Suchergebnisse

Significance: In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling. Recent Advances: Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function. Critical Issues: Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward. Future Directions: Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapies. © 2013, Mary Ann Liebert, Inc. ; Spanish Government (CSD2007-00020); (RosasNet, Consolider-Ingenio 2010 programme); CP07/00143 (Miguel Servet programme); PS09/00101; PI10/02136 ; Peer Reviewed

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions. ; DFG (German Research Foundation), by Spanish Government grants PI15/00107 and PT13/0001/0024 (partially funded by the European Union ERDF) ; Peer Reviewed

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer. ; This work was funded by grants from the Spanish "Ministerio de Ciencia, Innovación y Universidades" (MICINN) and ERDF/FEDER funds, SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, RTI2018-093864-B-I00 to M.M., SAF2017-83043-R and B2017/BMD-3724 to S·C., PI15/00107 to A.M.R, the University of the Basque Country UPV/EHU grant GIU16/62) to J.l.R.S. and M.B.R.L., and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT to M.M. ; Peer reviewed

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer. ; This work was funded by grants from the Spanish "Ministerio de Ciencia, Innovación y Universidades" (MICINN) and ERDF/FEDER funds, SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, RTI2018-093864-B-I00 to M.M., SAF2017-83043-R and B2017/BMD-3724 to S·C., PI15/00107 to A.M.R, the University of the Basque Country UPV/EHU grant GIU16/62) to J.l.R.S. and M.B.R.L., and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT to M.M.

Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to 'deactive' form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na/H antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia. ; This research has been financed by Spanish Government grants (partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R.), SAF2015-71521-REDC (Consolredox network, to A.M.-R.), PI12/00875 and PI15/00107 (to A.M.-R), and SAF2013-32223 (to M.G.L.), by a grant from the Fundación Domingo Martínez (Ayuda a la Investigación 2014. Área de Biomedicina y Salud), by Swiss National Science Foundation (SNF) grant 310030_124970/1 to A.B, by a travel grant from the Instituto de Investigación Sanitaria Princesa (Ayuda para estancia breve 2012, to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). P.H.-A. and I.B. are recipients of predoctoral FPU fellowships (AP2010-1219 and AP2012-5621) from the Spanish Government, E.N. is recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM; FPI-UAM2012), and A.M.-R. and J.E. are supported by the I3SNS or "Miguel Servet" programmes (CES12/005 and CP14/00008; ISCIII, Spanish Government; partially funded by FEDER/ERDF). ; Peer Reviewed

Funding: The authors acknowledge the kind gift of HC7 cells by Dr. Fred H. Gage (The Salk Institute for Biological Studies). The excellent assistance of Pedro Marques Vilela in graphical artwork is acknowledged by the authors. Ana Santos and Ana Lourenço were supported by Foundation for Science and Technology, I.P. (FCT, Portugal; fellowships SFRH/BD/ 77903/2011 and SFRH/BD/79308/2011) and are PhD students of the PhD programme in Biomedical Sciences of the University of Algarve. Antonio Martínez-Ruiz is supported by the ISCIII from the Spanish Government (IS3SNS programme, partially funded by FEDER/ERDF). Inês Araújo is supported by FEDER/ERDF funds via Programa Operacional Factores de Competitividade (COMPETE), by national funds via FCT (grants PTDC/NEU-OSD/0473/2012, PTDC/QUI-QFI/ 29319/2017) and by regional funds via CRESC ALGARVE 2020 (grant UID/BIM/04773/2019), by the Algarve Biomedical Center (ABC) and Loulé Municipality. This work was supported by the COST action BM1005 (ENOG: European Network on Gasotransmitters), by the Spanish Government (grants PS09/00101, PI12/00875 and PI15/ 00107 from ISCIII and RTI2018-094203-B-I00 from AEI; co-financed by FEDER/ERDF) and by the Spanish-Portuguese Integrated Action grant PRI-AIBPT-2011-1015/E-10/12. The Proteomics Service of the CBMSO is a member of ProteoRed (PRB3-ISCIII), and is supported by grant PT13/0001/0024 of Spanish Government (cofinanced by FEDER/ ERDF). ; Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R–SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC. ; publishersversion ; published

Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R–SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC. ; Spanish Government (grants PS09/00101, PI12/00875 and PI15/00107 from ISCIII and RTI2018-094203-B-I00 from AEI; co-financed by FEDER/ERDF) and by the Spanish-Portuguese Integrated Action grant PRI-AIBPT-2011-1015/E-10/12.

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction. ; This work has been supported by grants from CONICET (PIP 0202 to M.C.T), Universidad Nacional de Mar del Plata and Agencia Nacional de Promoción Científica y Tecnológica, -Argentina (PICT 1167 to M.C.T and PICT 2421 to C.A.C); from the Spanish Government (PS09/00101 and PI12/00875 to A.M.R., cofinanced by the European Union ERDF), and by the Argentinian-Spanish Integrated Action ES/11/02 / PRIAIBAR- 2011-0782. A.M.R. is supported by the I3SNS programme (ISCIII, Spanish Government). The Proteomics Service of the CBMSO is a member of Proteored (PRB2-ISCIII), and is supported by grants PT13/ 0001/0024 and PT17/0019/0018 of Spanish Government (cofinanced by the European Union ERDF). Work in the laboratory of M.E is supported by grants from the National Institutes of Health (NIH) GM43644, The Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation, USA