RESUMO
As a consequence of impaired glucose or fatty acid metabolism, bioenergetic stress in skeletal muscles may trigger myopathy and rhabdomyolysis. Genetic mutations causing loss of function of the LPIN1 gene frequently lead to severe rhabdomyolysis bouts in children, though the metabolic alterations and possible therapeutic interventions remain elusive. Here, we show that lipin1 deficiency in mouse skeletal muscles is sufficient to trigger myopathy. Strikingly, muscle fibers display strong accumulation of both neutral and phospholipids. The metabolic lipid imbalance can be traced to an altered fatty acid synthesis and fatty acid oxidation, accompanied by a defect in acyl chain elongation and desaturation. As an underlying cause, we reveal a severe sarcoplasmic reticulum (SR) stress, leading to the activation of the lipogenic SREBP1c/SREBP2 factors, the accumulation of the Fgf21 cytokine, and alterations of SR-mitochondria morphology. Importantly, pharmacological treatments with the chaperone TUDCA and the fatty acid oxidation activator bezafibrate improve muscle histology and strength of lipin1 mutants. Our data reveal that SR stress and alterations in SR-mitochondria contacts are contributing factors and potential intervention targets of the myopathy associated with lipin1 deficiency.
Assuntos
Estresse do Retículo Endoplasmático/genética , Doenças Musculares/genética , Fosfatidato Fosfatase/genética , Retículo Sarcoplasmático/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Chaperonas Moleculares/farmacologia , Chaperonas Moleculares/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/tratamento farmacológico , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/patologia , Ácido Tauroquenodesoxicólico/uso terapêuticoRESUMO
BACKGROUND AND OBJECTIVE: Premature skin ageing, and skin hyperpigmentation are influenced by exogenous factors, such as ultraviolet radiation and blue light. In this study, we assess the protective effect of a sunscreen (TDF® Blu Voile Sunscreen) in protecting the skin against the harmful effects of blue light irradiation in vivo and through the in situ quantitative and qualitative evaluation of protein carbonylation in human skin explants. METHODOLOGY: The protective effect of the test product against blue light was first evaluated ex vivo on human skin explants. The treated and non-treated explants were exposed to 14 J/cm2 of blue light 460 nm following which the protein carbonylation was evaluated by in situ epifluorescence imaging and separation by high-resolution gel electrophoresis. To determine whether the test product could also protect against the immediate and persistent pigmenting effect of blue light, two randomized in vivo studies were conducted, which included respectively 17 subjects with a skin phototype of IV and V (Fitzpatrick classification) and 22 subjects with a skin phototype of IV, V, and VI (Fitzpatrick classification). The duration of the study for each subject was 2 days (D1 and D2) for immediate observations and 5 days (D1-D5) for persistent observations. Specific zones on the subjects' back were either left non-treated or treated with the test product and were then exposed to a unique dose of blue light 415 nm. The onset of pigmentation between the treated and exposed zones was then assessed relative to the non-exposed treated zone through colorimetric measurements of the Individual Typology Angle (ITAo ). RESULTS: Human skin explants treated with test product showed significantly lower levels of accumulated carbonylated proteins, with a protection of 82%, following exposure to blue light 460 nm. Findings of the in vivo studies also indicated that the test product presented significantly better protective efficacy against immediate and persistent pigmentation induced by blue light 415 nm. CONCLUSION: Hence, it can be concluded that the test product can protect against the oxidative stress as well as the immediate and persistent pigmentation induced by blue light.
CONTEXTE ET OBJECTIF: Le vieillissement prématuré de la peau et l'hyperpigmentation cutanée sont influencés par des facteurs exogènes, tels que les rayons ultraviolets et la lumière bleue. Dans cette étude, nous évaluons l'effet protecteur d'un écran solaire (TDF® Blu Voile Sunscreen) en matière de protection de la peau contre les effets nocifs de l'irradiation à la lumière bleue in vivo et par l'évaluation quantitative et qualitative in situ de la carbonylation des protéines dans des explants cutanés humains. MÉTHODOLOGIE: L'effet protecteur du produit testé contre la lumière bleue a d'abord été évalué ex vivo sur des explants cutanés humains. Les explants traités et non traités ont été exposés à 14 J/cm2 de lumière bleue à 460 nm, après quoi la carbonylation des protéines a été évaluée par imagerie par épifluorescence in situ et séparation par électrophorèse sur gel à haute résolution. Afin de déterminer si le produit testé pouvait également protéger contre la pigmentation immédiate et persistante dues à lumière bleue, deux études in vivo randomisées incluant respectivement 17 sujets ayant un phototype cutané IV et V (classification de Fitzpatrick) et 22 sujets ayant un phototype cutané IV, V et VI (classification de Fitzpatrick) ont été menées. La durée de l'étude pour chaque sujet était de 2 jours (J1 et J2) pour les observations immédiates et de 5 jours (J1 à J5) pour les observations persistantes. Des zones spécifiques du dos des sujets ont été laissées non traitées ou bien traitées avec le produit testé, et ont ensuite été exposées à une dose unique de lumière bleue à 415 nm. L'apparition de la pigmentation entre les zones traitées et exposées a ensuite été évaluée par rapport à la zone traitée non exposée par des mesures colorimétriques de l'angle typologique individuel (Individual Typology Angle, ITAo). RÉSULTATS: Les explants cutanés humains traités avec le produit testé ont montré des taux significativement plus faibles de protéines carbonylées accumulées, avec une protection de 82 %, après une exposition à la lumière bleue à 460 nm. Les résultats des études in vivo ont également indiqué que le produit testé présentait une efficacité protectrice significativement meilleure contre la pigmentation immédiate et persistante induite par la lumière bleue à 415 nm. CONCLUSION: Par conséquent, on peut conclure que le produit testé peut protéger contre le stress oxydatif ainsi que contre la pigmentation immédiate et persistante induite par la lumière bleue.
Assuntos
Hiperpigmentação , Protetores Solares , Humanos , Luz , Pele/efeitos da radiação , Pigmentação da Pele , Protetores Solares/farmacologia , Raios UltravioletaRESUMO
Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance is a major contributor to the age-associated accumulation of damaged proteins that is believed to participate to the age-related decline in cellular function. In this context, quantitative proteomics approaches, including 2-D gel electrophoresis (2-DE)-based methods, represent powerful tools for monitoring the extent of protein oxidative modifications at the proteome level and for identifying the targeted proteins, also referred as to the "oxi-proteome." Previous studies have identified proteins targeted by oxidative modifications during replicative senescence of human WI-38 fibroblasts and myoblasts and have been shown to represent a restricted set within the total cellular proteome that fall in key functional categories, such as energy metabolism, protein quality control, and cellular morphology. To provide mechanistic support into the role of oxidized proteins in the development of the senescent phenotype, untargeted metabolomic profiling is also performed for young and senescent myoblasts and fibroblasts. Metabolomic profiling is indicative of energy metabolism impairment in both senescent myoblasts and fibroblasts, suggesting a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts and fibroblasts.
Assuntos
Envelhecimento , Senescência Celular , Estresse Oxidativo , Proteoma/metabolismo , Animais , Metabolismo Energético , Fibroblastos/metabolismo , Humanos , Redes e Vias Metabólicas , Mioblastos/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Proteômica/métodos , ProteostaseRESUMO
Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes.
Assuntos
Músculo Esquelético , Distrofia Miotônica , Proteínas Serina-Treonina Quinases/genética , Animais , Núcleo Celular/metabolismo , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiopatologia , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , Miotonina Proteína Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisms involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty acid ß-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid ß-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation end product-modified proteins, including 6 enzymes involved in the fatty acid ß-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid ß-oxidation and TCA/urea cycle enzymes.
Assuntos
Envelhecimento/patologia , Biomarcadores/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteômica , Envelhecimento/metabolismo , Animais , Western Blotting , Feminino , Glicosilação , Mitocôndrias Hepáticas/patologia , Oxirredução , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial BidimensionalRESUMO
Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10 days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365 nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D(2)O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.
Assuntos
Cristalinas/metabolismo , Glucose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Western Blotting , Bovinos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , OxirreduçãoRESUMO
Skeletal muscle ageing is characterized by a progressive and dramatic loss of muscle mass and strength leading to decreased muscular function resulting in muscle weakness which is often referred to as sarcopenia. Following the standardisation of "omics" approaches to study the genome (genomics) and the transcriptome (transcriptomics), the study of the proteins encoded by the genome, referred to as proteomics, is a tremendous challenge. Unlike the genome, the proteome varies in response to many physiological or pathological factors. In addition, the proteome is orders of magnitude more complex than the transcriptome due to post-translational modifications, protein oxidation and limited protein degradation. Proteomic studies, including the analysis of protein abundance as well as post-translational modified proteins have been shown to provide valuable information to unravel the key molecular pathways implicated in complex biological processes, such as tissue and organ ageing. In this article, we will describe proteomic approaches for the analysis of protein abundance as well as the specific protein targets for oxidative damage upon oxidative stress and/or during skeletal muscle ageing.
Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Envelhecimento/genética , Animais , Citoesqueleto/fisiologia , Metabolismo Energético/fisiologia , Europa (Continente) , Humanos , Modelos Animais , Proteínas Musculares/genética , Estresse Oxidativo/fisiologia , Sarcopenia/fisiopatologiaRESUMO
Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.
Assuntos
Ferritinas/genética , Ferritinas/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Ferro/metabolismo , Mutação , Apoferritinas/química , Apoferritinas/genética , Apoferritinas/metabolismo , Precipitação Química , Ferritinas/química , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Polimerização , SolubilidadeRESUMO
Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be ß-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.
Assuntos
Carboidratos/química , Glicoproteínas/química , Lectinas/química , Neuraminidase/química , Trypanosoma cruzi/enzimologia , Cristalografia por Raios X , Glicoproteínas/fisiologia , Hidrólise , Lactose/química , Modelos Moleculares , Neuraminidase/fisiologia , Estrutura Terciária de Proteína , alfa-Fetoproteínas/químicaRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.
Assuntos
Fibras Musculares de Contração Rápida/metabolismo , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Fenótipo , Análise de Variância , Animais , Western Blotting , Perfilação da Expressão Gênica , Glicólise/fisiologia , Imuno-Histoquímica , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/patologia , Atrofia Muscular/etiologia , Distrofia Muscular Oculofaríngea/complicações , Proteína I de Ligação a Poli(A)/genética , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Increased protein carbonylation is a hallmark of oxidative stress, protein homeostasis dysregulation and aging in the nervous system and skin. Sensory neurons interact with skin cells and are involved in skin homeostasis. We have previously reported that the 5-octanoyl salicylic acid (C8-SA), a salicylic acid derivative, increased C. elegans lifespan and delayed the accumulation of carbonylated proteins, through the stimulation of autophagy. OBJECTIVES: In this study we aimed to investigate if C8-SA protects human sensory neurons and human skin from extrinsic oxidative stressors as an approach to delay skin aging. METHODS: In vitro reconstituted human epidermis innervated with hiPSc-derived human sensory neurons, as well as ex vivo human organotypic full skin models were used. The fully differentiated sensory neurons were pretreated with C8-SA before oxidative stress induction. Skin explants were maintained in culture and treated topically with C8-SA before the application of urban pollutants. Carbonylated proteins were detected using amino-oxy functionalized fluorophores and quantified. Chaperone mediated autophagy was monitored with LAMP2A immunofluorescence. Inflammation, ROS detoxification and autophagy were assessed by RT-PCR. RESULTS: C8-SA prevented the accumulation of carbonylated proteins, both in human sensory neurons and skin explants. C8-SA stimulated chaperone-mediated autophagy and modulated NRF2 antioxidant response genes, as well as catalase enzymatic activity. CONCLUSIONS: C8-SA acts at two levels to protect skin against oxidative stress: 1) it prevents protein oxidation by stimulating endogenous antioxidant defense and 2) it increases the clearance of oxidized proteins by stimulating chaperone-mediated autophagy. These results suggest that C8-SA maintains skin health in urban polluted environments.
Assuntos
Caenorhabditis elegans , Ácido Salicílico , Animais , Caenorhabditis elegans/metabolismo , Humanos , Estresse Oxidativo , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Células Receptoras Sensoriais/metabolismo , Pele/metabolismoRESUMO
Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 A resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.
Assuntos
Apoferritinas/química , Apoferritinas/genética , Ferro/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Doenças Neurodegenerativas/genética , Sequência de Aminoácidos , Animais , Apoferritinas/metabolismo , Bovinos , Ceruloplasmina/metabolismo , Precipitação Química/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Ferro/farmacologia , Minerais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Oxirredução , Peptídeos/metabolismo , Porosidade , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Deleção de SequênciaRESUMO
Increased iron levels and iron-mediated oxidative stress play an important role in the pathogenesis of many neurodegenerative diseases. The finding that mutations in the ferritin light polypeptide (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy (HF) provided a direct connection between abnormal brain iron storage and neurodegeneration. HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic ferritin inclusion bodies in glia and neurons throughout the CNS and in tissues of multiple organ systems. Here we report that the expression in transgenic mice of a human FTL cDNA carrying a thymidine and cytidine insertion at position 498 (FTL498-499InsTC) leads to the formation of nuclear and cytoplasmic ferritin inclusion bodies. As in HF, ferritin inclusions are seen in glia and neurons throughout the CNS as well as in cells of other organ systems. Our studies show histological, immunohistochemical, and biochemical similarities between ferritin inclusion bodies found in transgenic mice and in individuals with HF. Expression of the transgene in mice leads to a significant decrease in motor performance and a shorter life span, formation of ferritin inclusion bodies, misregulation of iron metabolism, accumulation of ubiquitinated proteins, and incorporation of elements of the proteasome into inclusions. This new transgenic mouse represents a relevant model of HF in which to study the pathways that lead to neurodegeneration in HF, to evaluate the role of iron mismanagement in neurodegenerative disorders, and to evaluate potential therapies for HF and related neurodegenerative diseases.
Assuntos
Ferritinas/genética , Expressão Gênica/genética , Sobrecarga de Ferro/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Animais , Apoferritinas , Comportamento Animal , Encéfalo/patologia , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Atividade Motora/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologiaRESUMO
Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone-protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain.
Assuntos
Apoferritinas/genética , Apoferritinas/fisiologia , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/metabolismo , Ferro/metabolismo , Estresse Oxidativo/genética , Animais , Química Encefálica/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Homeostase/genética , Homeostase/fisiologia , Imuno-Histoquímica , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ferroproteínas não Heme/metabolismoRESUMO
Accumulation of oxidatively damaged proteins is a hallmark of cellular and organismal ageing, and is also a phenotypic feature shared by both replicative senescence and stress-induced premature senescence of human fibroblasts. Moreover, proteins that are building up as oxidized (i.e. the "Oxi-proteome") during ageing and age-related diseases represent a restricted set of cellular proteins, indicating that certain proteins are more prone to oxidative carbonylation and subsequent intracellular accumulation. The occurrence of specific carbonylated proteins upon oxidative stress induced premature senescence of WI-38 human fibroblasts and their follow-up identification have been addressed in this study. Indeed, it was expected that the identification of these proteins would give insights into the mechanisms by which oxidatively damaged proteins could affect cellular function. Among these proteins, some are belonging to the cytoskeleton while others are mainly involved in protein quality control and/or biosynthesis as well as in redox and energy metabolism, the impairment of which has been previously associated with cellular ageing. Interestingly, the majority of these carbonylated proteins were found to belong to functional interaction networks pointing to signalling pathways that have been implicated in the oxidative stress response and subsequent premature senescence.
Assuntos
Senescência Celular , Fibroblastos/metabolismo , Carbonilação Proteica , Proteoma/metabolismo , Linhagem Celular , Fibroblastos/patologia , HumanosRESUMO
Accumulation of oxidized proteins is a hallmark of cellular and organismal aging. Adult muscle stem cell (or satellite cell) replication and differentiation is compromised with age contributing to sarcopenia. However, the molecular events related to satellite cell dysfunction during aging are not completely understood. In the present study we have addressed the potential impact of oxidatively modified proteins on the altered metabolism of senescent human satellite cells. By using a modified proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during the replicative senescence of satellite cells. Inactivation of the proteasome appeared to be a likely contributor to the accumulation of such damaged proteins. Metabolic and functional analyses revealed an impaired glucose metabolism in senescent cells. A metabolic shift leading to increased mobilization of non-carbohydrate substrates such as branched chain amino acids or long chain fatty acids was observed. Increased levels of acyl-carnitines indicated an increased turnover of storage and membrane lipids for energy production. Taken together, these results support a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts.
Assuntos
Metabolismo Energético/fisiologia , Glicólise/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Células Cultivadas , Eletroforese em Gel Bidimensional , Humanos , Estresse Oxidativo , Carbonilação ProteicaRESUMO
Sarcopenia corresponds to the degenerative loss of skeletal muscle mass, quality, and strength associated with ageing and leads to a progressive impairment of mobility and quality of life. However, the cellular and molecular mechanisms involved in this process are not completely understood. A hallmark of cellular and tissular ageing is the accumulation of oxidatively modified (carbonylated) proteins, leading to a decreased quality of the cellular proteome that could directly impact on normal cellular functions. Although increased oxidative stress has been reported during skeletal muscle ageing, the oxidized protein targets, also referred as to the 'oxi-proteome' or 'carbonylome', have not been characterized yet. To better understand the mechanisms by which these damaged proteins build up and potentially affect muscle function, proteins targeted by these modifications have been identified in human rectus abdominis muscle obtained from young and old healthy donors using a bi-dimensional gel electrophoresis-based proteomic approach coupled with immunodetection of carbonylated proteins. Among evidenced protein spots, 17 were found as increased carbonylated in biopsies from old donors comparing to young counterparts. These proteins are involved in key cellular functions such as cellular morphology and transport, muscle contraction and energy metabolism. Importantly, impairment of these pathways has been described in skeletal muscle during ageing. Functional decline of these proteins due to irreversible oxidation may therefore impact directly on the above-mentioned pathways, hence contributing to the generation of the sarcopenic phenotype.
Assuntos
Envelhecimento , Eletroforese em Gel Bidimensional , Músculo Esquelético/metabolismo , Proteoma/análise , Espectrometria de Massas em Tandem , Idoso , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Estresse Oxidativo , Carbonilação ProteicaRESUMO
Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.
Assuntos
Proteínas de Ligação a DNA/genética , Mitocôndrias/metabolismo , Neoplasias/genética , Proteínas Quinases/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Animais , Sobrevivência Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA/genética , Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Mitocôndrias/patologia , Células-Tronco Embrionárias Murinas/metabolismo , Neoplasias/metabolismo , Proteínas Quinases/biossíntese , Pirimidinas/biossíntese , Proteínas Repressoras , Estresse Fisiológico/genética , Fatores de Transcrição/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Ubiquitina-Proteína LigasesRESUMO
OBJECTIVE: To develop a reliable ELISA by use of a unique antigen preparation for serum IgG quantification after vaccination against Campylobacter fetus in cattle. ANIMALS: Twenty-six 24-month-old virgin Hereford heifers and a naturally infected Hereford bull. PROCEDURES: 5 antigens were prepared from a cell suspension of C fetus. Antigen preparations were the same as those reported in the literature, with the exception of antigens that were obtained by detergent solubilization of a C fetus cell suspension. For each antigen preparation, the optimal ELISA conditions for its immobilization were determined. Biotinylated antibodies against bovine immunoglobulins were obtained and used in the ELISA. Two groups of heifers were inoculated with commercial vaccines according to manufacturers' instructions. A control group was included. The immune response of vaccinated heifers and controls was followed for 6 months. RESULTS: Detergent solubilized C fetus antigens resulted in better ELISA performance than other antigen preparations. Antigens were optimally immobilized at neutral pH and low ionic strength. All antigen preparations saturated the well with the same amount of protein. The vaccination schedule that advised a booster resulted in higher antibody titers, which were sustained over a longer period than the other schedule. CONCLUSIONS AND CLINICAL RELEVANCE: In the vaccination of cattle against C fetus, the ELISA we have developed may be used to evaluate serum antibody concentrations in response to various vaccines and vaccination schedules. Our results indicate that it is advisable to include a booster in the immunization protocol.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Campylobacter/veterinária , Campylobacter fetus/imunologia , Doenças dos Bovinos/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Vacinação/veterinária , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/normas , Infecções por Campylobacter/imunologia , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/prevenção & controle , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Immunoblotting/veterinária , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Vacinação/métodosRESUMO
Proteins are involved in key cellular functions and our health and wellness depends on their quality. Accumulation of oxidatively damaged proteins is a hallmark of deleterious processes such increased oxidative stress, chronic inflammation, ageing and age-related diseases. Thus, quantifying and identifying oxidized proteins is a biomarker of choice for monitoring biological ageing and/or the efficiency of anti-oxidant, ant-inflammatory and anti-ageing therapies. However, the absence of reliable tools for analyses has inhibited its establishment as the gold standard for measuring the efficacy of anti-ageing and age related diseases interventions. Herein, we present a novel proteomics technology, named Oxi-DIGE?, which provides a significant improvement in terms of specificity, reproducibility and statistical support for proteomic analysis of carbonylated proteins. In Oxi-DIGE, protein carbonyls are labelled with fluorescent hydrazide probes that bind specifically to carbonyl groups in proteins. Experimental groups (e.g. control and experimental samples) are labelled with different flurophore-binded hydrazides that fluoresce light at different wavelengths, producing different colour fluorescence. Thus samples from different experimental groups are co-resolved on a single 2D gel. Increased accuracy is provided due to: (i) reduced false positives by using an exogenous synthetic fluorescent tag; (ii) multiplexing, that is the possibility to run multiple samples on the same gel, (iii) the use of an internal standard on each gel which eliminates inter-gel variations and provides an increased statistical confidence. In addition, the resolution of the carbonyl groups is improved, forming distinct spots that can be identified by mass spectrometry. ?Patent Application (M. Baraibar, R. Ladouce., B. Friguet, A method for detecting and/or quantifying carbonylated proteins (WO/2012/175519) filed by UPMC and referring to the technology described in this abstract.