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1.
J Biol Chem ; 297(4): 101204, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34543622

RESUMO

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

2.
EMBO Rep ; 22(10): e51991, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34351705

RESUMO

Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe class of PBDs-Zellweger spectrum disorder (ZSD)-is caused by mutations in peroxin genes that result in both non-functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter-organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.

3.
Neurogenetics ; 22(4): 297-312, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34345994

RESUMO

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

4.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360575

RESUMO

Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Proteínas/química , Proteínas/fisiologia , Animais , Humanos
5.
Redox Biol ; 45: 102054, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34198070

RESUMO

Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.


Assuntos
Comportamento Exploratório , Atividade Motora , Oxirredutases/genética , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nestina , Neurônios
6.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281173

RESUMO

Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.


Assuntos
Neoplasias da Mama/metabolismo , Epóxido Hidrolases/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Epóxido Hidrolases/genética , Compostos de Epóxi/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Deleção de Genes , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo
7.
Biol Chem ; 402(8): 925-935, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34261205

RESUMO

Reactive oxygen species are produced by a number of stimuli and can lead both to irreversible intracellular damage and signaling through reversible post-translational modification. It is unclear which factors contribute to the sensitivity of cysteines to redox modification. Here, we used statistical and machine learning methods to investigate the influence of different structural and sequence features on the modifiability of cysteines. We found several strong structural predictors for redox modification. Sensitive cysteines tend to be characterized by higher exposure, a lack of secondary structure elements, and a high number of positively charged amino acids in their close environment. Our results indicate that modified cysteines tend to occur close to other post-translational modifications, such as phosphorylated serines. We used these features to create models and predict the presence of redox-modifiable cysteines in human mitochondrial complex I as well as make novel predictions regarding redox-sensitive cysteines in proteins.

8.
Artigo em Inglês | MEDLINE | ID: mdl-34312900

RESUMO

AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

9.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836590

RESUMO

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.


Assuntos
Carcinógenos/toxicidade , NADPH Oxidase 4/genética , Neoplasias/induzido quimicamente , Animais , Núcleo Celular/metabolismo , Citosol/metabolismo , Dano ao DNA , Instabilidade Genômica , Camundongos , NADPH Oxidase 4/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Oxirredução , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais
10.
Redox Biol ; 41: 101951, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33831709

RESUMO

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.


Assuntos
Apoptose , Isotiocianatos , Animais , Humanos , Camundongos , Mitocôndrias , Ratos , Espécies Reativas de Oxigênio , Sulfóxidos
11.
J Immunol ; 206(8): 1890-1900, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33731338

RESUMO

Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP-null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome, but also display immune system alterations. However, the molecular mechanisms and signaling pathways underlying immunological changes in CLPP-null mice remain unclear. In this study, we report the steady-state activation of type I IFN signaling and antiviral gene expression in CLPP-deficient cells and tissues, resulting in marked resistance to RNA and DNA virus infection. Depletion of the cyclic GMP-AMP (cGAS)-stimulator of IFN genes (STING) DNA sensing pathway reduces steady-state IFN-I signaling and abrogates the broad antiviral phenotype of CLPP-null cells. Moreover, we report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene expression, implicating mtDNA stress as the driver of IFN-I signaling in CLPP-null mice. Our work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have implications for understanding Perrault syndrome and other human diseases involving CLPP dysregulation.


Assuntos
Interferon beta , Nucleotidiltransferases , Animais , DNA Mitocondrial/genética , Endopeptidase Clp/genética , Humanos , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Nucleotídeos Cíclicos , Nucleotidiltransferases/metabolismo , Peptídeo Hidrolases
12.
Free Radic Biol Med ; 168: 155-167, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33789124

RESUMO

Previous studies suggested that reactive oxygen species (ROS) produced by NADPH oxidase 4 (Nox4) affect the processing of neuropathic pain. However, mechanisms underlying Nox4-dependent pain signaling are incompletely understood. In this study, we aimed to identify novel Nox4 downstream interactors in the nociceptive system. Mice lacking Nox4 specifically in sensory neurons were generated by crossing Advillin-Cre mice with Nox4fl/fl mice. Tissue-specific deletion of Nox4 in sensory neurons considerably reduced mechanical hypersensitivity and neuronal action potential firing after peripheral nerve injury. Using a proteomic approach, we detected various proteins that are regulated in a Nox4-dependent manner after injury, including the small calcium-binding protein S100A4. Immunofluorescence staining and Western blot experiments confirmed that S100A4 expression is massively up-regulated in peripheral nerves and dorsal root ganglia after injury. Furthermore, mice lacking S100A4 showed increased mechanical hypersensitivity after peripheral nerve injury and after delivery of a ROS donor. Our findings suggest that S100A4 expression is up-regulated after peripheral nerve injury in a Nox4-dependent manner and that deletion of S100A4 leads to an increased neuropathic pain hypersensitivity.


Assuntos
Neuralgia , Traumatismos dos Nervos Periféricos , Animais , Gânglios Espinais , Hiperalgesia/genética , Camundongos , NADPH Oxidase 4/genética , Neuralgia/genética , Traumatismos dos Nervos Periféricos/genética , Proteômica , Proteína A4 de Ligação a Cálcio da Família S100 , Regulação para Cima
13.
Biochim Biophys Acta Bioenerg ; 1862(7): 148411, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722514

RESUMO

Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.


Assuntos
Bases de Dados Factuais , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Software , Humanos , Proteoma/análise
15.
J Antimicrob Chemother ; 76(3): 626-634, 2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33201995

RESUMO

OBJECTIVES: This study analysed the novel carbapenem-hydrolysing class D ß-lactamase OXA-822 identified in the clinical Acinetobacter calcoaceticus isolate AC_2117. METHODS: WGS was employed for identification of ß-lactamases. Micro-broth dilution was used for evaluation of antibiotic susceptibility of AC_2117 and transformants containing blaOXA-822. After heterologous purification of OXA-822, OXA-359 and OXA-213, enzyme kinetics were determined using spectrometry. The effect of OXA-822 upon meropenem treatment was analysed in the Galleria mellonella in vivo infection model. RESULTS: OXA-822 is a member of the intrinsic OXA-213-like family found in A. calcoaceticus and Acinetobacter pittii. Amino acid sequence similarity to the nearest related OXA-359 was 97%. Production of OXA-822, OXA-359 and OXA-213 in Acinetobacter baumannii ATCC® 19606T resulted in elevated MICs for carbapenems (up to 16-fold). Penicillinase activity of the purified OXA-822 revealed high KM values, in the millimolar range, combined with high turnover numbers. OXA-822 showed the highest affinity to carbapenems, but affinity to imipenem was ∼10-fold lower compared with other carbapenems. Molecular modelling revealed that imipenem does not interact with a negatively charged side chain of OXA-822, as doripenem does, leading to the lower affinity. Presence of OXA-822 decreased survival of infected Galleria mellonella larvae after treatment with meropenem. Only 52.7% ±â€Š7.7% of the larvae survived after 24 h compared with 90.9% ±â€Š3.7% survival in the control group. CONCLUSIONS: The novel OXA-822 from a clinical A. calcoaceticus isolate displayed penicillinase and carbapenemase activity in vitro, elevated MICs in different species and decreased carbapenem susceptibility in A. baumannii in vivo.

16.
Circulation ; 143(9): 935-948, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33307764

RESUMO

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.

17.
Methods Mol Biol ; 2192: 269-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230779

RESUMO

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.


Assuntos
Espectrometria de Massas/métodos , Mitocôndrias/química , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Espectrometria de Massas em Tandem/métodos
18.
Basic Res Cardiol ; 115(6): 75, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33258989

RESUMO

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.

19.
Nat Commun ; 11(1): 6425, 2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33349630

RESUMO

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here we report a regenerative paradigm that we call enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons compared to EE or a conditioning injury alone, propelling axon growth well beyond the spinal injury site. Mechanistically, we established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI leading to improved functional recovery. These studies provide a paradigm that drives the regenerative ability of sensory neurons offering a potential redox-dependent regenerative model for mechanistic and therapeutic discoveries.


Assuntos
Regeneração Nervosa , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Transdução de Sinais , Traumatismos da Medula Espinal/fisiopatologia , Animais , Axônios/patologia , Axotomia , Gânglios Espinais/patologia , Camundongos Endogâmicos C57BL , NADPH Oxidase 2/metabolismo , Crescimento Neuronal , Plasticidade Neuronal , Oxirredução , Fosforilação , Regiões Promotoras Genéticas/genética , Proteína Quinase C/metabolismo , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Nervo Isquiático/fisiopatologia , Regulação para Cima
20.
EMBO Mol Med ; 12(11): e12619, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32969598

RESUMO

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.

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