The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1.

Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

Discovery and structural analysis of Eph receptor tyrosine kinase inhibitors.

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC(50)s of 40nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the 'DFG-out' inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes.

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.

DNA methyl transferase 1: regulatory mechanisms and implications in health and disease.

DNA methylation serves as the principal form of post-replicative epigenetic modification. It is intricately involved in gene regulation and silencing in eukaryotic cells, making significant contributions to cell phenotype. Much of it is mitotically inherited; some is passed on from one filial generation to the next. Establishment and maintenance of DNA methylation patterns in mammals is governed by three catalytically active DNA methyltransferases - DNMT3a, DNMT3b and DNMT1. While the first two are responsible mainly for de novo methylation, DNMT1 maintains the methylation patterns by preferentially catalyzing S-adenosyl methionine-dependant transfer of a methyl group to cytosine at hemimethylated CpG sites generated as a result of semi-conservative DNA replication. DNMT1 contains numerous regulatory domains that fine-tune associated catalytic activities, deregulation of which is observed in several diseases including cancer. In this minireview, we analyze the regulatory mechanisms of various sub-domains of DNMT1 protein and briefly discuss its pathophysiological and pharmacological implications. A better understanding of DNMT1 function and structure will likely reveal new applications in the treatment of associated diseases.

Human neutrophil peptides upregulate expression of COX-2 and endothelin-1 by inducing oxidative stress.

Polymorphonuclear leukocytes (PMNs) play an important role during inflammation in cardiovascular diseases. Human neutrophil peptides (HNPs) are released from PMN granules upon activation and are conventionally involved in microbial killing. Recent studies suggested that HNPs may be involved in the pathogenesis of vascular abnormality by modulating inflammatory responses and vascular tone. Since HNPs directly interact with endothelium upon release from PMNs in the circulation, we tested the hypothesis that the stimulation with HNPs of endothelial cells modulates the expression of vasoactive by-products through altering cyclooxygenase (COX) activity. When human umbilical vein endothelial cells were stimulated with purified HNPs, we observed a time- and dose-dependent increase in the expression of COX-2, whereas COX-1 levels remained unchanged. Despite an increased expression of COX-2 at the protein level, HNPs did not significantly enhance the COX-2 activity, thus the production of the prostaglandin PGI2. HNPs significantly induced the release of endothelin-1 (ET-1) as well as the formation of nitrotyrosine. The HNP-induced COX-2 and ET-1 production was attenuated by the treatment with the oxygen free radical scavenger N-acetyl-L-cysteine and the inhibitors of p38 MAPK and NF-kappaB, respectively. The angiontensin II pathway did not seem to be involved in the HNP-induced upregulation of COX-2 and ET-1 since the use of the angiotensin-converting enzyme inhibitor enalapril had no effect in this context. In conclusion, HNP may play an important role in the pathogenesis of inflammatory cardiovascular diseases by activating endothelial cells to produce vasoactive by-products as a result of oxidative stress.

Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways.

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.