Transcriptional Regulation of Cystathionine-{gamma}-lyase in Endothelial cells by NADPH oxidase 4-dependent Signaling [Gene Regulation]

November 30th, 2015 by

The gasotransmitter, hydrogen sulfide (H2S) is recognised as an important mediator of endothelial cell homeostasis and function, which impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species (ROS), H2O2. However, the cellular source of H2O2, and the redox-dependent molecular signaling pathway which modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor (HRI) kinase /eIF2α /Activating Transcription Factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE, to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic (eNox4 Tg) mice, compared to wild-type littermate controls. Using wire myography we demonstrate that eNox4 Tg mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with the CSE inhibitor, Propargyl-glycine. We therefore conclude that positive transcriptional regulation of CSE by Nox4 in endothelial cells results in increased H2S production, and contributes to the regulation of vascular tone.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in vitro Reconstitution of Lipid II Synthesis Pathway [Enzymology]

November 30th, 2015 by

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been amongst the first drug targets, compounds directed against the membrane integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi and Escherichia coli as well as of Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B.subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY/nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane associated protein MurG As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin and tunicamycin.

Binding Affinities among DNA Helicase-Primase, DNA Polymerase and Replication Intermediates in the Replisome of Bacteriophage T7 [Enzymology]

November 30th, 2015 by Zhang, H., Tang, Y., Lee, S.-J., Wei, Z., Cao, J., Richardson, C. C.

The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed the loop is released and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7 the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase-DNA complex are much faster at a nick DNA than the release from an ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.
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Uracil-DNA glycosylase UNG promotes Tet-mediated DNA demethylation [Gene Regulation]

November 30th, 2015 by

In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in ESCs and iPSCs, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. In order to search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation.

RUTBC1 Functions as a GTPase-acitvating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes [Membrane Biology]

November 30th, 2015 by Marubashi, S., Shimada, H., Fukuda, M., Ohbayashi, N.

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed to regulate the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. The same as other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and GTP-bound active form, and the cycle is thought to be regulated by an activating enzyme GEF (guanine nucleotide exchange factor) and an inactivating enzyme GAP (GTPase-activating protein), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has never been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes, and we discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicated that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.

The orphan G protein-coupled receptor GPR17 negatively regulates oligodendrocyte differentiation via G{alpha}i/o and its downstream effector molecules [Neurobiology]

November 30th, 2015 by

Recent studies have recognized G protein-coupled receptors (GPCRs) as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan GPCR that has been identified as oligodendroglial maturation inhibitor since its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small-molecule agonist MDL29,951 decreases myelin basic protein (MBP) expression levels mainly by triggering the Gαi/o signaling pathway which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element‐binding protein (CREB). In addition, we show that GPR17 activation also diminishes MBP abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.
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Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities [Protein Synthesis and Degradation]

November 30th, 2015 by

Serpins regulate coagulation and inflammation, binding serine proteases in suicide inhibitory complexes. Target proteases cleave the serpin reactive center loop (RCL) scissile P1-P1′ bond resulting in serpin-protease suicide inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases while mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon gamma receptor deficient mice (IFNγR-/-). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the RCL produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing R or RN, not RM, with 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking R or RN with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b+ monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in PAI-1 serpin deficient transplants. Plaque reduction correlated with mononuclear cell activation..In a separate study Serp-1 peptide, S-7 improved survival in the MHV68 vasculitis model whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.
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The Transcription Factor Bach2 is Phosphorylated at Multiple Sites in Murine B Cells But a Single Site Prevents Its Nuclear Localization [Gene Regulation]

November 30th, 2015 by

The transcription factor Bach2 regulates the immune system at multiple points including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells is still unclear. Here we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in mature B cell line BAL17. Among the sites identified, serine 535 (S535) was critical for the regulation of Bach2 since a single mutation of S535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas S509 played an auxiliary role. Bach2 repressor activity was enhanced by the S535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells.
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SIRT3 Deacetylates Ceramide Synthases: Implications for Mitochondrial Dysfunction and Brain Injury [Molecular Bases of Disease]

November 30th, 2015 by

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2 and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2 and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, 2 and 6 are hyper-acetylated in the mitochondria of SIRT3-null mice and SIRT3 directly deacetylates the ceramide synthases in a NAD+-dependent manner that increases enzyme activity. Investigation of SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron-transport chain at the level of Complex III, attenuated mitochondrial outer membrane permeabilization, decreased ROS generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide which could inhibit Complex III, leading to increased ROS generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke.

Systemic CNS-targeted Delivery of NPY Reduces Neurodegeneration and Increases Neural Precursor Cell Proliferation in a Mouse Model of Alzheimer’s Disease [Cell Biology]

November 30th, 2015 by

Neuropeptide Y (NPY) is one of the most abundant protein transmitters in the central nervous system with roles in a variety of biological functions including: food intake, cardiovascular regulation, cognition, seizure activity, circadian rhythms and neurogenesis. Reduced NPY and NPY receptor expression is associated with numerous neurodegenerative disorders including Alzheimer's disease (AD). To determine if replacement of NPY could ameliorate some of the neurodegenerative and behavioral pathology associated with AD, we generated a lentiviral vector expressing NPY fused to a brain transport peptide (apoB) for wide-spread CNS delivery in an APP-tg mouse model of AD. The recombinant NPY-apoB effectively reversed neurodegenerative pathology and behavioral deficits although it had no effect on accumulation of Aβ. The subgranular zone of the hippocampus showed a significant increase in proliferation of neural precursor cells (NPC) without further differentiation into neurons. The neuroprotective and neurogenic effects of NPY-apoB appeared to involve signaling via ERK and Akt through the NPYR1 and NPYR2 receptors. Thus, widespread CNS targeted delivery of NPY appears to be effective at reversing the neuronal and glial pathology associated with Aβ accumulation while also increasing NPC proliferation. Overall, increased delivery of NPY to the CNS for AD might be an effective therapy especially if combined with an anti-Aβ therapeutic.
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