Search

13 p.-6 fig.-1 tab., It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions., This work was supported in part by Ministerio de Educación y Ciencia Grants BFU2008-04084/BMC and BFU2011-30456, European Union Grant LSHMCT- 2006-518153, and CIBERER Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (an initiative of the ISCIII Instituto de SaludCarlos III) (to J. S.), Comunidad de Madrid Grants S-GEN-0269-2006 and S2010/BMD-2402 MITOLAB-CM (to J. S., E. R., and A. S.), by ISCIII Grant PI080610 (to A. delA), and an institutional grant from the Fundación Ramon Areces to the Centro de Biología Molecular Severo Ochoa.

13 pags, 6 figs, 2 tabs, Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)8 barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His249-Glu250, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by Spanish Ministry of Education and Science Grant BIO2007-67708-C04-04

Mitochondrial alterations are detected in most neurodegenerative disorders and may contribute to the dysfunction and demise of neuronal cells. Because glycogen synthase kinase-3 (GSK-3) is considered to be a critical factor in regulating neuronal cell survival and death, we studied the effects of modulating GSK-3 activity in cultured neurons treated with the mitochondrial inhibitor, rotenone. Interestingly, chronic inhibition of GSK-3 protects against rotenone-induced apoptosis in cultured neuronal cells. In an attempt to elucidate the molecular mechanisms underlying this neuroprotection, we demonstrated that chronic inhibition of GSK-3 reprograms the metabolism of neuronal cells, leading to an enhancement of glycolysis. This effect was accompanied by the induction and accumulation of hexokinase II (HKII) in the mitochondria. Interfering with either the mitochondrial binding of HKII or HKII expression significantly diminished the neuroprotection evoked by GSK-3 inhibition, and importantly, HKII overexpression is sufficient to protect against rotenone-induced cell death. Thus, mitochondrial HKII is a promoter of neuronal survival under the regulation of GSK-3. Furthermore, the neuroprotective effect of HKII may be relevant to neurodegenerative diseases in which glucose hypometabolism and mitochondrial dysfunction are prominent features., This work was supported in part by the Spanish Ministry of Education and Science (Grants SAF 2003-06782 and SAF2006-12782-C03-02) and the Autonomous Government of Madrid (Comunidad Autonoma de Madrid, CAM) through the NEURODEGMODELS project S-SAL-0202-2006. The Centre for Biomedical Research on Rare Diseases (Centro de Investigacion Biomedica en Red sobre Enfermedades Raras) is an initiative supported by the Instituto de Salud Carlos III.

CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic tripeptidyl-peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminopeptidases, probably ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes. This work was supported in part by grants from Spanish Ministerio de Educación y Ciencia and from Instituto de Salud Carlos III (to M. D. V.), by a grant from Spanish Ministerio de Educación y Ciencia (to L. C. A.), by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa, and by a grant from Comunidad de Madrid (to M. D. V. and L. C. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage. This work was supported by grants from the Programa Ramón y Cajal, Comunidad de Madrid, Instituto de Salud Carlos III, Fundación FIPSE, and Fondo de Investigaciones Sanitarias de la SS (to D. L.) and from the Ministerio de Educación y Ciencia, Comunidad de Madrid, and Instituto de Salud Carlos III (to M. D. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases., This work was supported by grants from ViaLactiaBiosciences Ltd. (New Zealand), the BMBF BiotechGenoMik Programme (to P. N. G. and O. V. G.), European Community Project MERG-CT-2004-505242 “BIOMELI,” Spanish Ministry of Education and Science (VEM2004-08559) and Comunidad de Madrid Project (GR/AMB/0690/2004), Gobierno Vasco (to M. Z.), a European Community Marie Curie fellowship, a Spanish Ministerio de Ciencia y Tecnología-Ramón y Cajal contract (to M. F.), and Madrid Autonomic Community and European Social Funds (to M. P.).

Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells. This work was supported by grants from European Union, Ministerio de Educación y Ciencia, Comunidad de Madrid, Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa en Sindrome de Inmunodeficiencia Adquirida (SIDA) del Fondo de Investigaciones Sanitarias (to M. D. V.), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación para la Investigación y la Prevención del Sindrome de Inmunodeficiencia Adquirida en España (to D. L.), and by European Commission Grant QLK2-CT-2001-01167 (to P. M. V. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

© 2014 by The American Society for Biochemistry and Molecular Biology Inc. Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosisdriven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys477-His444-Asp388, rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solventfilled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates., This work was supported, in whole or in part, by National Institutes of Health Grant DE09761/DE/NIDCR. This work was also supported by Polish National Science Center Grant UMO-2012/04/A/NZ1/00051; Ministry of Science and Higher Education of Poland Grant 2137/7.PR-EU/2011/2; European Union Grants FP7-HEALTH-2010-261460 “Gums&Joints,” FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER,” and POIG.02.01.00-12-064/08; Spanish Ministry of Economy and Competitiveness Grants BFU2012-32862 and BIO2013-49320-EXP; Spanish Ministry of Science and Education Grant CSD2006-00015; and National Government of Catalonia Grant 2014SGR9. Funding for data collection was provided in part by the European Synchrotron Radiation Facility (Grenoble, France)

A selective primary culture of fetal rat hepatocytes was established in our laboratory in order to elucidate the molecular mechanisms of action of different factors and conditions on insulin-like growth factor (IGF)-I and -II gene expression during the perinatal period of the rat. In this model we report that, in a serum-free condition and the presence of non-stimulatory doses of insulin, 5–20 mM glucose evoked an increase of IGF-I and -II mRNA abundance. Glucose regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the carbohydrate. The experiment with 2-deoxyglucose suggests that glucose 6-phosphate, but not its further metabolism, is necessary for the induction of IGF transcript abundance in cultured fetal hepatocytes. Finally, the glucose-induced rise in IGF-II mRNA, the main IGF in fetal stages, was mediated by stimulation of gene transcription and increased transcript stability. The results support the idea that IGFs belong to a family of genes that are positively regulated by glucose., This work was supported by Direccion General de Investigacion, Ciencia y Tecnologia, Ministerio de Educacion y Ciencia, Spain, Grants PB94-0030 and PM 97-0017 and by Comunidad Autonoma de Madrid Grant 08.5/0009/1997.

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by Ministerio de Educación y Ciencia, Spain, Grants BFU2004-04591 (to R. G.) and BFU2005-09071 (to J. J. A.), Instituto de Salud Carlos III, Redes de centros RCMN Grant C03/08 (to R. G. and J. J. A.), Temáticas Grant G03/011 (to R. G.), the Centro de Investigación Biomédica en Red de Enfermedades Raras, and National Institutes of Health Grant GM45295 (to L. S. K.).