ACS DIVISION OF BIOLOGICAL CHEMISTRY

December 14th, 2015 by

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine cocktail containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of Connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.

December 14th, 2015 by

December 14th, 2015 by

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December 11th, 2015 by

ZMYND8, a newly identified component of transcriptional coregulator network, was found to interact with Nucleosome Remodelling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting NuRD complex to damaged chromatin for repressing transcription and promoting double-strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with selective epigenetic marks H3.1K36Me2/H4K16Ac. Further, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the All Trans Retinoic Acid (ATRA)-responsive ones, through its modified histone binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation competent RNA Polymerase II phosphorylated at Ser5 in a DNA template dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone binding ability.

December 11th, 2015 by

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hy-perplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD+-dependent protein deacetylase sirtuin-1 (SIRT1), a key regulator of mammalian metabo-lism, maintains proper metabolic functions in many tissues counteracting obesity. Here we re-port that differentiated adipocytes are hyperplas-tic when SIRT1 is stably knocked down in mouse 3T3-L1 preadipocytes. This phenotype is associ-ated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveals that the c-Myc pathway is altered to drive en-hanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Re-markably, differentiating SIRT1-silenced preadi-pocytes exhibit enhanced mitotic clonal expansion (MCE) accompanied by reduced levels of p27, as well as elevated levels of CCAAT/enhancer-binding protein beta (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and en-hanced proliferation phenotype are also found to be SIRT1-dependent in proliferating MEFs and differentiating human SW872 preadipocytes. Re-ducing both SIRT1 and c-Myc expression in 3T3-L1 simultaneously do not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. Better understanding of the molecu-lar mechanisms of adipocyte hyperplasia will open new venues towards understanding obesity.

December 11th, 2015 by

The degree of phosphorylation and phosphoethanolaminylation of lipid A on Neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of N. meningitidis without disease. Invasive strains were classified as Groups 1, 2 and 3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with 3 phosphates and 1 phosphoethanolamine were detected in all LOS analyzed. LOS from Groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared to LOS from Group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared to carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multi-faceted effects on homeostatic innate immune responses which have critical impact on the pathophysiology of meningococcal infections.

December 11th, 2015 by

December 11th, 2015 by