158, Cys163, and Cys149) [453].
These findings show that the disruption of critical disulfide bonds by S-
homocysteinylation unmasks an RNA-binding pocket in hnRNP-E1 and optimizes
its binding to FR-

μ

mRNA cis-element required for the FR upregulation. Further-
more, these data suggest that hnRNP-E1 may function as a physiologically relevant
cellular sensor of folate deficiency [453].
In addition to FR-α mRNA, S-homocysteinylated hnRNP-E1 also binds to cis-
elements of other mRNAs, including 15-lipooxygenase mRNA, human papilloma-
virus 16 (HPV16) L2 mRNA, tyrosine hydroxylase mRNA, and the neuronal
intermediate neurofilament middle molecular mass (neurofilament-M) mRNA.
That these interactions are likely to occur in vivo has been demonstrated by
increased accumulation of tyrosine hydroxylase and neurofilament-M in the adrenal
medulla and the cerebellum, respectively, in brains of murine fetuses (gestation day
17) whose mothers were fed folate-deficient diet prior to and during pregnancy
[453]. Other studies have shown that the translation of these mRNAs is also
activated by the conventional reducing agent 2-mercaptoethanol or DTT.
Human papillomavirus (HPV) 16 is known to transform epithelial tissues to
cancer in the presence of several cofactors, but there is insufficient evidence that
poor nutrition (folate deficiency) has any such role. Because physiological folate
deficiency leads to S-homocysteinylation of hnRNP-E1 and activates a nutrition-
sensitive (Hcy-responsive) posttranscriptional RNA operon that includes interac-
tion with HPV16 L2 mRNA, the functional consequences of folate deficiency on
HPV16 in immortalized HPV16-harboring human (BC-1-Ep/SL) keratinocytes and
HPV16-organotypic rafts have been investigated [454]. In addition to interacting
with HPV16 L2 mRNA cis-element, S-homocysteinylated hnRNP-E1 also binds
(with greater affinity) to another HPV16 57-nucleotide poly(U)-rich cis-element in
the early polyadenylation element (upstream of L2L1 genes). These interactions
lead to reductions of both L1 and L2 mRNA and proteins (but not HPV16 E6 and
E7) in vitro, while in cultured keratinocytes, HPV16-low-folate-organotypic rafts
develop in physiological low-folate medium. (Intracellular Hcy increases from
7

α

M in low-folate medium.) Furthermore, HPV16-low-
folate-organotypic rafts contain fewer HPV16 viral particles, a similar HPV16
DNA viral load, and a much greater extent of integration of HPV16 DNA into
genomic DNA, compared with HPV16-high-folate-organotypic rafts. Subcutane-
ous implantation of 18-day-old HPV16-low-folate-organotypic rafts into folate-
replete immunodeficient mice transforms this benign keratinocyte-derived raft
tissue into an aggressive HPV16-induced cancer within 12 weeks. Taken together,
these results establish a likely molecular linkage between poor folate nutrition and
HPV16 and predict that nutritional folate and/or vitamin B 12 deficiency, which are
both common worldwide, is likely to alter the natural history of HPV16 infections
and also warrant serious consideration of B vitamins as reversible cofactors in
oncogenic transformation of HPV16-infected tissues to cancer [454].

μ M in high-folate to 20 μ