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Autophagy refers to the natural cellular process by which cells degrade and recycle their own damaged or dysfunctional cellular components. It is an essential mechanism for maintaining cellular homeostasis removing toxic substances and providing energy during times of stress or nutrient deprivation. When autophagy is dysregulated or impaired, it can have detrimental effects on cell function and overall health. Studying autophagy in skin exposed to pollutants can provide valuable insights into the cellular mechanisms underlying pollutant-induced skin damage. Proteomic methods, which involve the large-scale analysis of proteins, can be employed to investigate the changes in protein expression associated with biological processes including autophagy. Here, we thus describe a method where LC-MS/MS was applied to identify the deregulated proteins in pollutant exposed-skin. Using bioinformatics and statistical analysis, we extracted the qualitative and quantitative information for proteins involved in autophagy. These deregulated proteins were then validated by immunohistochemistry (IHC). These methods help to understand how the pollutants affect the autophagy process.
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In intensive care units, COVID-19 viral pneumonia patients (VPP) present symptoms similar to those of other patients with Nonviral infection (NV-ICU). To better manage VPP, it is therefore interesting to better understand the molecular pathophysiology of viral pneumonia and to search for biomarkers that may clarify the diagnosis. The secretome being a set of proteins secreted by cells in response to stimuli represents an opportunity to discover new biomarkers. The objective of this study is to identify the secretomic signatures of VPP with those of NV-ICU. Plasma samples and clinical data from NV-ICU (n = 104), VPP (n = 30) or healthy donors (HD, n = 20) were collected at Nantes Hospital (France) upon admission. Samples were enriched for the low-abundant proteins and analyzed using nontarget mass spectrometry. Specifically deregulated proteins (DEP) in VPP versus NV-ICU were selected. Combinations of 2 to 4 DEPs were established. The differences in secretome profiles of the VPP and NV-ICU groups were highlighted. Forty-one DEPs were specifically identified in VPP compared to NV-ICU. We describe five of the best combinations of 3 proteins (complement component C9, Ficolin-3, Galectin-3-binding protein, Fibrinogen alpha, gamma and beta chain, Proteoglycan 4, Coagulation factor IX and Cdc42 effector protein 4) that show a characteristic receptor function curve with an area under the curve of 95.0%. This study identifies five combinations of candidate biomarkers in VPP compared to NV-ICU that may help distinguish the underlying causal molecular alterations.
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Biomarcadores , COVID-19 , Unidades de Terapia Intensiva , Humanos , COVID-19/diagnóstico , COVID-19/complicações , COVID-19/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Biomarcadores/sangue , Idoso , Proteômica/métodos , SARS-CoV-2 , Adulto , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Pneumonia Viral/sangue , França/epidemiologiaRESUMO
The skin is the outermost protective barrier of the human body. Its role is to protect against different physical, chemical, biological and environmental stressors. The vast majority of studies have focused on investigating the effects of single environmental stressors on skin homeostasis and the induction of several skin disorders, such as cancer or ageing. On the other hand, much fewer studies have explored the consequences of the co-exposure of skin cells to two or more stressors simultaneously, which is much more realistic. In the present study, we investigated, using mass-spectrometry-based proteomic analysis, the dysregulated biological functions in skin explants after their co-exposure to ultraviolet radiation (UV) and benzo[a]pyrene (BaP). We observed that several biological processes were dysregulated, among which autophagy appeared to be significantly downregulated. Furthermore, immunohistochemistry analysis was carried out to validate the downregulation of the autophagy process further. Altogether, the output of this study provides an insight into the biological responses of skin to combined exposure to UV + BaP and highlights autophagy as a potential target that might be considered in the future as a novel candidate for pharmacological intervention under such stress conditions.
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Benzo(a)pireno , Raios Ultravioleta , Humanos , Benzo(a)pireno/toxicidade , Raios Ultravioleta/efeitos adversos , Proteômica , Pele/efeitos da radiação , AutofagiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with ß-catenin. We found that the Wnt/ß-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing ß-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/ß-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased ß-catenin stabilization. In conclusion, in this study, we identified ß-catenin as a new nucleolin interactor and suggest that the Wnt/ß-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.
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Conventional chemotherapy used against cancer is mostly limited due to their non-targeted nature, affecting normal tissue and causing undesirable toxic effects to the affected tissue. With the aim of improving these treatments both therapeutically and in terms of their safety, numerous studies are currently being carried out using nanoparticles (NPs) as a vector combining tumor targeting and carrying therapeutic tools. In this context, it appears that nucleolin, a molecule over-expressed on the surface of tumor cells, is an interesting therapeutic target. Several ligands, antagonists of nucleolin of various origins, such as AS1411, the F3 peptide and the multivalent pseudopeptide N6L have been developed and studied as therapeutic tools against cancer. Over the last ten years or so, numerous studies have been published demonstrating that these antagonists can be used as tumor targeting agents with NPs from various origins. Focusing on nucleolin ligands, the aim of this article is to review the literature recently published or under experimentation in our research team to evaluate the efficacy and future development of these tools as anti-tumor agents.
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Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Peptídeos/uso terapêutico , Fosfoproteínas/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Expressão Gênica , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Humanos , Ligantes , Terapia de Alvo Molecular/métodos , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oligodesoxirribonucleotídeos/química , Fragmentos de Peptídeos/química , Peptídeos/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nanomedicina Teranóstica/métodos , NucleolinaRESUMO
Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from Mecp2-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from Mecp2 KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of Mecp2 KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.
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Astrócitos/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas do Tecido Nervoso/metabolismo , Neurossecreção , Síndrome de Rett/metabolismo , Animais , Astrócitos/fisiologia , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Masculino , Camundongos , Proteômica , Síndrome de Rett/genética , Síndrome de Rett/fisiopatologiaRESUMO
Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Hipóxia Celular , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Neoplasias Pulmonares/metabolismo , Oxigênio/química , Células A549 , Apoptose , Humanos , Lisina/química , Aprendizado de Máquina , Redes e Vias Metabólicas , Fosforilação Oxidativa , Proteoma/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.
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Venenos Elapídicos/química , Peptídeos/química , Peptídeos/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Cromatografia por Troca Iônica , Dissulfetos/química , Egito , Venenos Elapídicos/farmacologia , Elapidae , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cauda do Espermatozoide/química , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Espectrometria de Massas em TandemRESUMO
The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.
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The modulation by amino acids of muscle secretome is largely unknown. In this study, we investigate the effect of hyperaminoacidemia or specific amino acids (citrulline or leucine) on protein synthesis and secretome in myotubes. All conditions stimulate muscle protein synthesis, and secretome is differently modulated depending of the amino acids considered. In conclusion, the activation of protein synthesis by amino acids induces different modulations of the muscle secretome, proposing a new role of amino acids in the regulation of muscle function.
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Aminoácidos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Animais , Células Cultivadas , Citrulina/metabolismo , Meios de Cultura/metabolismo , Leucina/metabolismo , Masculino , Camundongos , Biossíntese de Proteínas , ProteomaRESUMO
KDAC inhibitors (KDACi) overcome gefitinib primary resistance in non-small cell lung cancer (NSCLC) including mutant-KRAS lung adenocarcinoma. To identify which proteins are involved in the restoration of this sensitivity and to provide new therapeutic targets for mutant-KRAS lung adenocarcinoma, we performed an iTRAQ quantitative proteomic analysis after subcellular fractionation of H358-NSCLC treated with gefitinib and KDACi (TSA/NAM) versus gefitinib alone. The 86 proteins found to have been significantly dysregulated between the two conditions, were mainly involved in cellular metabolism and cell transcription processes. As expected, the pathway related to histone modifications was affected by the KDACi. Pathways known for controlling tumor development and (chemo)-resistance (miRNA biogenesis/glutathione metabolism) were affected by the KDACi/gefitinib treatment. Moreover, 57 dysregulated proteins were upstream of apoptosis (such as eEF1A2 and STAT1) and hence provide potential therapeutic targets. The inhibition by siRNA of eEF1A2 expression resulted in a slight decrease in H358-NSCLC viability. In addition, eEF1A2 and STAT1 siRNA transfections suggested that both STAT1 and eEF1A2 prevent AKT phosphorylation known for enhancing gefitinib resistance in NSCLC. Therefore, altogether our data provide new insights into proteome regulations in the context of overcoming the NSCLC resistance to gefitinib through KDACi in H358 KRAS mutated and amphiregulin-overexpressing NSCLC cells.
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Adenocarcinoma de Pulmão/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Mutação , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
INTRODUCTION: Acetylation is a widely occurring post-translational modification (PTM) of proteins that plays a crucial role in many cellular physiological and pathological processes. Over the last decade, acetylation analyses required the development of multiple methods to target individual acetylated proteins, as well as to cover a broader description of acetylated proteins that comprise the acetylome. Areas covered: This review discusses the different types of acetylation (N-ter/K-/O-acetylation) and then describes some major strategies that have been reported in the literature to detect, enrich, identify and quantify protein acetylation. The review highlights the advantages and limitations of these strategies, to guide researchers in designing their experimental investigations and analysis of protein acetylation. Finally, this review highlights the main applications of acetylomics (proteomics based on mass spectrometry) for understanding physiological and pathological conditions. Expert opinion: Recent advances in acetylomics have enhanced knowledge of the biological and pathological roles of protein acetylation and the acetylome. Besides, radiolabeling and western blotting remain also techniques-of-choice for targeted protein acetylation. Future challenges in acetylomics to analyze the N-ter and K-acetylome will most likely require enrichment/fractionation, MS instrumentation and bioinformatics. Challenges also remain to identify the potential biological roles of O-acetylation and cross-talk with other PTMs.
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Proteoma/análise , Proteômica/métodos , Acetilação , Espectrometria de Massas , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen. OBJECIVE: We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey. METHODS: Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from 1D-gel were analyzed by MALDI-TOF/TOF. RESULTS: Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen. CONCLUSION: This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time.
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Alérgenos/imunologia , Antígenos de Plantas/imunologia , Morus/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Testes de Provocação Nasal , Proteômica , Rinite Alérgica Sazonal/sangue , Rinite Alérgica Sazonal/diagnóstico , Testes Cutâneos , Adulto JovemRESUMO
BACKGROUND: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. METHODS: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. RESULTS: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. CONCLUSIONS: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.
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Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.
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Humanos , Animais , Elapidae , Fármacos para a Fertilidade Masculina , Motilidade dos Espermatozoides , Sêmen , Venenos Elapídicos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Reações BioquímicasRESUMO
Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)
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Animais , Masculino , Ratos , Motilidade dos Espermatozoides , Espermatozoides/química , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/uso terapêutico , Fosfolipases A2 , Acetilcolinesterase , Espectrometria de Massas em Tandem/métodos , Fracionamento Químico/métodos , CamundongosRESUMO
High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.
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Corantes/química , Peptídeos/análise , Proteoma/análise , Fracionamento Químico , Cromatografia Líquida de Alta Pressão/métodos , Cor , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Peso Molecular , Albumina Sérica/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging.
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Proteínas/metabolismo , Envelhecimento da Pele/patologia , Tubulina (Proteína)/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Humanos , MasculinoRESUMO
Besides their main contribution as substrates for protein synthesis, amino acids as signaling molecules could exert some regulatory functions on protein synthesis and/or proteolysis that have been emphasized in a number of recent studies. Several publications have highlighted supplemental roles of those amino acids in protein metabolism as well as in immunity, heat shock response, or apoptosis processes. In this way, via their regulatory properties, selected amino acids (such as leucine, glutamine, arginine, citrulline, or methionine) directly influence the proteome. In this review, we are proposing an overview of the regulation of the proteome by amino acids in mammals.
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Arginina/metabolismo , Citrulina/metabolismo , Glutamina/metabolismo , Leucina/metabolismo , Metionina/metabolismo , Biossíntese de Proteínas/fisiologia , Proteoma/metabolismo , Animais , Humanos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17ß-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca(2+)-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca(2+)-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (K d) of this interaction.