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While estrogen receptor (ER) profile plays an important role in response to estrogens, receptor coregulators act as critical determinants of signaling. Although the clinical effects of ovarian hormones on various normal and pathological processes are an active area of research, the exact signaling effects on, for example, the vessel wall, are incompletely understood. Hence, we sought to discover proteins that associate with ER[beta], the isoform that shows upregulated mRNA expression after arterial injury. Using a yeast two-hybrid screen we identified NM23-H2, a multifaceted metastasis suppressor candidate protein, as an ER[beta]-associated protein. Although NM23-H2 was immunodetected in arteries from young subjects (27 [+ or -] 6 yr, 14 men and 6 women) with benign intimal hyperplasia, expression was diminished in fatty streaks/atheromas and altogether absent in advanced atherosclerotic lesions. Both nm23-H2 mRNA and protein were expressed by vascular cells in vitro. Treatment with 17[beta]-estradiol and an ER[beta]-selective agonist, diarylpropionitrile, increased protein expression of NM23-H2; an effect that was not seen with an ERa-selective agonist, propylpyrazole-triol. Estrogen also prompted nuclear localization of NM23-H2 protein in human coronary smooth muscle cells (SMCs). An in vitro mimic of inflammation decreased the expression of NM23-H2 in SMCs, which was restored on addition of estrogen and dependent on the estrogen receptor. In summary, we report the novel association of NM23-H2 with ER[beta] and show for the first time its expression in vascular cells and demonstrate regulation of its expression and localization by estrogen. In that the abundance of NM23-H2 diminishes with both the advancement of atherosclerosis and inflammation, this ER[beta]-associated protein may play an important role in mediating the vasculoprotective effects of estrogens. atherosclerosis; receptors

Research has been conducted on the human NM23-H2 protein, a transcription regulator which binds and cleaves DNA. The role of the nucleotide-binding site in the DNA cleavage reaction has been investigated and the details are reported.

NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the e-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic pocket involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected.

The ubiquitously expressed nucleoside diphosphate kinases (Nm23/NDPK/Awd) are a large family of multifunctional enzymes implicated in nucleic acid metabolism and in normal and abnormal development. Here, we describe the generation and characterization of NDPK A- and B-deficient ( Nme1−/−/ Nme2−/−) mice in which >95% of the enzyme activity is eliminated. These mice are undersized, die perinatally, and exhibit a spectrum of hematological phenotypes including severe anemia, impaired maturation of erythrocytes, and abnormal hematopoiesis in the liver and bone marrow. Flow cytometric analysis of developing Nme1−/−/ Nme2−/− erythroid cells indicated that the major iron transport receptor molecule TfR1 is attenuated concomitant with a reduction of intracellular iron, suggesting that TfR1 is a downstream target of NDPKs and that reduced iron in Nme1−/−/ Nme2−/− erythroblasts is inhibiting their development. We conclude that Nm23/NDPKs play critical roles in definitive erythroid development. Our novel mouse model also links erythropoiesis and nucleotide metabolism. Developmental Dynamics 238:775-787, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]