Although recent studies highlight the importance of histone modifications and ATP-dependent chromatin remodelling in DNA double-strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser-139 (S139ph) after DNA damage, binds to c-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to c-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with c-H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to c-H2AX nucleosomes. S139ph stimulates the H3 acetylation on c-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on c-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, c-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed.

Curcumin is a natural phenolic component of yellow curry spice, which is used in some cultures for the treatment of diseases associated with oxidative stress and inflammation. Curcumin has been reported to be capable of preventing the death of neurons in animal models of neurodegenerative disorders, but its possible effects on developmental and adult neuroplasticity are unknown. In the present study, we investigated the effects of curcumin on mouse multi-potent neural progenitor cells (NPC) and adult hippocampal neurogenesis. Curcumin exerted biphasic effects on cultured NPC; low concentrations stimulated cell proliferation, whereas high concentrations were cytotoxic. Curcumin activated extracellular signal-regulated kinases (ERKs) and p38 kinases, cellular signal transduction pathways known to be involved in the regulation of neuronal plasticity and stress responses. Inhibitors of ERKs and p38 kinases effectively blocked the mitogenic effect of curcumin in NPC. Administration of curcumin to adult mice resulted in a significant increase in the number of newly generated cells in the dentate gyrus of hippocampus, indicating that curcumin enhances adult hippocampal neurogenesis. Our findings suggest that curcumin can stimulate developmental and adult hippocampal neurogenesis, and a biological activity that may enhance neural plasticity and repair.

Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (c-H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA-PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB-surrounding chromatin via interaction with c-H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for c-H2AX in efficient DSB repair, these results suggest that the SWI/ SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin.

SummaryThe emergence and dissemination of extended-spectrum (ES) b -lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by thirdgeneration b -lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C b -lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES b -lactamase from Enterobacter cloacae GC1, the W -loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 Å resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of blactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 b -lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES b -lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C b -lactamases to extend their substrate spectrum.

It has been reported that interferon-gamma (IFN-gamma) facilitates differentiation of PC-12 cells and murine adult neural stem cells. Here we show that IFN-gamma promotes the differentiation of C17.2 neural progenitor cells (NPC) into a neuronal phenotype characterized by neurite outgrowth and the expression of the neuronal marker protein beta-III tubulin. IFN-gamma induced an increase in the activity c-jun N-terminal kinase (JNK) without affecting activities of extracellular signal-regulated kinases (ERKs 1 and 2). An inhibitor of JNK blocked the ability of IFN-gamma to promote differentiation of NPC into neurons, whereas an inhibitor of ERKs 1 and 2 did not. Our findings show that the pro-inflammatory cytokine, IFN-gamma has the potential to stimulate neurogenesis, suggesting roles for this cytokine in development and repair of the nervous system.

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys 184 , His 185 , and Asp 213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His 85 , His 122 , and Glu 90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8 -12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDSpolyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.Every organism responds to a sudden increase in the environmental temperature by the overexpression of a set of highly conserved heat shock proteins (1, 2). Most of these heat shock proteins function either as molecular chaperones, assisting in protein folding and renaturation or as proteases which degrade proteins that are beyond rescue.Hsp31, 1 the hchA gene product (formerly known as YedU), is a heat-inducible homodimeric protein of 31-kDa subunits, which was recently shown to exhibit molecular chaperone activity (3, 4). It promotes the functional folding of citrate synthase, ␣-glucosidase, and alcohol dehydrogenase. It also prevents the aggregation at 43°C of citrate synthase and alcohol dehydrogenase and interacts specifically with unfolded proteins (3, 4). Although Hsp31 does not exhibit any ATPase activity, some of its chaperone activities are partially inhibited by ATP (3, 4). The crystal structure of Hsp31 was solved at 1.6 Å resolution and revealed a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded protein substrates and expla...

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