The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.     

Susceptibility to Crohn’s disease is mediated by <Emphasis Type="Italic">KIR</Emphasis>2DL2/<Emphasis Type="Italic">KIR</Emphasis>2DL3 heterozygosity and the HLA-C ligand

In the present study, we investigated the relationship between the KIR loci and the genes encoding their HLA ligands and genetic susceptibility to Crohn’s disease (CD). Analyses of the interactions between KIR3DL1, KIR2DL1, KIR2DL2, and KIR2DL3 with their respective HLA ligands indicate that there is a protective effect for KIR2DL2 in the absence of its HLA ligand C1. Given that KIR2DL2 and KIR2DL3 segregate as alleles, we compared their genotypic distributions to expectations under Hardy–Weinberg Equilibrium (HWE) with regard to the HLA ligand C1 status. While all the genotypic distributions conform to expectations under HWE in controls, in C2 ligand homozygous cases there is significant deviation from HWE, with a reduction of KIR2DL2, KIR2DL3 heterozygotes. KIR2DL2, KIR2DL3 heterozygosity is the only genotypic combination that confers protection from CD. In addition to the protective effect (OR = 0.44, CI = 0.22–0.87; p = 0.018) observed in C2 ligand homozygotes, the KIR2DL2, KIR2DL3 genotype is predisposing (OR = 1.34, CI = 1.03–4.53; p = 0.031) in the presence of C1 ligand. A test for trend of HLA class I C ligand group genotypes with KIR2DL2, KIR2DL3 heterozygosity in cases and controls indicates that C1, C2 ligand group heterozygotes have an intermediate effect on predisposition. These results show for the first time that disease susceptibility may be related to heterozygosity at a specific KIR locus, and that HLA ligand genotype influences the relative effect of the KIR genotype.     

Discovery of Novel Proteasome Inhibitors Using a High-Content Cell-Based Screening System

The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.     

IBD-Associated TL1A Gene (TNFSF15) Haplotypes Determine Increased Expression of TL1A Protein

Background

The recently identified member of the TNF superfamily TL1A (TNFSF15) increases IFN-γ production by T cells in peripheral and mucosal CCR9+ T cells. TL1A and its receptor DR3 are up-regulated during chronic intestinal inflammation in ulcerative colitis and Crohn''s disease (CD). TL1A gene haplotypes increase CD susceptibility in Japanese, European, and US cohorts.

Methodology and Principal Findings

Here we report that the presence of TL1A gene haplotype B increases risk in Jewish CD patients with antibody titers for the E. coli outer membrane porin C (OmpC+) (Haplotype B frequency in Jewish CD patients: 24.9% for OmpC negative and 41.9% for OmpC positive patients, respectively, P≤0.001). CD14+ monocytes isolated from Jewish OmpC+ patients homozygous for TL1A gene haplotype B express higher levels of TL1A in response to FcγR stimulation, a known inducing pathway of TL1A, as measured by ELISA. Furthermore, the membrane expression of TL1A is increased on peripheral monocytes from Jewish but not non-Jewish CD patients with the risk haplotype.

Conclusions and Significance

These findings suggest that TL1A gene variation exacerbates induction of TL1A in response to FcγR stimulation in Jewish CD patients and this may lead to chronic intestinal inflammation via overwhelming T cell responses. Thus, TL1A may provide an important target for therapeutic intervention in this subgroup of IBD patients.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53

The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.