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1.
Biochem J ; 480(3): 197-217, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-36625375

RESUMO

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. During infection in vivo, Candida albicans must adapt to host microenvironments and this adaptive response is crucial for the survival of this organism, as it facilitates the effective assimilation of alternative carbon sources others than glucose. We performed a global proteomic analysis on the global changes in protein abundance in response to changes in micronutrient levels, and, in parallel, explored changes in the intracellular redox and metabolic status of the cells. We show here that each of the carbon sources considered - glucose, acetate and lactate - induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive response involving the adaptation of metabolic pathways, but also a complete remodeling of thiol-dependent antioxidant defenses. Protein S-thiolation and the overproduction of reduced glutathione are two components of the response to high glucose concentration. In the presence of acetate, glutathione-dependent oxidative stress occurs, reduced thiol groups bind to proteins, and glutathione is exported out of the cells, these changes probably being triggered by an increase in glutathione-S-transferases. Overall, our results suggest that the role of cellular redox status regulation and defenses against oxidative stress, including the thiol- and glutathione-dependent response, in the adaptive response of C. albicans to alternative carbon sources should be reconsidered.


Assuntos
Candida albicans , Carbono , Candida albicans/metabolismo , Carbono/metabolismo , Proteômica , Proteínas Fúngicas/metabolismo , Oxirredução , Glutationa/metabolismo , Glucose/metabolismo , Acetatos/metabolismo
2.
J Proteome Res ; 22(3): 996-1002, 2023 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-36748112

RESUMO

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[12C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[12C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae, as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[12C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[12C]-labeled bacteria (Escherichia coli) and the worm proteome analyzed for 12C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of 12C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics.


Assuntos
Caenorhabditis elegans , Proteoma , Animais , Humanos , Caenorhabditis elegans/genética , Proteoma/análise , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Linhagem Celular , Isótopos , Marcação por Isótopo/métodos
3.
J Cell Mol Med ; 27(15): 2228-2238, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37415307

RESUMO

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins.


Assuntos
Síndrome de Down , Trombocitopenia , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Síndrome de Down/metabolismo , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Hemorragia/metabolismo , Trombocitopenia/metabolismo , Quinases Dyrk
4.
Hum Mol Genet ; 29(12): 1969-1985, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32068834

RESUMO

Vitamin B12 or cobalamin (Cbl) metabolism can be affected by genetic defects leading to defective activity of either methylmalonyl-CoA mutase or methionine synthase or both enzymes. Patients usually present with a wide spectrum of pathologies suggesting that various cellular processes could be affected by modifications in gene expression. We have previously demonstrated that these genetic defects are associated with subcellular mislocalization of RNA-binding proteins (RBP) and subsequent altered nucleo-cytoplasmic shuttling of mRNAs. In order to characterize the possible changes of gene expression in these diseases, we have investigated global gene expression in fibroblasts from patients with cblC and cblG inherited disorders by RNA-seq. The most differentially expressed genes are strongly associated with developmental processes, neurological, ophthalmologic and cardiovascular diseases. These associations are consistent with the clinical presentation of cblC and cblG disorders. Multivariate analysis of transcript processing revaled splicing alterations that led to dramatic changes in cytoskeleton organization, response to stress, methylation of macromolecules and RNA binding. The RNA motifs associated with this differential splicing reflected a potential role of RBP such as HuR and HNRNPL. Proteomic analysis confirmed that mRNA processing was significantly disturbed. This study reports a dramatic alteration of gene expression in fibroblasts of patients with cblC and cblG disorders, which resulted partly from disturbed function of RBP. These data suggest to evaluate the rescue of the mislocalization of RBP as a potential strategy in the treatment of severe cases who are resistant to classical treatments with co-enzyme supplements.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Oxirredutases/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Processamento Alternativo/genética , Linhagem Celular , Proteína Semelhante a ELAV 1/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Proteômica , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia
6.
Genomics ; 113(6): 4352-4360, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34793950

RESUMO

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.


Assuntos
Percepção de Quorum , Saccharomycetales , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteômica , Saccharomycetales/genética , Saccharomycetales/metabolismo
7.
J Proteome Res ; 20(3): 1476-1487, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33573382

RESUMO

Simple light isotope metabolic labeling (SLIM labeling) is an innovative method to quantify variations in the proteome based on an original in vivo labeling strategy. Heterotrophic cells grown in U-[12C] as the sole source of carbon synthesize U-[12C]-amino acids, which are incorporated into proteins, giving rise to U-[12C]-proteins. This results in a large increase in the intensity of the monoisotope ion of peptides and proteins, thus allowing higher identification scores and protein sequence coverage in mass spectrometry experiments. This method, initially developed for signal processing and quantification of the incorporation rate of 12C into peptides, was based on a multistep process that was difficult to implement for many laboratories. To overcome these limitations, we developed a new theoretical background to analyze bottom-up proteomics data using SLIM-labeling (bSLIM) and established simple procedures based on open-source software, using dedicated OpenMS modules, and embedded R scripts to process the bSLIM experimental data. These new tools allow computation of both the 12C abundance in peptides to follow the kinetics of protein labeling and the molar fraction of unlabeled and 12C-labeled peptides in multiplexing experiments to determine the relative abundance of proteins extracted under different biological conditions. They also make it possible to consider incomplete 12C labeling, such as that observed in cells with nutritional requirements for nonlabeled amino acids. These tools were validated on an experimental dataset produced using various yeast strains of Saccharomyces cerevisiae and growth conditions. The workflows are built on the implementation of appropriate calculation modules in a KNIME working environment. These new integrated tools provide a convenient framework for the wider use of the SLIM-labeling strategy.


Assuntos
Proteoma , Proteômica , Sequência de Aminoácidos , Marcação por Isótopo , Espectrometria de Massas
8.
J Pathol ; 248(3): 291-303, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30734924

RESUMO

The pathomechanisms that associate a deficit in folate and/or vitamin B12 and the subsequent hyperhomocysteinemia with pathological brain ageing are unclear. We investigated the homocysteinylation of microtubule-associated proteins (MAPs) in brains of patients with Alzheimer's disease or vascular dementia, and in rats depleted in folate and vitamin B12, Cd320 KO mice with selective B12 brain deficiency and H19-7 neuroprogenitors lacking folate. Compared with controls, N-homocysteinylated tau and MAP1 were increased and accumulated in protein aggregates and tangles in the cortex, hippocampus and cerebellum of patients and animals. N-homocysteinylation dissociated tau and MAPs from ß-tubulin, and MS analysis showed that it targets lysine residues critical for their binding to ß-tubulin. N-homocysteinylation increased in rats exposed to vitamin B12 and folate deficit during gestation and lactation and remained significantly higher when they became 450 days-old, despite returning to normal diet at weaning, compared with controls. It was correlated with plasma homocysteine (Hcy) and brain expression of methionine tRNAsynthetase (MARS), the enzyme required for the synthesis of Hcy-thiolactone, the substrate of N-homocysteinylation. Experimental inactivation of MARS prevented the N-homocysteinylation of tau and MAP1, and the dissociation of tau and MAP1 from ß-tubulin and PSD95 in cultured neuroprogenitors. In conclusion, increased N-homocysteinylation of tau and MAP1 is a mechanism of brain ageing that depends on Hcy concentration and expression of MARS enzyme. Its irreversibility and cumulative occurrence throughout life may explain why B12 and folate supplementation of the elderly has limited effects, if any, to prevent pathological brain ageing and cognitive decline. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Doença de Alzheimer/patologia , Demência Vascular/patologia , Hiper-Homocisteinemia/patologia , Proteínas tau/metabolismo , Envelhecimento/fisiologia , Doença de Alzheimer/metabolismo , Animais , Autopsia/métodos , Encéfalo/metabolismo , Encéfalo/patologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Demência Vascular/metabolismo , Feminino , Humanos , Camundongos Knockout , Ratos
9.
Mol Cell Proteomics ; 16(11): 2017-2031, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28821603

RESUMO

Many quantitative proteomics strategies rely on in vivo metabolic incorporation of amino acids with modified stable isotope profiles into proteins. These methods give rise to multiple ions for each peptide, with possible distortion of the isotopolog distribution, making the overall analytical process complex. We validated an alternative strategy, simple light isotope metabolic labeling (SLIM-labeling), which alleviates many of these problems. SLIM-labeling is based on the in vivo reduction of the isotopic composition of proteins using metabolic precursors with a unique light isotope composition to label all amino acids. This brings a new dimension to in-depth, high resolution MS-based quantitative proteomics. Here, we describe a 12C-based SLIM-labeling strategy using U-[12C]-glucose as the metabolic precursor of all amino acids in the pathogenic yeast Candida albicans Monoisotopic ion intensity increased exponentially following 12C enrichment, substantially improving peptide identification scores and protein sequence coverage in bottom-up analyses. Multiplexing samples of 12C composition varying from natural abundance (98.93%) to 100% makes it possible to address relative quantification issues, keeping all the critical information for each peptide within a single isotopolog cluster. We applied this method to measure, for the first time, protein turnover at the proteome scale in Candida albicans and its modulation by inhibitors of the proteasome and vacuolar protein degradation systems.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Marcação por Isótopo/métodos , Proteômica/métodos , Isótopos de Carbono/metabolismo , Proteoma , Espectrometria de Massas em Tandem
10.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt A): 997-1009, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29329987

RESUMO

Friedreich's ataxia (FRDA) represents the most frequent type of autosomal-recessively inherited ataxia and is caused by the deficiency of frataxin, a mitochondrial protein. It is known that frataxin-deficiency leads to alterations in cellular and mitochondrial iron metabolism and impacts in the cell physiology at several levels. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. Currently, cellular antioxidant defense is dysregulated when frataxin is deficient, which exacerbates oxidative damage in FRDA. Moreover, alterations in lipid metabolism have been observed in several models of the disease. To better understand the biochemical sequelae of frataxin reduction, global protein expression analysis was performed using quantitative proteomic experiments in Friedreich's ataxia patient-derived B-lymphocytes as compared to controls. We were able to confirm a subset of changes in these cells and importantly, we observed previously unreported signatures of protein expression. Among the novel protein signatures that we have identified, the decrease in CHCHD4 might partly explain some aspects of the molecular pathogenesis of FRDA. The identification of a core set of proteins changing in the FRDA pathogenesis is a useful tool in trying to decipher the function(s) of frataxin in order to clarify the mitochondrial metabolic disease process.


Assuntos
Linfócitos B/metabolismo , Ataxia de Friedreich/metabolismo , Proteoma/metabolismo , Proteômica , Linfócitos B/patologia , Ataxia de Friedreich/patologia , Humanos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial
11.
Mol Cell Proteomics ; 15(7): 2308-23, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27125826

RESUMO

Protein glycolysation is an essential posttranslational modification in eukaryotic cells. In pathogenic yeasts, it is involved in a large number of biological processes, such as protein folding quality control, cell viability and host/pathogen relationships. A link between protein glycosylation and apoptosis was established by the analysis of the phenotypes of oligosaccharyltransferase mutants in budding yeast. However, little is known about the contribution of glycosylation modifications to the adaptive response to apoptosis inducers. The cysteine protease metacaspase Mca1p plays a key role in the apoptotic response in Candida albicans triggered by the quorum sensing molecule farnesol. We subjected wild-type and mca1-deletion strains to farnesol stress and then studied the early phase of apoptosis release in quantitative glycoproteomics and glycomics experiments on cell-free extracts essentially devoid of cell walls. We identified and characterized 62 new glycosylated peptides with their glycan composition: 17 N-glycosylated, 45 O-glycosylated, and 81 additional sites of N-glycosylation. They were found to be involved in the control of protein folding, cell wall integrity and cell cycle regulation. We showed a general increase in the O-glycosylation of proteins in the mca1 deletion strain after farnesol challenge. We identified 44 new putative protein substrates of the metacaspase in the glycoprotein fraction enriched on concanavalin A. Most of these substrates are involved in protein folding or protein resolubilization and in mitochondrial functions. We show here that key Mca1p substrates, such as Cdc48p or Ssb1p, involved in degrading misfolded glycoproteins and in the protein quality control system, are themselves differentially glycosylated. We found putative substrates, such as Bgl2p (validated by immunoblot), Srb1p or Ugp1p, that are involved in the biogenesis of glycans. Our findings highlight a new role of the metacaspase in amplifying cell death processes by affecting several critical protein quality control systems through the alteration of the protein glycosylation machinery.Data are available via ProteomeXchange with identifier PXD003677.


Assuntos
Candida albicans/efeitos dos fármacos , Caspases/genética , Farneseno Álcool/farmacologia , Proteínas Fúngicas/metabolismo , Proteômica/métodos , Apoptose , Candida albicans/genética , Candida albicans/metabolismo , Caspases/metabolismo , Ciclo Celular , Proteínas Fúngicas/genética , Deleção de Genes , Glicômica/métodos , Glicosilação , Dobramento de Proteína
12.
Biochem J ; 474(7): 1175-1193, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28167699

RESUMO

The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H2O2, or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell.


Assuntos
Glutationa/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Diamida/farmacologia , Ontologia Genética , Dissulfeto de Glutationa/farmacologia , Peróxido de Hidrogênio/farmacologia , Ferro/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Oxirredução , Proteômica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Proc Natl Acad Sci U S A ; 112(47): 14652-7, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26553998

RESUMO

In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.


Assuntos
Ritmo Circadiano , Ferritinas/metabolismo , Homeostase , Ferro/metabolismo , Água do Mar/microbiologia , Western Blotting , Precipitação Química , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , Ferritinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Homeostase/efeitos dos fármacos , Homeostase/genética , Homeostase/efeitos da radiação , Ferro/farmacologia , Proteínas de Ligação ao Ferro/metabolismo , Cinética , Luz , Espectrometria de Massas , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Fitoplâncton/efeitos dos fármacos , Fitoplâncton/genética , Fitoplâncton/crescimento & desenvolvimento , Fitoplâncton/metabolismo , Transcriptoma/genética
14.
J Biol Chem ; 291(35): 18072-83, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27402852

RESUMO

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.


Assuntos
Monofosfato de Adenosina/química , Glicogênio Fosforilase Encefálica/química , Proteínas do Tecido Nervoso/química , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Glicogênio Fosforilase Encefálica/genética , Glicogênio Fosforilase Encefálica/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Doença de Lafora/genética , Doença de Lafora/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos
15.
Hum Mol Genet ; 24(17): 4916-32, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26060189

RESUMO

Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.


Assuntos
Proteínas Sanguíneas/metabolismo , Conectina/metabolismo , Distrofias Musculares/metabolismo , Proteômica , Adolescente , Adulto , Animais , Biomarcadores , Estudos de Casos e Controles , Criança , Pré-Escolar , Conectina/sangue , Creatina Quinase , Modelos Animais de Doenças , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos mdx , Distrofias Musculares/sangue , Distrofias Musculares/terapia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/metabolismo , Proteômica/métodos , Resultado do Tratamento , Adulto Jovem
16.
Mol Cell Proteomics ; 14(1): 93-108, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25348831

RESUMO

Manipulating the apoptotic response of Candida albicans may help in the control of this opportunistic pathogen. The metacaspase Mca1p has been described as a key protease for apoptosis in C. albicans but little is known about its cleavage specificity and substrates. We therefore initiated a series of studies to describe its function. We used a strain disrupted for the MCA1 gene (mca1Δ/Δ) and compared its proteome to that of a wild-type isogenic strain, in the presence and absence of a known inducer of apoptosis, the quorum-sensing molecule farnesol. Label-free and TMT labeling quantitative proteomic analyses showed that both mca1 disruption and farnesol treatment significantly affected the proteome of the cells. The combination of both conditions led to an unexpected biological response: the strong overexpression of proteins implicated in the general stress. We studied sites cleaved by Mca1p using native peptidomic techniques, and a bottom-up approach involving GluC endoprotease: there appeared to be a "K/R" substrate specificity in P1 and a "D/E" specificity in P2. We also found 77 potential substrates of Mca1p, 13 of which validated using the most stringent filters, implicated in protein folding, protein aggregate resolubilization, glycolysis, and a number of mitochondrial functions. An immunoblot assay confirmed the cleavage of Ssb1p, a member of the HSP70 family of heat-shock proteins, in conditions where the metacaspase is activated. These various results indicate that Mca1p is involved in a limited and specific proteolysis program triggered by apoptosis. One of the main functions of Mca1p appears to be the degradation of several major heat-shock proteins, thereby contributing to weakening cellular defenses and amplifying the cell death process. Finally, Mca1p appears to contribute significantly to the control of mitochondria biogenesis and degradation. Consequently, Mca1p may be a link between the extrinsic and the intrinsic programmed cell death pathways in C. albicans.


Assuntos
Apoptose/fisiologia , Candida albicans/metabolismo , Caspases/metabolismo , Proteínas Fúngicas/metabolismo , Candida albicans/genética , Caspases/genética , Farneseno Álcool/farmacologia , Proteínas Fúngicas/genética , Proteoma , Serina Endopeptidases/metabolismo
17.
BMC Genomics ; 17: 319, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-27142620

RESUMO

BACKGROUND: Low iron bioavailability is a common feature of ocean surface water and therefore micro-algae developed original strategies to optimize iron uptake and metabolism. The marine picoeukaryotic green alga Ostreococcus tauri is a very good model for studying physiological and genetic aspects of the adaptation of the green algal lineage to the marine environment: it has a very compact genome, is easy to culture in laboratory conditions, and can be genetically manipulated by efficient homologous recombination. In this study, we aimed at characterizing the mechanisms of iron assimilation in O. tauri by combining genetics and physiological tools. Specifically, we wanted to identify and functionally characterize groups of genes displaying tightly orchestrated temporal expression patterns following the exposure of cells to iron deprivation and day/night cycles, and to highlight unique features of iron metabolism in O. tauri, as compared to the freshwater model alga Chalamydomonas reinhardtii. RESULTS: We used RNA sequencing to investigated the transcriptional responses to iron limitation in O. tauri and found that most of the genes involved in iron uptake and metabolism in O. tauri are regulated by day/night cycles, regardless of iron status. O. tauri lacks the classical components of a reductive iron uptake system, and has no obvious iron regulon. Iron uptake appears to be copper-independent, but is regulated by zinc. Conversely, iron deprivation resulted in the transcriptional activation of numerous genes encoding zinc-containing regulation factors. Iron uptake is likely mediated by a ZIP-family protein (Ot-Irt1) and by a new Fea1-related protein (Ot-Fea1) containing duplicated Fea1 domains. The adaptation of cells to iron limitation involved an iron-sparing response tightly coordinated with diurnal cycles to optimize cell functions and synchronize these functions with the day/night redistribution of iron orchestrated by ferritin, and a stress response based on the induction of thioredoxin-like proteins, of peroxiredoxin and of tesmin-like methallothionein rather than ascorbate. We briefly surveyed the metabolic remodeling resulting from iron deprivation. CONCLUSIONS: The mechanisms of iron uptake and utilization by O. tauri differ fundamentally from those described in C. reinhardtii. We propose this species as a new model for investigation of iron metabolism in marine microalgae.


Assuntos
Clorófitas/metabolismo , Eucariotos/metabolismo , Ferro/metabolismo , Fitoplâncton/metabolismo , Adaptação Biológica , Clorófitas/classificação , Clorófitas/genética , Análise por Conglomerados , Cobre/metabolismo , Eucariotos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Compostos de Ferro/metabolismo , Oxirredução , Fotoperíodo , Filogenia , Fitoplâncton/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Estresse Fisiológico , Transcriptoma
18.
Hum Mol Genet ; 23(4): 968-79, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24105471

RESUMO

Friedreich's ataxia (FRDA), the most common hereditary ataxia, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and a high risk of diabetes. FRDA is caused by abnormally low levels of frataxin, a highly conserved mitochondrial protein. Drosophila has been previously successfully used to model FRDA in various cell types, including neurons and glial cells. Here, we report the development of a Drosophila cardiac model of FRDA. In vivo heart imaging revealed profound impairments in heart function in frataxin-depleted Drosophila, including a strong increase in end-systolic and end-diastolic diameters and a decrease in fractional shortening (FS). These features, reminiscent of pathological phenotypes in humans, are fully rescued by complementation with human frataxin, suggesting conserved cardiac functions of frataxin between the two organisms. Oxidative stress is not a major factor of heart impairment in frataxin-depleted flies, suggesting the involvement of other pathological mechanisms notably mitochondrial respiratory chain (MRC) dysfunction. Accordingly, we report that methylene blue (MB), a compound known to act as an alternative electron carrier that bypasses mitochondrial complexes I-III, was able to prevent heart dysfunction. MB also partially rescued the phenotype when administered post-symptomatically. Analysis of MB derivatives demonstrates that only compounds with electron carrier properties are able to prevent the heart phenotype. Thus MB, a compound already used for several clinical applications, appears promising for the treatment of the heart dysfunctions that are a major cause of death of FRDA patients. This work provides the grounds for further evaluation of MB action in mammals.


Assuntos
Cardiotônicos/farmacologia , Ataxia de Friedreich/tratamento farmacológico , Azul de Metileno/farmacologia , Animais , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos , Ataxia de Friedreich/patologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Azul de Metileno/uso terapêutico , Interferência de RNA , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Frataxina
19.
Hum Mol Genet ; 22(22): 4591-601, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23825108

RESUMO

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Anemia Megaloblástica/genética , Proteínas de Transporte/metabolismo , Isoformas de Proteínas/metabolismo , Deficiência de Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Hidroxocobalamina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredutases , Ligação Proteica/genética , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/genética
20.
J Pathol ; 234(4): 452-63, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24979321

RESUMO

Cirrhosis is a lesion at risk of hepatocellular carcinoma (HCC). Identifying mechanisms associated with the transition from cirrhosis to HCC and characterizing biomarkers of cirrhosis at high risk of developing into cancer are crucial for improving early diagnosis and prognosis of HCC. We used MALDI imaging to compare mass spectra obtained from tissue sections of cirrhosis without HCC, cirrhosis with HCC, and HCC, and a top-down proteomics approach to characterize differential biomarkers. We identified a truncated form of monomeric ubiquitin lacking the two C-terminal glycine residues, Ubi(1-74), the level of which increased progressively, from cirrhosis without HCC to cirrhosis with HCC to HCC. We showed that kallikrein-related peptidase 6 (KLK6) catalysed the production of Ubi(1-74) from monomeric ubiquitin. Furthermore, we demonstrated that KLK6 was induced de novo in cirrhosis and increased in HCC in parallel with accumulation of Ubi(1-74). We investigated in vitro the possible consequences of Ubi(1-74) accumulation and demonstrated that Ubi(1-74) interferes with the normal ubiquitination machinery in what is likely to be a kinetic process. Our data suggest that de novo KLK6 expression during early liver carcinogenesis may induce production of Ubi(1-74) by post-translational modification of ubiquitin. Given the deleterious effect of Ubi(1-74) on protein ubiquitination and the major role of ubiquitin machinery in maintenance of cell homeostasis, Ubi(1-74) might severely impact a number of critical cellular functions during transition from cirrhosis to cancer. Ubi(1-74) and KLK6 may serve as markers of cancer risk in patients with cirrhosis.


Assuntos
Carcinoma Hepatocelular/patologia , Calicreínas/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Ubiquitina/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Máquina de Vetores de Suporte
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