Structure of HsdS Subunit from Thermoanaerobacter tengcongensis Sheds Lights on Mechanism of Dynamic Opening and Closing of Type I Methyltransferase

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission

Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons

Diacerhein attenuates the inflammatory response and improves survival in a model of severe sepsis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Introduction: Hyperglycemia and insulin resistance have been associated with a worse outcome in sepsis. Although tight glycemic control through insulin therapy has been shown to reduce morbidity and mortality rates, the effect of intensive insulin therapy in patients with severe sepsis is controversial because of the increased risk of serious adverse events related to hypoglycemia. Recently, knowledge about diacerhein, an anthraquinone drug with powerful antiinflammatory properties, revealed that this drug improves insulin sensitivity, mediated by the reversal of chronic subclinical inflammation. The aim of the present study was to evaluate whether the antiinflammatory effects of diacerhein after onset of sepsis-induced glycemic alterations is beneficial and whether the survival rate is prolonged in this situation. Methods: Diffuse sepsis was induced by cecal ligation and puncture surgery (CLP) in male Wistar rats. Blood glucose and inflammatory cytokine levels were assessed 24 hours after CLP. The effect of diacerhein on survival of septic animals was investigated in parallel with insulin signaling and its modulators in liver, muscle, and adipose tissue. Results: Here we demonstrated that diacerhein treatment improves survival during peritoneal-induced sepsis and inhibits sepsis-induced insulin resistance by improving insulin signaling via increased insulin-receptor substrate-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation. Diacerhein also decreases the activation of endoplasmic reticulum stress signaling that involves upregulation of proinflammatory pathways, such as the I kappa B kinase and c-Jun NH2-terminal kinase, which blunts insulin-induced insulin signaling in liver, muscle, and adipose tissue. Additionally, our data show that this drug promoted downregulation of proinflammatory signaling cascades that culminate in transcription of immunomodulatory factors such interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha. Conclusions: This study demonstrated that diacerhein treatment increases survival and attenuates the inflammatory response with a significant effect on insulin sensitivity. On the basis of efficacy and safety profile, diacerhein represents a novel antiinflammatory therapy for management of insulin resistance in sepsis and a potential approach for future clinical trials.164Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq