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1.
PNAS Nexus ; 3(6): pgae210, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38881840

RESUMEN

Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS operational. At least two spatially distinct mitochondrial subpopulations facilitate OXPHOS in cardiomyocytes, i.e. subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Their intracellular localization below the sarcolemma or buried deep between the sarcomeres suggests different oxygen availability. Here, we studied SSM and IFM isolated from piglet hearts and found significantly lower activities of electron transport chain enzymes and F1FO-ATP synthase in IFM, indicative for compromised energy metabolism. To test the contribution of oxygen availability to this outcome, we ventilated piglets under hyperbaric hyperoxic (HBO) conditions for 240 min. HBO treatment raised OXPHOS enzyme activities in IFM to the level of SSM. Complexome profiling analysis revealed that a high proportion of the F1FO-ATP synthase in the IFM was in a disassembled state prior to the HBO treatment. Upon increased oxygen availability, the enzyme was found to be largely assembled, which may account for the observed increase in OXPHOS complex activities. Although HBO also induced transcription of genes involved in mitochondrial biogenesis, a full proteome analysis revealed only minimal alterations, meaning that HBO-mediated tissue remodeling is an unlikely cause for the observed differences in OXPHOS. We conclude that a previously unrecognized oxygen-regulated mechanism endows cardiac OXPHOS with spatiotemporal plasticity that may underlie the enormous metabolic and contractile adaptability of the heart.

2.
Neuropathol Appl Neurobiol ; 47(7): 1060-1079, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-33974284

RESUMEN

AIMS: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1-/- SNCAA53T double mutant mice. METHODS: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1-/- SNCAA53T double mutant mice beyond 15 months of age. RESULTS: Similar to PD patients, Pink1-/- SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. CONCLUSIONS: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.


Asunto(s)
Mutación/genética , Enfermedad de Parkinson/genética , Proteínas Quinasas/genética , alfa-Sinucleína/genética , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Mitocondrias/genética , Mitocondrias/metabolismo , Neuronas/patología , Enfermedad de Parkinson/patología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Proteínas Quinasas/deficiencia , alfa-Sinucleína/metabolismo
3.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33836590

RESUMEN

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2 By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.


Asunto(s)
Carcinógenos/toxicidad , NADPH Oxidasa 4/genética , Neoplasias/inducido químicamente , Animales , Núcleo Celular/metabolismo , Citosol/metabolismo , Daño del ADN , Inestabilidad Genómica , Ratones , NADPH Oxidasa 4/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Oxidación-Reducción , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal
4.
J Clin Invest ; 131(6)2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33465056

RESUMEN

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Proteínas del Choque Térmico HSP40/genética , Mutación , Atrofia Óptica Hereditaria de Leber/genética , Atrofia Óptica Hereditaria de Leber/metabolismo , Adolescente , Adulto , Línea Celular , Preescolar , Complejo I de Transporte de Electrón/química , Femenino , Técnicas de Inactivación de Genes , Genes Recesivos , Proteínas del Choque Térmico HSP40/deficiencia , Proteínas del Choque Térmico HSP40/metabolismo , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , Penetrancia , Fenotipo , Subunidades de Proteína , Especies Reactivas de Oxígeno/metabolismo , Adulto Joven
5.
Circulation ; 143(9): 935-948, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33307764

RESUMEN

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.


Asunto(s)
Cadenas beta de Integrinas/química , Compuestos de Sulfhidrilo/química , Animales , Cromatografía Líquida de Alta Presión , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Cisteína/química , Disulfuros/análisis , Disulfuros/química , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Sulfuro de Hidrógeno/farmacología , Cadenas beta de Integrinas/metabolismo , Mecanotransducción Celular , Ratones , Resistencia al Corte , Espectrometría de Masas en Tándem , Vasodilatación/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismo
6.
Methods Mol Biol ; 2192: 269-285, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33230779

RESUMEN

Complexome profiling combines blue native gel electrophoresis (BNE) and quantitative mass spectrometry to define an entire protein interactome of a cell, an organelle, or a biological membrane preparation. The method allows the identification of protein assemblies with low abundance and detects dynamic processes of protein complex assembly. Applications of complexome profiling range from the determination of complex subunit compositions, assembly of single protein complexes, and supercomplexes to comprehensive differential studies between patients or disease models. This chapter describes the workflow of complexome profiling from sample preparation, mass spectrometry to data analysis with a bioinformatics tool.


Asunto(s)
Espectrometría de Masas/métodos , Mitocondrias/química , Electroforesis en Gel de Poliacrilamida Nativa/métodos , Línea Celular Tumoral , Cromatografía Liquida/métodos , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Péptidos/química , Péptidos/metabolismo , Espectrometría de Masas en Tándem/métodos
7.
Basic Res Cardiol ; 115(6): 75, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-33258989

RESUMEN

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.


Asunto(s)
Plaquetas/enzimología , Calpaína/sangre , Micropartículas Derivadas de Células/enzimología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 2/enzimología , Angiopatías Diabéticas/enzimología , Células Endoteliales/enzimología , Receptor PAR-1/metabolismo , Vasculitis/enzimología , Proteína ADAM17/metabolismo , Adulto , Animales , Calpaína/genética , Estudios de Casos y Controles , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/genética , Receptor de Proteína C Endotelial/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptor PAR-1/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vasculitis/sangre , Vasculitis/genética
8.
EMBO Mol Med ; 12(11): e12619, 2020 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-32969598

RESUMEN

Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module.


Asunto(s)
Enfermedad de Leigh , Enfermedades Mitocondriales , Alelos , Niño , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Humanos , Enfermedad de Leigh/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Mutación
9.
PPAR Res ; 2020: 8253831, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32695149

RESUMEN

The peroxisome proliferator-activated receptor (PPARγ) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-γ- (IFNγ-) and interleukin-4- (IL4-) polarized macrophages (MΦ) initiates posttranslational modifications of PPARγ, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of MΦ, while the redox status of PPARγ was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPARγ were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFNγ, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPARγ-dependent reporter gene transactivation supporting a functional shift of PPARγ associated with the MΦ phenotype. These data suggest distinct mechanisms for regulating PPARγ function based on the redox state of MΦ.

10.
Neurogenetics ; 21(3): 187-203, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32342250

RESUMEN

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.


Asunto(s)
Proteasas ATP-Dependientes/genética , Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Endopeptidasa Clp/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Enfermedad de Moyamoya/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Citosol/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema Inmunológico , Inflamación , Interferón gamma/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Espectrometría de Masas , Ratones , Mutación , Poli I-C , Pliegue de Proteína , Proteoma , ARN/metabolismo , Transcriptoma
11.
Nat Commun ; 11(1): 1643, 2020 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-32242014

RESUMEN

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Animales , Complejo I de Transporte de Electrón/genética , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mioblastos/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(11): 5923-5930, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32123095

RESUMEN

Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44-/- mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMTΔ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMTΔ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.


Asunto(s)
Neoplasias de la Mama/metabolismo , Familia 2 del Citocromo P450/metabolismo , Linfangiogénesis/fisiología , Prostaglandinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Familia 2 del Citocromo P450/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Ácidos Grasos Insaturados/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangiogénesis/genética , Macrófagos , Ratones , Ratones Noqueados , Monocitos , Procesos Neoplásicos , Proteómica , Transducción de Señal , Receptores Toll-Like , Regulación hacia Arriba
13.
J Cell Mol Med ; 24(6): 3534-3548, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32040259

RESUMEN

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.


Asunto(s)
Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Transducción de Señal , Remodelación Ventricular , Animales , Biocatálisis , Transporte de Electrón , Matriz Extracelular/metabolismo , Masculino , Ratones , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Contracción Miocárdica , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/genética , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Miocardio/ultraestructura , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
14.
Biochim Biophys Acta Bioenerg ; 1861(2): 148137, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31825809

RESUMEN

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.


Asunto(s)
Aldehído Oxidasa/metabolismo , Ciona intestinalis/genética , Expresión Génica , Mitocondrias Cardíacas/enzimología , Especies Reactivas de Oxígeno/metabolismo , Aldehído Oxidasa/genética , Animales , Ciclo del Ácido Cítrico/genética , Transporte de Electrón/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Ratones , Mitocondrias Cardíacas/genética , Consumo de Oxígeno/genética , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo
15.
Am J Hum Genet ; 106(1): 92-101, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31866046

RESUMEN

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.


Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Fibroblastos/patología , Enfermedad de Leigh/etiología , Enfermedades Mitocondriales/etiología , Proteínas Mitocondriales/genética , Mutación , NADH Deshidrogenasa/genética , Alelos , Femenino , Fibroblastos/metabolismo , Humanos , Lactante , Enfermedad de Leigh/patología , Masculino , Enfermedades Mitocondriales/patología , Linaje , Fenotipo
16.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-31547314

RESUMEN

Research on healthy aging shows that lifespan reductions are often caused by mitochondrial dysfunction. Thus, it is very interesting that the deletion of mitochondrial matrix peptidase LonP1 was observed to abolish embryogenesis, while deletion of the mitochondrial matrix peptidase Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (ClpP) prolonged survival. To unveil the targets of each enzyme, we documented the global proteome of LonP1+/- mouse embryonal fibroblasts (MEF), for comparison with ClpP-/- depletion. Proteomic profiles of LonP1+/- MEF generated by label-free mass spectrometry were further processed with the STRING (Search tool for the retrieval of interacting genes) webserver Heidelberg for protein interactions. ClpP was previously reported to degrade Eral1 as a chaperone involved in mitoribosome assembly, so ClpP deficiency triggers the accumulation of mitoribosomal subunits and inefficient translation. LonP1+/- MEF also showed Eral1 accumulation, but no systematic effect on mitoribosomal subunits. In contrast to ClpP-/- profiles, several components of the respiratory complex-I membrane arm, of the glutathione pathway and of lysosomes were accumulated, whereas the upregulation of numerous innate immune defense components was similar. Overall, LonP1, as opposed to ClpP, appears to have no effect on translational machinery, instead it shows enhanced respiratory dysfunction; this agrees with reports on the human CODAS syndrome (syndrome with cerebral, ocular, dental, auricular, and skeletal anomalies) caused by LonP1 mutations.


Asunto(s)
Proteasas ATP-Dependientes/metabolismo , Transporte de Electrón , Fibroblastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Mitocondriales/metabolismo , Ribosomas Mitocondriales/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ratones , Mapas de Interacción de Proteínas , Proteoma/metabolismo
17.
EMBO J ; 38(17): e100938, 2019 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-31328803

RESUMEN

Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.


Asunto(s)
Sustitución de Aminoácidos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Piruvato Quinasa/metabolismo , Animales , Células Cultivadas , Células Endoteliales , Homeostasis , Humanos , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo III/genética , Oxidación-Reducción , Vía de Pentosa Fosfato , Unión Proteica
18.
Int J Mol Sci ; 20(12)2019 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-31248224

RESUMEN

The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Expresión Génica , Monocitos/metabolismo , Células Mieloides/metabolismo , Animales , Aterosclerosis/patología , Metilación de ADN , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Macrófagos/metabolismo , Metionina/metabolismo , Ratones , Ratones Noqueados , Especificidad de Órganos , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología
19.
J Cardiovasc Transl Res ; 12(5): 478-487, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-30963423

RESUMEN

We have shown previously that during myocardial ischemia/reperfusion (MI/R), toll-like receptor 2 (TLR2) signaling regulates connexin 43 (Cx43) subcellular localization and function and dampens arrhythmia formation. We aimed to identify sites capable of TLR2-dependent redox modification within Cx43. Post-ischemic TLR2-/- or wild-type (WT) mouse hearts were analyzed by OxICAT. Cx43 was mutated to exclude redox modification and transfected into HL-1 cardiomyocytes (CM) that were challenged with a TLR2 agonist. We identified Cys260 of Cx43 to be susceptible to reversible oxidation MI/R; TLR2-/- leads to reduced H2O2 production in post-ischemic isolated mitochondria and subsequently reduced oxidation of Cx43 at Cys260. Cx43 was dephosphorylated in WT, while phosphorylation was preserved in TLR2-/-. Mutation of Cx43 (C260A) and lentiviral transfection in HL-1 CM accelerated pacemaker activity and reduced activity after TLR2 ligand stimulation. We here provide evidence for TLR2-dependent reversible oxidation of Cx43 at Cys260, which led to decreased Cx43 phosphorylation and affected CM pacemaker frequency and intercellular communication.


Asunto(s)
Arritmias Cardíacas/metabolismo , Conexina 43/metabolismo , Frecuencia Cardíaca , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Receptor Toll-Like 2/metabolismo , Potenciales de Acción , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Comunicación Celular , Línea Celular , Conexina 43/deficiencia , Conexina 43/genética , Cisteína , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/patología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/patología , Oxidación-Reducción , Fosforilación , Transducción de Señal , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética
20.
Redox Biol ; 21: 101125, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30716538

RESUMEN

AIM: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. RESULTS: TGFß1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. INNOVATION & CONCLUSION: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.


Asunto(s)
Espectrometría de Masas , NADPH Oxidasa 4/metabolismo , Oxidación-Reducción , Animales , Expresión Génica , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , NADPH Oxidasa 4/genética , NADPH Oxidasa 5/genética , NADPH Oxidasa 5/metabolismo , Peroxirredoxinas/metabolismo , Podocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismo
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