High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erb{beta} Leads to its Degradation and Indirectly Regulates its Interaction with Nuclear Receptor Corepressor [Metabolism]

December 15th, 2015 by Carter, E. L., Gupta, N., Ragsdale, S. W.

Rev-erbα and Rev-erbβ are heme-binding nuclear receptors (NRs) that repress the transcription of genes involved in regulating metabolism, inflammation and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases affinity of the ligand-binding domain (LBD) of Rev-erbs for NCoR1 peptides. One explanation for this discrepancy is that the LBD and NCoR1 peptides used for in vitro studies cannot replicate key features of the full-length proteins used in cellular studies. However, combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbβ (FLRev-erbβ) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme- replete FLRev-erbβ:DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbβ-NCoR1 complex. The interaction between FLRev-erbβ and NCoR1 as well as Rev-erbβ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating degradation of Rev-erbβ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10-6 s-1) for heme dissociation from Rev-erbβ, rules out a prior proposal that Rev-erbβ acts as an intracellular heme sensor.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erb{beta} Leads to its Degradation and Indirectly Regulates its Interaction with Nuclear Receptor Corepressor [Metabolism]

Nerve Growth Factor is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs [Molecular Bases of Disease]

December 14th, 2015 by

Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity, but more recently were found to be activated by endogenous alarmins such as fragmented extra-cellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, BDNF and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2 and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity blocked TLR2-driven NGF upregulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.

Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 [Molecular Bases of Disease]

December 14th, 2015 by Chen, Q., Lu, M., Monks, B. R., Birnbaum, M. J.

Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, while the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was significantly decreased in livers with insulin signaling disrupted by ablation of insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

Low Level Pro-Inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide Mediated Protein Kinase C{delta} [Signal Transduction]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Low Level Pro-Inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide Mediated Protein Kinase C{delta} [Signal Transduction]

Inhibition of MAP Kinase-Interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing both a 5′-Terminal Cap and Hairpin [RNA]

December 14th, 2015 by Korneeva, N. L., Song, A., Gram, H., Edens, M. A., Rhoads, R. E.

The mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular-signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eIF4G and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that a MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-untranslated region (UTR). Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m7GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Inhibition of MAP Kinase-Interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing both a 5′-Terminal Cap and Hairpin [RNA]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Selective Recognition of H3.1K36 dimethylation / H4K16 acetylation facilitates the regulation of ATRA-responsive genes by putative chromatin reader ZMYND8 [Cell Biology]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis are Correlated with Pathogenicity and Carriage [Lipids]

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).
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Progesterone-induced acrosome exocytosis requires sequential involvement of calcium-independent iPLA2[beta] and group X sPLA2 [Developmental Biology]