Farnesoid X receptor protects against kidney injury in uninephrectomized obese mice [Metabolism]

December 11th, 2015 by Gai, Z., Gui, T., Hiller, C., Kullak-Ublick, G. A.

Activation of the farnesoid X receptor (FXR) has indicated a therapeutic potential for this nuclear bile acid receptor in the prevention of diabetic nephropathy and obesity-induced renal damage. Here, we investigated the protective role of FXR against kidney damage induced by obesity in mice that had undergone uninephrectomy, a model resembling the clinical situation of kidney donation by obese individuals. Mice fed a high-fat diet developed the core features of metabolic syndrome, with subsequent renal lipid accumulation and renal injury, including glomerulosclerosis, interstitial fibrosis, and albuminuria. The effects were accentuated by uninephrectomy. In human renal biopsies, staining of 4-hydroxynonenal (4-HNE), glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), markers of ER stress, was more prominent in the proximal tubules of 15 obese compared with 16 non-obese patients. In mice treated with the FXR agonist obeticholic acid (OCA), renal injury, renal lipid accumulation, apoptosis and changes in lipid peroxidation were attenuated. Moreover, disturbed mitochondrial function was ameliorated and the mitochondrial respiratory chain recovered following OCA treatment. Culturing renal proximal tubular cells with free fatty acid (FFA) and FXR agonists showed that FXR activation protected cells from FFA-induced oxidative stress and ER stress, as denoted by a reduction in the level of ROS staining and Grp78 immunostaining, respectively. Several genes involved in glutathione metabolism were induced by FXR activation in the remnant kidney, which was consistent with a decreased glutathione disulfide / glutathione ratio. In summary, FXR activation maintains endogenous glutathione homeostasis and protects the kidney in uninephrectomized mice from obesity-induced injury.

SIRT1 Limits Adipocyte Hyperplasia Through c-Myc Inhibition [Metabolism]

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.

Selective Recognition of H3.1K36 dimethylation / H4K16 acetylation facilitates the regulation of ATRA-responsive genes by putative chromatin reader ZMYND8 [Cell Biology]

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.
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Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis are Correlated with Pathogenicity and Carriage [Lipids]

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.
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Progesterone-induced acrosome exocytosis requires sequential involvement of calcium-independent iPLA2[beta] and group X sPLA2 [Developmental Biology]

December 11th, 2015 by

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR) but the molecular identity of PLA2 has remained elusive. Here we have tested the role of two intracellular (iPLA2β and cPLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR while both iPLA2β and group X sPLA2 are involved in P4-induced AR. cPLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 and Ca2+ pathways to achieve AR. At low P4 concentration, sperm achieving early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm achieving late AR (20-30 min post-P4) rely on both iPLA2β and group X sPLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, PLA2s being key actors at low physiological P4 concentrations (≤ 2 μM) but not at higher P4 concentrations (>10 μM).
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The insect peptide CopA3 increases colonic epithelial cell proliferation and mucosal barrier function to prevent inflammatory responses in the gut [Cell Biology]

December 11th, 2015 by

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. Epidermal growth factor (EGF) enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide, CopA3, promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin A-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the DSS-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21Cip1/Waf1. Taken together, our findings indicate that the insect peptide, CopA3, prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.
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Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacteylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B Cell Acute Lymphoblastic Leukemia [Signal Transduction]

December 11th, 2015 by

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements (UREs) of its target genes and regulates their transcription via chromatin remodeling, Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 tri-methylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the URE of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic Casein Kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of H3K27me3 and decreased H3K9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. Presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.
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Structural and functional analyses reveal insights into the molecular properties of the E. coli Z ring stabilizing protein, ZapC [Cell Biology]

December 11th, 2015 by

In Escherichia coli, cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multi-protein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins are the Z-ring associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8 kDa ZapC, in particular, shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Angstrom and performed genetic and biochemical studies. ZapC is a monomer with a heretofore-unseen fold composed of two domains, an N-terminal alpha-beta region and a C-terminal twisted beta-barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC's function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail and the presence of two adjacent pockets suggests possible mechanisms for ZapC mediated FtsZ bundling.
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Uncovering the early assembly mechanism for amyloidogenic {beta}2-microglobulin using cross-linking and native mass spectrometry [Computational Biology]

December 11th, 2015 by Hall, Z., Schmidt, C., Politis, A.

β2-microglobulin (β2m), a key component of the major histocompatibility class I complex , can aggregate into fibrils with severe clinical consequences. As such, investigating structural aspects of the formation of oligomeric intermediates of β2m and their subsequent progression towards fibrillar aggregates is of great importance. However β2m aggregates are challenging targets in structural biology, primarily due to their inherent transient and heterogeneous nature. Here we study the oligomeric distributions and structures of the early intermediates of amyloidogenic β2m and its truncated variant ΔN6-β2m. We established compact oligomers for both variants by integrating advanced mass spectrometric techniques with available electron microscopy maps and atomic-level structures from NMR spectroscopy and X-ray crystallography. Our results revealed a stepwise assembly mechanism by monomer addition and domain swapping, for the oligomeric species of (ΔN6)-β2m. The observed structural similarity and common oligomerization pathway between the two variants is likely to enable ΔN6-β2m to cross-seed β2m fibrillation and allow the formation of mixed fibrils. We further determined the key subunit interactions in ΔN6-β2m tetramer, revealing the importance of a domain-swapped hinge region for formation of higher-order oligomers. Overall, we deliver new mechanistic insights into β2m aggregation paving the way for future studies on the mechanisms and cause of amyloid fibrillation.
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Wilms tumor suppressor, WT1, cooperates with microRNA-26a and microRNA-101 to suppress translation of the Polycomb protein, EZH2, in mesenchymal stem cells [RNA]

December 10th, 2015 by

Hereditary forms of Wilms arise from developmentally arrested clones of renal progenitor cells with biallelic mutations of WT1; recently, it has been found that Wilms tumors may also be associated with biallelic mutations in DICER1 or DROSHA, crucial for miRNA biogenesis. We have previously shown that a critical role for WT1 during normal nephrogenesis is to suppress transcription of the Polycomb group protein, EZH2, thereby de-repressing genes in the differentiation cascade. Here we show that WT1 also suppresses translation of EZH2. All major WT1 isoforms induce an array of miRNAs, which target the 3[prime] UTR of EZH2 and other Polycomb associated transcripts. We show that the WT1(+KTS) isoform binds to the 5[prime] UTR of EZH2 and interacts directly with the miRNA-containing RISC to enhance post-transcriptional inhibition. These observations suggest a novel mechanism through which WT1 regulates the transition from resting stem cell to activated progenitor cell during nephrogenesis. Our findings also offer a plausible explanation for the fact that Wilms tumors can arise either from loss of WT1 or loss of miRNA processing enzymes.
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