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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Substrate selectivity of lysophospholipid transporter LplT involved in membrane phospholipid remodeling in Escherichia coli [Membrane Biology]

November 26th, 2015 by Lin, Y., Bogdanov, M., Tong, S., Guan, Z., Zheng, L.

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lyso-PE recycling in gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the E.coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E.coli spheroplasts. Our results show that lyso-PE and lyso-PG, but not lyso-PC, are taken up by LplT for reacylation by acyltransferase/acyl-ACP synthetase (Aas) on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated head group. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate tri-acylated cardiolipin by Aas, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lyso-PA and its substrate binding was not inhibited by either orthophosphate or glycerol-3-phosphate, indicating that either a glycerol or ethanolamine head group is the chemical determinant for substrate recognition. Diacyl forms of PE, PG or the tetra-acylated form of cardiolipin could not serve as competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a sideways sliding mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.
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Solution Structure of the HIV-1 Intron Splicing Silencer and its Interactions with the UP1 Domain of hnRNP A1 [Gene Regulation]

November 25th, 2015 by Jain, N., Morgan, C. E., Rife, B. D., Salemi, M., Tolbert, B. S.

Splicing patterns in HIV-1 are maintained through cis regulatory elements that recruit antagonistic host RNA binding proteins. The activity of the 3 prime acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b) and an exonic splicing enhancer (ESE3). Since HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate the UP1 domain of hnRNP A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA binding protein.

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin [Metabolism]

November 24th, 2015 by

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with GSH. Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 were able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH, and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 M-1s-1) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knockout mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.

Characterization of Microfibrillar-Associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-Binding Protein Involved in Elastic Fiber Formation [Glycobiology and Extracellular Matrices]

November 24th, 2015 by

Microfibrillar-associated protein 4 (MFAP4) is an extracellular glycoprotein found in elastic fibers with not clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin, fibrillin-1 and -2 as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues F241 and S203 in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.
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Hyaluronan rafts on airway epithelial cells [Glycobiology and Extracellular Matrices]

November 24th, 2015 by Abbadi, A., Lauer, M., Swaidani, S., Wang, A., Hascall, V.

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain-hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables, but occur in distinct sheets or rafts, that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has 2 heavy chains (HC1 and HC2) on its chondroitin sulfate (CS) chain. The transesterification transfer of HCs from CS to HA is mediated by tumor-necrosis-factor-induced-gene 6 (TSG-6), which is upregulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage (BAL) of naιve mice, and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.