Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcino-Embryonic Antigen-related Cell Adhesion Molecule 2 [Metabolism]

November 19th, 2015 by

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with Ceacam2 null mutation (Cc2-/-). The current studies investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in beta cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-beta cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2-/- islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2-/- mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared to wild type, Cc2-/- mice also showed a higher insulin excursion during oral glucose tolerance test. Pretreating with exendin (9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag entero-endocrine cells was increased with siRNA-mediated Ceacam2 downregulation in parallel to increase in Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1 mediated mechanism, independent of confounding metabolic factors.
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Mitochondrial Ribosomal Protein L12 is required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis During Import [Cell Biology]

November 19th, 2015 by

To translate the thirteen mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease (MPP) and a second inefficient or regulated cleavage by mitochondrial intermediary protease (MIP). We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation and longevity pathways to which MRPL12 has been linked.
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Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction [Metabolism]

November 19th, 2015 by

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and L-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which comprises many kinds of enzymes including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(D-alanyl)-L-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-L-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S→N acyl transfer in the intermediate, resulting in peptide formation. Step (ii) is identical to the corresponding reaction in native chemical ligation (NCL), a method of chemical peptide synthesis, whereas step (i) is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in NCL. Together with these findings, the same mechanism-dependent formation of N-acyl-compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate, thus, not only the corresponding peptides but also new types of amide compounds can be formed.

Leishmania donovani encodes a functional selenocysteinyl-tRNA synthase [Gene Regulation]

November 19th, 2015 by Manhas, R., Gowri, V. S., Madhubala, R.

The synthesis of selenocysteine, the 21st amino acid occurs on its transfer RNA (tRNA), tRNASec. tRNASec is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNASec and selenophosphate, to form Sec-tRNASec in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from L. donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ∆selA deletion in E. coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins; which expressed only in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed same sensitivity towards the wild-type and the null mutants suggesting its effect is not through binding to selenoproteins. The 3-D structural comparison indicates that the human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different

Cooperative interactions of oligosaccharide and peptide moieties of a glycopeptide derived from IgE with galectin-9 [Immunology]

November 18th, 2015 by

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N-terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the carboxy-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of oligosaccharides released with peptide-N-glycosidase F contained asialo- and mono-sialylated bi/tri-antennary complex-type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, while the nonbinding fraction contained monosialylated and disialylated biantennary complex-type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.

The A-kinase anchoring protein GSKIP regulates GSK3{beta} activity and controls palatal shelf fusion in mice [Developmental Biology]

November 18th, 2015 by

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind protein kinase A (PKA). A member of this family is Glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knockout mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day E18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser9 starting early in development (E 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5 GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.

{beta}-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting AKT [Signal Transduction]

November 18th, 2015 by

Recent studies reveal that multifunctional protein β-arrestin 2 (Arrb2) modulates cell apoptosis. Survival and various aspects of liver injury were investigated in WT and Arrb2 KO mice after bile duct ligation (BDL). We found that deficiency of Arrb2 enhances survival and attenuates hepatic injury and fibrosis. Following BDL, Arrb2 deficient mice as compared to WT controls displayed a significant reduction of hepatocyte apoptosis as demonstrated by the TUNNEL assay. Following BDL, the levels of phospho-Akt and phospho-GSK3β (glycogen synthase kinase 3β) in the livers were significantly increased in Arrb2 KO compared to WT mice while p-p38 increased in WT but not in Arrb2 deficient mice. Inhibition of GSK3β following BDL decreases hepatic apoptosis and decreased p-p38 in WT mice, but not in Arrb2 KO mice. Activation of Fas receptor with Jo2 reduces phospho-Akt and increases apoptosis in WT cells and WT mice but not in Arrb2 deficient cells and Arrb2 deficient mice. Consistent with direct interaction of Arrb2 with and regulating Akt phosphorylation, the expression of a full length or N terminus but not the C terminus of Arrb2 reduces Akt phosphorylation and coimmuoprecipates with Akt. These results reveal that the protective effect of deficiency of Arrb2 is due to loss of negative regulation of Akt due to BDL and decreased downstream GSK3β and p38 MAPK signaling pathways.

S-Adenosyl-L-methionine modulates CO and NO{middle dot} binding to the human H2S-generating enzyme cystathionine {beta}-synthase [Enzymology]

November 18th, 2015 by Vicente, J. B., Colaco, H. G., Sarti, P., Leandro, P., Giuffre, A.

Cystathionine β-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed by a pyridoxal 5′-phosphate (PLP)-binding catalytic domain, flanked by two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-L-methionine (AdoMet). CO or NO· binding at the ferrous heme negatively modulate the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Herein we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (≈5-fold) and faster association (≈10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO· binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (≈2-fold higher affinity) as compared to CO. Importantly, CO and NO· binding were unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme towards inhibition by exogenous ligands, like CO and NO·. This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically-relevant effectors.
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Lifeguard inhibits Fas ligand-mediated endoplasmic reticulum-calcium release mandatory for apoptosis in type II apoptotic cells [Neurobiology]

November 18th, 2015 by

Death receptors (DRs) are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a DR antagonist mainly expressed in the nervous system that specifically blocks Fas Ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons, and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-inmunoprecipiation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the Endoplasmic Reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis, and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation, and we found that LFG inhibits calcium release from the ER, a process which correlates with LFG blockage of cytochrome C release to the cytosol and caspases activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.
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The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases [Molecular Bases of Disease]

November 18th, 2015 by

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain triggering G-protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves' disease, hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves' disease and other dysfunctions of GPHRs.
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