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1.
bioRxiv ; 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38645260

RESUMO

Ergothioneine (EGT) is a diet-derived, atypical amino acid that accumulates to high levels in human tissues. Reduced EGT levels have been linked to age-related disorders, including neurodegenerative and cardiovascular diseases, while EGT supplementation is protective in a broad range of disease and aging models in mice. Despite these promising data, the direct and physiologically relevant molecular target of EGT has remained elusive. Here we use a systematic approach to identify how mitochondria remodel their metabolome in response to exercise training. From this data, we find that EGT accumulates in muscle mitochondria upon exercise training. Proteome-wide thermal stability studies identify 3-mercaptopyruvate sulfurtransferase (MPST) as a direct molecular target of EGT; EGT binds to and activates MPST, thereby boosting mitochondrial respiration and exercise training performance in mice. Together, these data identify the first physiologically relevant EGT target and establish the EGT-MPST axis as a molecular mechanism for regulating mitochondrial function and exercise performance.

2.
Curr Opin Chem Biol ; 79: 102435, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38382148

RESUMO

Cysteines are amenable to a diverse set of modifications that exhibit critical regulatory functions over the proteome and thereby control a wide range of cellular processes. Proteomic technologies have emerged as a powerful strategy to interrogate cysteine modifications across the proteome. Recent advancements in enrichment strategies, multiplexing capabilities and increased analytical sensitivity have enabled deeper quantitative cysteine profiling, capturing a substantial proportion of the cysteine proteome. This is complemented by a rapidly growing repertoire of analytical strategies illuminating the diverse landscape of cysteine modifications. Cysteine chemoproteomics technologies have evolved into a powerful strategy to facilitate the development of covalent drugs, opening unprecedented opportunities to target the extensive undrugged proteome. Herein we review recent technological and scientific advances that shape the cysteine proteomics field.


Assuntos
Cisteína , Compostos de Sulfidrila , Cisteína/metabolismo , Proteoma/metabolismo , Proteômica , Oxirredução
3.
bioRxiv ; 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38293190

RESUMO

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III 2 (termed type 2) was identified with a ∼25° rotation of CIII 2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.

4.
bioRxiv ; 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38260676

RESUMO

Zinc is an essential micronutrient that regulates a wide range of physiological processes, principally through Zn 2+ binding to protein cysteine residues. Despite being critical for modulation of protein function, for the vast majority of the human proteome the cysteine sites subject to regulation by Zn 2+ binding remain undefined. Here we develop ZnCPT, a comprehensive and quantitative mapping of the zinc-regulated cysteine proteome. We define 4807 zinc-regulated protein cysteines, uncovering protein families across major domains of biology that are subject to either constitutive or inducible modification by zinc. ZnCPT enables systematic discovery of zinc-regulated structural, enzymatic, and allosteric functional domains. On this basis, we identify 52 cancer genetic dependencies subject to zinc regulation, and nominate malignancies sensitive to zinc-induced cytotoxicity. In doing so, we discover a mechanism of zinc regulation over Glutathione Reductase (GSR) that drives cell death in GSR-dependent lung cancers. We provide ZnCPT as a resource for understanding mechanisms of zinc regulation over protein function.

5.
Nature ; 616(7958): 790-797, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36921622

RESUMO

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular , Ciclo Celular , Ácido Láctico , Humanos , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ácido Láctico/metabolismo , Mitose
6.
Redox Biol ; 59: 102600, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36630820

RESUMO

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.


Assuntos
Lesões Encefálicas , Isquemia Encefálica , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Camundongos , Animais , Humanos , Isquemia , Traumatismo por Reperfusão/terapia , Traumatismo por Reperfusão/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/metabolismo , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/terapia , Acidente Vascular Cerebral/metabolismo , Ácido Succínico/metabolismo , Trombectomia
7.
Redox Biol ; 55: 102429, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35961099

RESUMO

Mitochondria-targeted H2S donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing H2S that decreases oxidative damage. However, the rate of H2S release by current donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases H2S within mitochondria. MitoPerSulf is quickly taken up by mitochondria, where it reacts with endogenous thiols to generate a persulfide intermediate that releases H2S. MitoPerSulf is acutely protective against cardiac IR injury in mice, due to the acute generation of H2S that inhibits respiration at cytochrome c oxidase thereby preventing mitochondrial superoxide production by lowering the membrane potential. Mitochondria-targeted agents that rapidly generate H2S are a new class of therapy for the acute treatment of IR injury.

8.
Redox Biol ; 54: 102368, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35749842

RESUMO

Cell models of cardiac ischemia-reperfusion (IR) injury are essential to facilitate understanding, but current monolayer cell models poorly replicate the in vivo IR injury that occurs within a three-dimensional tissue. Here we show that this is for two reasons: the residual oxygen present in many cellular hypoxia models sustains mitochondrial oxidative phosphorylation; and the loss of lactate from cells into the incubation medium during ischemia enables cells to sustain glycolysis. To overcome these limitations, we incubated isolated adult mouse cardiomyocytes anoxically while inhibiting lactate efflux. These interventions recapitulated key markers of in vivo ischemia, notably the accumulation of succinate and the loss of adenine nucleotides. Upon reoxygenation after anoxia the succinate that had accumulated during anoxia was rapidly oxidized in association with extensive mitochondrial superoxide/hydrogen peroxide production and cell injury, mimicking reperfusion injury. This cell model will enable key aspects of cardiac IR injury to be assessed in vitro.


Assuntos
Miócitos Cardíacos , Traumatismo por Reperfusão , Animais , Modelos Animais de Doenças , Metabolismo Energético , Hipóxia/metabolismo , Isquemia/metabolismo , Lactatos/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Ácido Succínico/metabolismo
9.
J Am Chem Soc ; 144(15): 6791-6801, 2022 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-35380814

RESUMO

Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature.


Assuntos
Complexo I de Transporte de Elétrons , Elétrons , Animais , Catálise , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Mamíferos/metabolismo , NAD/metabolismo , Oxirredução
10.
Cell Chem Biol ; 29(4): 636-649.e14, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-34739852

RESUMO

Mammalian complex I can adopt catalytically active (A-) or deactive (D-) states. A defining feature of the reversible transition between these two defined states is thought to be exposure of the ND3 subunit Cys39 residue in the D-state and its occlusion in the A-state. As the catalytic A/D transition is important in health and disease, we set out to quantify it by measuring Cys39 exposure using isotopic labeling and mass spectrometry, in parallel with complex I NADH/CoQ oxidoreductase activity. To our surprise, we found significant Cys39 exposure during NADH/CoQ oxidoreductase activity. Furthermore, this activity was unaffected if Cys39 alkylation occurred during complex I-linked respiration. In contrast, alkylation of catalytically inactive complex I irreversibly blocked the reactivation of NADH/CoQ oxidoreductase activity by NADH. Thus, Cys39 of ND3 is exposed in complex I during mitochondrial respiration, with significant implications for our understanding of the A/D transition and the mechanism of complex I.


Assuntos
Complexo I de Transporte de Elétrons , NAD , Animais , Catálise , Complexo I de Transporte de Elétrons/metabolismo , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Respiração
11.
Elife ; 102021 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-34939929

RESUMO

The Tricarboxylic Acid (TCA) Cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology, and amino acid homeostasis.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Fumarato Hidratase/metabolismo , Succinato Desidrogenase/metabolismo , Aminoácidos/metabolismo , Animais , Células Cultivadas , Ciclo do Ácido Cítrico/genética , Rim/metabolismo , Metaboloma , Camundongos , Oxirredução , Interferência de RNA
12.
Nat Commun ; 12(1): 707, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514727

RESUMO

Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia-reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the "deactive" state, usually formed only after prolonged inactivity. Despite its tendency to adopt the "deactive" state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.


Assuntos
Complexo I de Transporte de Elétrons/ultraestrutura , Mitocôndrias/patologia , Traumatismo por Reperfusão Miocárdica/patologia , NADH Desidrogenase/genética , Espécies Reativas de Oxigênio/metabolismo , Substituição de Aminoácidos , Animais , Microscopia Crioeletrônica , DNA Mitocondrial/genética , Modelos Animais de Doenças , Resistência à Doença/genética , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Preparação de Coração Isolado , Leucina/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Traumatismo por Reperfusão Miocárdica/genética , NAD/metabolismo , NADH Desidrogenase/metabolismo , NADH Desidrogenase/ultraestrutura , Oxirredução , Mutação Puntual , Prolina/genética
13.
Clin Transl Sci ; 13(3): 539-546, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32027446

RESUMO

The objective of this study was to determine the effect of concomitant alcohol intake on the bioavailability of oxycodone from an oxycodone once-daily (OOD) formulation and an oxycodone twice-daily (OTD) formulation. A phase I, open-label, randomized, crossover alcohol interaction study in 20 healthy volunteers under fasting conditions was conducted. Participants received five treatments, OOD with 240 mL of 0%, 20%, or 40% alcohol; and OTD with 240 mL of 0% or 40% alcohol. Pharmacokinetic parameters did not differ between participants taking OOD with water or with 240 mL of 20% alcohol. There was a slight increase in overall oxycodone absorption from OOD with 40% alcohol but no increase in peak absorption. Oxycodone absorption from OTD showed peak and overall increases with 40% alcohol but maintained a prolonged-release profile. Although it is recommended that alcohol be avoided while taking opioids, there was no evidence of alcohol-induced dose dumping in these oxycodone formulations.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Etanol/efeitos adversos , Interações Alimento-Droga , Oxicodona/administração & dosagem , Administração Oral , Adolescente , Adulto , Consumo de Bebidas Alcoólicas/sangue , Área Sob a Curva , Disponibilidade Biológica , Estudos Cross-Over , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Esquema de Medicação , Etanol/administração & dosagem , Etanol/farmacocinética , Jejum/sangue , Feminino , Absorção Gastrointestinal/efeitos dos fármacos , Meia-Vida , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Oxicodona/farmacocinética , Comprimidos , Adulto Jovem
14.
Free Radic Biol Med ; 147: 37-47, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31811922

RESUMO

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and as a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo.


Assuntos
Espectrometria de Massas em Tandem , Ubiquinona , Cromatografia Líquida , Mitocôndrias/metabolismo , Oxirredução , Ubiquinona/metabolismo
15.
Free Radic Biol Med ; 134: 678-687, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30731114

RESUMO

Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.


Assuntos
Modelos Animais de Doenças , Hormese , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Paraquat/farmacologia , Superóxidos/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Herbicidas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar
16.
J Mol Cell Cardiol ; 123: 88-91, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30118790

RESUMO

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischemic tissue is essential for survival, it also initiates cell death through generation of mitochondrial reactive oxygen species (ROS). Recent work has revealed a novel pathway underlying ROS production at reperfusion in vivo in which the accumulation of succinate during ischemia and its subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting ischemic succinate accumulation, or slowing succinate metabolism at reperfusion, have been shown to be cardioprotective against IR injury. Here, we determined whether ischemic preconditioning (IPC) contributes to cardioprotection by altering kinetics of succinate accumulation and oxidation during IR. Mice were subjected to a 30-minute occlusion of the left anterior descending coronary artery followed by reperfusion, with or without a protective IPC protocol prior to sustained ischemia. We found that IPC had no effect on ischemic succinate accumulation with both control and IPC mice having profound increases in succinate compared to normoxia. Furthermore, after only 1-minute reperfusion succinate was rapidly metabolised returning to near pre-ischemic levels in both groups. We conclude that IPC does not affect ischemic succinate accumulation, or its oxidation at reperfusion.


Assuntos
Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/metabolismo , Oxirredução , Ácido Succínico/metabolismo , Análise de Variância , Animais , Modelos Animais de Doenças , Metabolismo Energético , Masculino , Metaboloma , Metabolômica/métodos , Camundongos , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , Espécies Reativas de Oxigênio/metabolismo
17.
Am J Hum Genet ; 102(4): 557-573, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576218

RESUMO

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.


Assuntos
Domínio Catalítico/genética , Metaloendopeptidases/genética , Mutação/genética , Degeneração Neural/genética , Criança , Pré-Escolar , Derme/patologia , Transporte de Elétrons , Feminino , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Ferro-Enxofre/genética , Imageamento por Ressonância Magnética , Masculino , Mitocôndrias/metabolismo , Linhagem , Proto-Oncogene Mas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Peptidase de Processamento Mitocondrial
18.
Mol Biol Cell ; 28(8): 997-1002, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-28228553

RESUMO

Approximately 70% of mitochondrial precursor proteins are imported from the cytosol via N-terminal presequences, which are cleaved upon exposure to the mitochondrial processing protease MPP in the matrix. Cleaved presequence peptides then need to be efficiently degraded, and impairment of this clearance step, for example, by amyloid ß peptides, causes feedback inhibition of MPP, leading ultimately to accumulation of immature precursor proteins within mitochondria. Degradation of mitochondrial peptides is performed by Cym1 in yeast and its homologue, PreP, in humans. Here we identify the novel mitochondrial matrix protease Ste23 in yeast, a homologue of human insulin-degrading enzyme, which is required for efficient peptide degradation. Ste23 and Cym1 tightly cooperate to ensure the correct functioning of the essential presequence processing machinery.


Assuntos
Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Produtos Finais de Degradação Proteica/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Humanos , Metaloproteases/metabolismo , Mitocôndrias/enzimologia , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Proteólise , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Peptidase de Processamento Mitocondrial
19.
Knee Surg Sports Traumatol Arthrosc ; 24(4): 1180-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25895834

RESUMO

PURPOSE: Acute ankle sprains are frequently accompanied by syndesmotic injuries. These injuries are often overlooked in clinical examinations. The aim of this study was (1) to evaluate the incidence of syndesmotic injuries in acute ankle sprains using MRI, (2) to determine the accuracy of common clinical diagnostic tests, (3) to analyse their inter-rater reliability, and (4) to evaluate the role of clinical symptoms in the diagnosis of syndesmotic injuries. METHODS: A total of 100 patients with acute ankle sprain injury without associated fractures in plane radiographs were enrolled. The clinical assessment was performed by two independent examiners. Local findings, ankle ligament palpation, squeeze test, external rotation test, Drawer test, Cotton test, and the crossed-leg test (two examiners) were compared with MRI results (read by two blinded radiologists) as a reference standard. RESULTS: Ninety-six participants (57% male) met the inclusion criteria. MRI detected a ruptured anterior inferior tibiofibular ligament (AITFL) in 14 patients (15%); 9 partial tears and 5 complete tears were evident. Evidence of pain at rest was found to predict syndesmotic injuries most accurately (p = 0.039). The palpation test over the proximal fibula produced the highest inter-rater correlation (κ = 0.65), but the lowest sensitivity for syndesmotic injuries of 8%. All other clinical tests demonstrated moderate to fair inter-rater reliabilities (κ = 0.37-0.52). Low sensitivity values were found with all clinical tests (13.9-55.6%). CONCLUSION: In this study, clinical examination was insufficient to detect syndesmotic injuries in acute ankle sprains. MRI scanning revealed a syndesmotic lesion in 15% of patients. MRI scanning should be recommended in patients with ongoing pain at rest following ankle sprains. LEVEL OF EVIDENCE: I.


Assuntos
Traumatismos do Tornozelo/diagnóstico , Ligamentos Articulares/lesões , Imageamento por Ressonância Magnética , Palpação , Entorses e Distensões/diagnóstico , Adolescente , Adulto , Feminino , Humanos , Ligamentos Articulares/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Exame Físico , Reprodutibilidade dos Testes , Adulto Jovem
20.
Redox Biol ; 6: 157-168, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26225731

RESUMO

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.


Assuntos
Catalase/antagonistas & inibidores , Éter de Diematoporfirina/farmacologia , Regulação Neoplásica da Expressão Gênica , Fármacos Fotossensibilizantes/farmacologia , Transdução de Sinais , Oxigênio Singlete/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/genética , Caspase 8/metabolismo , Catalase/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Ácido Hipocloroso/metabolismo , Luz , Metaloporfirinas/farmacologia , Camundongos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Oxigênio Singlete/metabolismo , Sulfonas/farmacologia , Taurina/farmacologia , Receptor fas/genética , Receptor fas/metabolismo
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