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1.
Biomolecules ; 13(1)2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36671537

RESUMO

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.


Assuntos
Células Secretoras de Insulina , Proinsulina , Proinsulina/metabolismo , Dobramento de Proteína , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Prolina/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Insulina/metabolismo
2.
Cell Rep ; 42(1): 111997, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36656716

RESUMO

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.


Assuntos
Álcool Desidrogenase , Quinase 1 de Adesão Focal , Neoplasias , Humanos , Aldeído Oxirredutases/metabolismo , Quinase 1 de Adesão Focal/genética , Neoplasias/genética , Óxido Nítrico/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Microambiente Tumoral , Álcool Desidrogenase/metabolismo
3.
Sci Adv ; 8(13): eabj8360, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35353576

RESUMO

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.


Assuntos
Proteínas Cromossômicas não Histona , Receptores de Glucocorticoides , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Humanos , Receptores de Glucocorticoides/genética , Coesinas
4.
J Mol Biol ; 433(21): 167240, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34508725

RESUMO

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Epirregulina/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador alfa/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Epirregulina/química , Epirregulina/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/química , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Humanos , Ligantes , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteômica , Fator de Crescimento Transformador alfa/química , Fator de Crescimento Transformador alfa/genética , Ubiquitinação
5.
Cell Metab ; 33(8): 1685-1700.e9, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34237252

RESUMO

Liver fibrosis is a strong predictor of long-term mortality in individuals with metabolic-associated fatty liver disease; yet, the mechanisms underlying the progression from the comparatively benign fatty liver state to advanced non-alcoholic steatohepatitis (NASH) and liver fibrosis are incompletely understood. Using cell-type-resolved genomics, we show that comprehensive alterations in hepatocyte genomic and transcriptional settings during NASH progression, led to a loss of hepatocyte identity. The hepatocyte reprogramming was under tight cooperative control of a network of fibrosis-activated transcription factors, as exemplified by the transcription factor Elf-3 (ELF3) and zinc finger protein GLIS2 (GLIS2). Indeed, ELF3- and GLIS2-controlled fibrosis-dependent hepatokine genes targeting disease-associated hepatic stellate cell gene programs. Thus, interconnected transcription factor networks not only promoted hepatocyte dysfunction but also directed the intra-hepatic crosstalk necessary for NASH and fibrosis progression, implying that molecular "hub-centered" targeting strategies are superior to existing mono-target approaches as currently used in NASH therapy.


Assuntos
Redes Reguladoras de Genes , Hepatopatia Gordurosa não Alcoólica , Comunicação , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo
6.
Reprod Biol ; 20(3): 300-306, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32684427

RESUMO

The kappa-opioid receptor (KOR) is involved in the regulation of the fertilizing capacity of human sperm. Recently, a testicular-specific protein family, SPANX-A/D, has also been found to be involved in regulating this process. In order to determine if KOR has a role in the regulation of sperm fertility through the SPANX-A/D protein family, we activated the kappa opioid receptor adding its selective agonist, U50488H to normozoospermic human spermatozoa. Then, we performed immunofluorescence assays and immunoprecipitation experiments followed by LC-MS/MS. According to our results, KOR activation may cause the translocation of SPANX-A/D into the nucleus of human spermatozoa. Phosphoproteomic studies show that KOR does not cause phosphorylation changes in SPANX-A/D residues. However, interactome assays demonstrate that KOR activation provokes changes in SPANX-A/D potential interactors involved in sperm motility, energy metabolism and nuclear processes. Taking these results into account, KOR may regulate human sperm fertility through SPANX-A/D protein family, modifying its subcellular location and interactions. Although further studies are needed, this finding could help us describing the molecular mechanisms underlying sperm fertility as well as developing new strategies for treating infertility.


Assuntos
Proteínas Nucleares/metabolismo , Receptores Opioides kappa/metabolismo , Espermatozoides/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Analgésicos não Narcóticos/farmacologia , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espectrometria de Massas em Tandem
7.
Mol Cell ; 79(2): 332-341.e7, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32521225

RESUMO

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.


Assuntos
Ácido Aspártico Proteases/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Ubiquitina/metabolismo , Ácido Aspártico Proteases/genética , Sítios de Ligação , Sistemas CRISPR-Cas , Linhagem Celular , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Biossíntese de Proteínas , Proteólise
8.
Sci Rep ; 10(1): 5625, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221341

RESUMO

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. Present only in hominids, SPANX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. Combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SPANX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SPANX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SPANX-A/D family members, therefore, might be promising targets for sperm fertility management.


Assuntos
Proteínas Nucleares/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Células HEK293 , Células HeLa , Hominidae/metabolismo , Humanos , Masculino , Membrana Nuclear/metabolismo , Fosforilação/genética , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Espermátides/metabolismo , Testículo/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/genética
9.
Sci Rep ; 10(1): 1148, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980690

RESUMO

To improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (NASH), a multi-omics approach combining bulk RNA-sequencing based transcriptomics, quantitative proteomics and single-cell RNA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (DIO) NASH mice compared to chow-fed controls. Bulk RNA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRNA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of NASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of NASH demonstrating the cellular heterogeneity and complexity of NASH pathogenesis.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/etiologia , Proteômica/métodos , Transcriptoma , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/genética , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de RNA , Análise de Célula Única , Espectrometria de Massas em Tandem
10.
Mol Cell Proteomics ; 18(Suppl 1): S118-S131, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30622161

RESUMO

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.


Assuntos
Fosfoproteínas/metabolismo , Proteômica , Receptores Opioides kappa/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica , Canais de Cálcio/metabolismo , Humanos , Masculino , Fosforilação , Proteoma/metabolismo , Receptores Opioides kappa/agonistas
11.
Nat Struct Mol Biol ; 25(7): 631-640, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29967540

RESUMO

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.


Assuntos
Lisina/química , Ubiquitina/imunologia , Ubiquitina/metabolismo , Ubiquitinação , Especificidade de Anticorpos , Sítios de Ligação , Linhagem Celular , Humanos , Células Jurkat , Espectrometria de Massas , Proteoma/química , Proteoma/metabolismo , Ubiquitina/química
12.
J Proteome Res ; 17(1): 296-304, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29091453

RESUMO

Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.


Assuntos
Sítios de Ligação , Peptídeos/isolamento & purificação , Ubiquitina/metabolismo , Ubiquitinação , Linhagem Celular , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional
13.
J Proteomics ; 170: 28-42, 2018 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-28970102

RESUMO

Cystic Fibrosis (CF) is a recessively inherited disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR has a pivotal role in the onset of CF, and several proteins are involved in its homeostasis. To study CFTR interactors at protein species level, we used a functional proteomics approach combining 2D-DIGE, mass spectrometry and enrichment analysis. A human bronchial epithelial cell line with cystic fibrosis (CFBE41o-) and the control (16HBE14o-) were used for the comparison. 73 differentially abundant spots were identified and some validated by western-blot. Enrichment analysis highlighted molecular pathways in which ezrin, HSP70, endoplasmin and lamin A/C, in addition to CFTR, were considered central hubs in CFTR homeostasis. These proteins acquire different functions through post-translational modifications, emphasizing the importance of studying the CF proteome at protein species level. Moreover, serpin H1, prelamin A/C, protein-SET and cystatin-B were associated to CF, demonstrating the importance of heat shock response, cross-talk between the cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating in the pathophysiology of the disease. These results open new perspectives for the understanding of the proteostasis network, characteristic of CF pathology, and could provide a springboard for new therapeutic strategies. BIOLOGICAL SIGNIFICANCE: Homeostasis of CFTR is a dynamic process managed by multiple proteostatic pathways. The used gel-based proteomic approach and enrichment analysis pointed out protein species variations among Human Bronchial (16HBE14o-) and Cystic Fibrosis Bronchial Epithelial cell lines (CFBE41o-) and specific molecular mechanisms involved in CF. In particular, we have highlighted HSP70 (HSP7C), HSP90 (endoplasmin), ERM proteins (ezrin), and lamin-A/C as central hubs of the functional analysis. Moreover, for the first time we consider serpin H1, lamin A/C, protein-SET and cystatin-B important player in CF, affecting acute exacerbation, cytoskeleton reorganization, CFTR gating and chronic inflammation in CF. Due to the presence of different spots corresponding to the same protein, we focalize our attention on the idea that a "protein species discourse" is mandatory to well-define functional roles of proteins. Our approach has permitted to pay attention to the molecular mechanisms which regulate pathways directly or indirectly involved with CFTR defects: heat shock response, cross-talk between cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating. Our data could open new perspectives into the understanding of CF, identifying potential targets for drug treatments in order to alleviate Δ508CFTR membrane instability and consequently increase life expectancy for CF patients.


Assuntos
Fibrose Cística/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Linhagem Celular Transformada , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Proteoma/genética
14.
Mol Cell Proteomics ; 16(8): 1433-1446, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28572092

RESUMO

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.


Assuntos
Enzima Desubiquitinante CYLD/metabolismo , Receptores ErbB/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Ubiquitinação , Cromatografia Líquida , Enzima Desubiquitinante CYLD/genética , Células HeLa , Humanos , Fosforilação , Proteômica , Espectrometria de Massas em Tandem , Tirosina/metabolismo
15.
Sci Rep ; 6: 21607, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26899371

RESUMO

Spinal cord injury is characterized by acute cellular and axonal damage followed by aggressive inflammation and pathological tissue remodelling. The biological mediators underlying these processes are still largely unknown. Here we apply an innovative proteomics approach targeting the enriched extracellular proteome after spinal cord injury for the first time. Proteomics revealed multiple matrix proteins not previously associated with injured spinal tissue, including small proteoglycans involved in cell-matrix adhesion and collagen fibrillogenesis. Network analysis of transcriptomics and proteomics datasets uncovered persistent overexpression of extracellular alarmins that can trigger inflammation via pattern recognition receptors. In mechanistic experiments, inhibition of toll-like receptor-4 (TLR4) and the receptor for advanced glycation end-products (RAGE) revealed the involvement of alarmins in inflammatory gene expression, which was found to be dominated by IL1 and NFκΒ signalling. Extracellular high-mobility group box-1 (HMGB1) was identified as the likely endogenous regulator of IL1 expression after injury. These data reveal a novel tissue remodelling signature and identify endogenous alarmins as amplifiers of the inflammatory response that promotes tissue pathology and impedes neuronal repair after spinal cord injury.


Assuntos
Proteína HMGB1/biossíntese , Interleucina-1/biossíntese , Receptor para Produtos Finais de Glicação Avançada/biossíntese , Traumatismos da Medula Espinal/genética , Receptor 4 Toll-Like/biossíntese , Alarminas/biossíntese , Alarminas/genética , Animais , Junções Célula-Matriz/genética , Junções Célula-Matriz/patologia , Regulação da Expressão Gênica , Proteína HMGB1/genética , Ensaios de Triagem em Larga Escala , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-1/genética , Neurônios/metabolismo , Neurônios/patologia , Proteômica , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais , Traumatismos da Medula Espinal/patologia , Receptor 4 Toll-Like/genética
16.
Free Radic Biol Med ; 89: 931-43, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26475040

RESUMO

Free hemoglobin (Hb) triggered vascular damage occurs in many hemolytic diseases, such as sickle cell disease, with an unmet need for specific therapeutic interventions. Based on clinical observations the Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe intravascular hemolysis. In this study we present a mechanistic rationale for this paradigm based on a combined biochemical and cell biological approach directed at understanding the unique roles of Hp and Hx in Hb detoxification. Using a novel in vitro model of Hb triggered endothelial damage, which recapitulates the well-characterized pathophysiologic sequence of oxyHb(Fe(2+)) transformation to ferric Hb(Fe(3+)), free heme transfer from ferric Hb(Fe(3+)) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions ultimately lead to endothelial damage characterized by a specific gene expression pattern with reduced cellular ATP and monolayer disintegration. Quantitative analysis of key chemical and biological parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe(2+)) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protection defines a highly efficient backup scavenger system during conditions of large excess of free oxyHb(Fe(2+)) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Haptoglobinas/farmacologia , Hemoglobinas/farmacologia , Hemopexina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/química , Western Blotting , Células Cultivadas , Impedância Elétrica , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Oxirredução , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Free Radic Biol Med ; 85: 259-68, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25933590

RESUMO

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the ß-globin chain of the Hb:Hp complex, including decreased ßCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.


Assuntos
Radicais Livres/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Detecção de Spin , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Aminoácidos/química , Cromatografia Líquida , Espectroscopia de Ressonância de Spin Eletrônica , Haptoglobinas/química , Hemoglobinas/química , Humanos , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Oxirredução , Peroxidases/metabolismo
18.
J Proteome Res ; 14(2): 1089-100, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25566950

RESUMO

Beta-thalassemia results from mutations of the ß-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.


Assuntos
Modelos Animais de Doenças , Eritropoese , Proteoma , Transcriptoma , Transferrina/uso terapêutico , Talassemia beta/tratamento farmacológico , Animais , Camundongos , Camundongos Endogâmicos C57BL , Talassemia beta/genética , Talassemia beta/metabolismo
19.
Haematologica ; 100(5): 611-22, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25616571

RESUMO

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.


Assuntos
Apoproteínas/administração & dosagem , Deleção de Genes , Hemocromatose/genética , Hemocromatose/patologia , Transferrina/administração & dosagem , Globinas beta/genética , Animais , Transfusão de Sangue , Proteínas de Transporte de Cátions/sangue , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Índices de Eritrócitos/efeitos dos fármacos , Feminino , Expressão Gênica , Hemocromatose/metabolismo , Hemocromatose/terapia , Hepcidinas/sangue , Ferro/sangue , Ferro/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Esplenomegalia/tratamento farmacológico , Transferrina/metabolismo
20.
J Inorg Biochem ; 118: 94-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23142974

RESUMO

DIGE (difference in gel electrophoresis) proteomics is exploited here to gain insight into the molecular mechanisms of two established ruthenium-based antimetastatic agents, namely trans-[tetrachloro (DMSO) (imidazole)ruthenate(III)] (NAMI-A) and [Ru(η(6)-toluene)Cl(2)(PTA)] (RAPTA-T), where PTA is 1,3,5-triaza-7-phosphaadamantane. Following 24h exposure of A2780/S human ovarian carcinoma cells to pharmacologically relevant concentrations of either ruthenium compound, 2D-DIGE proteomic analysis evidenced only few differentially expressed proteins with respect to controls. Successive mass spectrometry measurements, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) or LC-ESI/MS-MS (liquid chromatography-electrospray ionization/multi-stage mass spectrometry), allowed identification of most altered protein spots, some of which were associated to perturbations in specific cellular functions. Direct insight into the cellular effects of the investigated metallodrugs is thus achieved. Notably, the patterns of protein alterations induced by NAMI-A and RAPTA-T are quite similar to each other while being deeply different from those of cisplatin. To the best of our knowledge this is the first proteomic study on human cancer cells investigating responses to antimetastatic ruthenium drugs. The key role of new "omic" approaches for deciphering the elusive and complex biochemical mechanisms through which anticancer metallodrugs produce their pharmacological effects is further documented.


Assuntos
Antineoplásicos/farmacologia , Dimetil Sulfóxido/análogos & derivados , Compostos Organometálicos/farmacologia , Proteoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Metástase Neoplásica , Proteômica , Compostos de Rutênio , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial Bidimensional , Regulação para Cima/efeitos dos fármacos
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