RESUMO
PURPOSE: The impact of tea constituents on the insulin-signaling pathway as well as their antidiabetic activity are still debated questions. Previous studies suggested that some tea components act as Protein Tyrosine Phosphatase 1B (PTP1B) inhibitors. However, their nature and mechanism of action remain to be clarified. This study aims to evaluate the effects of both tea extracts and some of their constituents on two main negative regulators of the insulin-signaling pathway, Low-Molecular-Weight Protein Tyrosine Phosphatase (LMW-PTP) and PTP1B. METHODS: The effects of cold and hot tea extracts on the enzyme activity were evaluated through in vitro assays. Active components were identified using gas chromatography-mass spectrometry (GC-MS) analysis. Finally, the impact of both whole tea extracts and specific active tea components on the insulin-signaling pathway was evaluated in liver and muscle cells. RESULTS: We found that both cold and hot tea extracts inhibit LMW-PTP and PTP1B, even if with a different mechanism of action. We identified galloyl moiety-bearing catechins as the tea components responsible for this inhibition. Specifically, kinetic and docking analyses revealed that epigallocatechin gallate (EGCG) is a mixed-type non-competitive inhibitor of PTP1B, showing an IC50 value in the nanomolar range. Finally, in vitro assays confirmed that EGCG acts as an insulin-sensitizing agent and that the chronic treatment of liver cells with tea extracts results in an enhancement of the insulin receptor levels and insulin sensitivity. CONCLUSION: Altogether, our data suggest that tea components are able to regulate both protein levels and activation status of the insulin receptor by modulating the activity of PTP1B.
Assuntos
Resistência à Insulina , Proteínas Tirosina Fosfatases , Receptor de Insulina , Chá , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Insulina/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Chá/químicaRESUMO
Diabetes mellitus (DM) represents a group of metabolic disorders that leads to acute and long-term serious complications and is considered a worldwide sanitary emergence. Type 2 diabetes (T2D) represents about 90% of all cases of diabetes, and even if several drugs are actually available for its treatment, in the long term, they show limited effectiveness. Most traditional drugs are designed to act on a specific biological target, but the complexity of the current pathologies has demonstrated that molecules hitting more than one target may be safer and more effective. The purpose of this review is to shed light on the natural compounds known as α-glucosidase and Protein Tyrosine Phosphatase 1B (PTP1B) dual-inhibitors that could be used as lead compounds to generate new multitarget antidiabetic drugs for treatment of T2D.
Assuntos
Inibidores Enzimáticos/química , Hipoglicemiantes/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , alfa-Glucosidases/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , alfa-Glucosidases/química , alfa-Glucosidases/genéticaRESUMO
Chemoresistance is the primary cause of chemotherapy failure. Compelling evidence shows that micro RNAs (miRNAs) contribute to reprogram cancer cells toward a resistant phenotype. We investigate the role of miRNAs in the response to acute treatment with 5-FU in colon cancer-resistant cells. We performed a global gene expression profile for the entire miRNA genome and found a change in the expression of four miRNAs following acute treatment with 5-FU. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. We show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, responsible for DNA damage repair. Moreover, miR-210-3p downregulation enhances oxidative phosphorylation (OXPHOS), increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. Altogether, these adaptations lead to increased cells survival following drug exposure. These evidence suggest that miR-210-3p downregulation following 5-FU sustains DNA damage repair and metabolic adaptation to counteract drug treatment.
Assuntos
Neoplasias do Colo/genética , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Antimetabólitos Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias do Colo/metabolismo , Dano ao DNA , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Células HT29 , HumanosRESUMO
LMW-PTP has been associated with the development of colorectal cancer (CRC) and with the resistance to chemotherapy in cancer cells. To clarify its role in vivo, we studied LMW-PTP expression in Pirc rats (F344/NTac-Apc am1137 ), genetically prone to CRC and resistant to apoptosis. In the morphologically normal mucosa (NM) of Pirc rats, a dramatic over-expression of LMW-PTP was found compared to wt rats (about 60 times higher). Moreover, LMW-PTP levels further increase in spontaneously developed Pirc colon tumors. To understand if and how LMW-PTP affects resistance to apoptosis, we studied CRC cell lines, sensitive (HT29 and HCT-116), or resistant (HT29R, HCT116R) to 5-Fluorouracil (5-FU): resistant cells over-express LMW-PTP. When resistant cells were challenged with morin, a polyphenol inhibiting LMW-PTP, a fast and dose-related down-regulation of LMW-PTP was observed. 5-FU and morin co-treatment dramatically decreased cell viability, increased apoptosis, and significantly impaired self-renewal ability of all the cancer cell lines we have studied. Similarly, we observed that, in Pirc rats, one-week morin administration (50 mg/kg) down-regulated LMW-PTP and restored the apoptotic response to 5-FU in the NM. Finally, administration of morin for a longer period led to a significant reduction in colon precancerous lesions, together with a down-regulation of LMW-PTP. Taken together, these results document the involvement of LMW-PTP in the process of CRC in vitro and in vivo. Morin treatment may be envisaged as a system to increase the sensitivity to chemotherapy and to prevent carcinogenesis.
Assuntos
Carcinogênese/patologia , Colo/patologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Flavonoides/farmacologia , Genes APC , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Carcinogênese/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Colo/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/etiologia , Técnicas In Vitro , Masculino , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Endogâmicos F344RESUMO
Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors.
Assuntos
Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Humanos , Peso Molecular , Fosfotirosina/genética , Fosfotirosina/metabolismo , Alinhamento de Sequência , Transdução de Sinais/genéticaRESUMO
Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells.
Assuntos
Proliferação de Células , Metabolismo dos Lipídeos , Neoplasias da Próstata/patologia , Transporte Proteico , Linhagem Celular Tumoral , Técnicas de Cocultura , Fibroblastos/patologia , Humanos , Masculino , Microambiente TumoralRESUMO
Cerato-platanin (CP) is the founder of a fungal protein family consisting in non-catalytic secreted proteins, which work as virulence factors and/or as elicitors of defense responses and systemic resistance, thus acting as PAMPs (pathogen-associated molecular patterns). Moreover, CP has been defined an expansin-like protein showing the ability to weaken cellulose aggregates, like the canonical plant expansins do. Here, we deepen the knowledge on CP PAMP activity by the use of a multi-disciplinary approach: proteomic analysis, VOC (volatile organic compound) measurements, and gas exchange determination. The treatment of Arabidopsis with CP induces a differential profile either in protein expression or in VOC emission, as well changes in photosynthetic activity. In agreement with its role of defense activator, CP treatment induces down-expression of enzymes related to primary metabolism, such as RuBisCO, triosephosphate isomerase, and ATP-synthase, and reduces the photosynthesis rate. Conversely, CP increases expression of defense-related proteins and emission of some VOCs. Interestingly, CP exposure triggered the increase in enzymes involved in GSH metabolism and redox homeostasis (glutathione S-transferase, thioredoxin, Cys-peroxiredoxin, catalase) and in enzymes related to the "glucosinolate-myrosinase" system, which are the premise for synthesis of defence compounds, such as camalexin and some VOCs, respectively. The presented results are in agreement with the accepted role of CP as a PAMP and greatly increase the knowledge of plant primary defences induced by a purified fungal elicitor.
Assuntos
Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Moléculas com Motivos Associados a Patógenos/metabolismo , Plantas/metabolismo , Plantas/microbiologia , Resistência à Doença , Genômica , Metabolômica , Doenças das Plantas/microbiologia , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Compostos Orgânicos Voláteis/metabolismoRESUMO
Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Osteoblastos/citologia , Receptores de Trombopoetina/fisiologia , Nicho de Células-Tronco/fisiologia , Irradiação Corporal Total , Animais , Becaplermina , Movimento Celular/fisiologia , Movimento Celular/efeitos da radiação , Proliferação de Células , Endotélio Vascular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos da radiação , Megacariócitos/metabolismo , Megacariócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas Proto-Oncogênicas c-sis/metabolismo , Transdução de Sinais , Trombopoetina/metabolismoRESUMO
BACKGROUND: Type-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs. METHODS: Selection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [(3)H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program. RESULTS: We demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a Ki in the µM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth. CONCLUSIONS: We have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only. GENERAL SIGNIFICANCE: Our study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.
Assuntos
Flavonoides/farmacologia , Hipoglicemiantes/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Animais , Apigenina/farmacologia , Proliferação de Células/efeitos dos fármacos , Glucose/biossíntese , Glicogênio/biossíntese , Células Hep G2 , Humanos , Insulina/metabolismo , Insulina/farmacologia , Camundongos , Células NIH 3T3RESUMO
The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.
Assuntos
Transplante de Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas , Fator de Crescimento Insulin-Like I/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Osso e Ossos/citologia , Movimento Celular , Proliferação de Células , Sobrevivência de Enxerto , Hematopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/fisiologia , Irradiação Corporal TotalRESUMO
Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.
Assuntos
Anidrases Carbônicas/metabolismo , Fibroblastos/metabolismo , Neoplasias/patologia , Microambiente Tumoral/fisiologia , Animais , Catálise , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Xenoenxertos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos SCID , Neoplasias/metabolismo , Bicarbonato de Sódio/metabolismoRESUMO
In the early stages of carcinogenesis, the transformed cells become "invisible" to the immune system. From this moment on, the evolution of the tumor depends essentially on the genotype of the primitive cancer cells and their subsequent genetic drift. The role of the immune system in blocking tumor progression from the earliest stages is largely underestimated because by the time tumors are clinically detectable, the immune system has already completely failed its task. Therefore, a clinical treatment capable of restoring the natural anti-tumor role of the immune system could prove to be the "ultimate weapon" against cancer. Herein, we propose a novel therapeutic approach for the treatment of solid cancer that exploits the capability of activated monocytes to transfer major histocompatibility complex I (MHC-I) molecules bound to antigenic peptides to cancer cells using microvesicles as cargo, making tumor cells target of a "natural" CD8+ T lymphocyte cytotoxic response.
RESUMO
5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.
Assuntos
Neoplasias Colorretais , Neoplasias , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/metabolismo , Fluoruracila/farmacologia , Humanos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Serina/metabolismoRESUMO
Cancer progression is supported by the cross-talk between tumor cells and the surrounding stroma. In this context, senescent cells in the tumor microenvironment contribute to the development of a pro-inflammatory milieu and the acquisition of aggressive traits by cancer cells. Anticancer treatments induce cellular senescence (therapy-induced senescence, TIS) in both tumor and non-cancerous cells, contributing to many detrimental side effects of therapies. Thus, we focused on the effects of chemotherapy on the stromal compartment of prostate and ovarian cancer. We demonstrated that anticancer chemotherapeutics, regardless of their specific mechanism of action, promote a senescent phenotype in stromal fibroblasts, resulting in metabolic alterations and secretion of paracrine factors, sustaining the invasive and clonogenic potential of both prostate and ovarian cancer cells. The clearance of senescent stromal cells, through senolytic drug treatment, reverts the malignant phenotype of tumor cells. The clinical relevance of TIS was validated in ovarian and prostate cancer patients, highlighting increased accumulation of lipofuscin aggregates, a marker of the senescent phenotype, in the stromal compartment of tissues from chemotherapy-treated patients. These data provide new insights into the potential efficacy of combining traditional anticancer strategies with innovative senotherapy to potentiate anticancer treatments and overcome the adverse effects of chemotherapy.
Assuntos
Neoplasias Ovarianas , Neoplasias da Próstata , Humanos , Masculino , Feminino , Neoplasias Ovarianas/genética , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Fenótipo , Microambiente TumoralRESUMO
Cancer-associated fibroblasts (CAFs) are one of the main components of the stromal compartment in the tumor microenvironment (TME) and the crosstalk between CAFs and cancer cells is essential for tumor progression and aggressiveness. Cancer cells mediate an activation process, converting normal fibroblasts into CAFs, that are characterized by modified expression of many proteins and increased production and release of microvesicles (MVs), extracellular vesicles generated by outwards budding from the cell membrane. Recent evidence underlined that the uptake of CAF-derived MVs changes the overall protein content of tumor cells. In this paper, we demonstrate that tumor activated fibroblasts overexpress Galectin-1 (Gal-1) and consequently release MVs containing increased levels of this protein. The uptake of Gal-1 enriched MVs by tumor cells leads to the upregulation of its intracellular concentration, that strongly affects cancer cell migration, while neither proliferation nor adhesion are altered. Accordingly, tumor cells co-cultured with fibroblasts silenced for Gal-1 have a reduced migratory ability. The present work reveals the key role of an exogenous protein, Gal-1, derived from activated fibroblasts, in cancer progression, and contributes to clarify the importance of MVs-mediated protein trafficking in regulating tumor-stroma crosstalk.
RESUMO
BACKGROUND: L-Homocysteine (Hcy) is a non-proteinogenic α-amino acid synthesized from dietary methionine. In healthy humans, high Hcy levels are a risk factor for cardiovascular diseases, stroke and type 2 diabetes. A recent study reports that Hcy reacts with Cys10 of transthyretin (TTR), generating a stable covalent adduct. However, to date the effect of S-homocysteinylation on TTR conformational stability remains unknown. METHODS: The effect of Hcy on the conformational properties of wt- and L55P-TTR were analysed using a set of biophysical techniques. The cytotoxicity of S-homocysteinylated L55P-TTR was also evaluated in the HL-1 cardiomyocyte cell line, while the effects of the assemblies on kinematic and dynamics properties of cardiac muscle cells were analysed in cardiomyocyte syncytia. RESULTS: We found that Hcy stabilizes tetrameric wt-TTR, while it destabilizes the tetrameric structure of the L55P mutant, promoting the accumulation of self-assembly-prone monomeric species. CONCLUSIONS: Our study demonstrated that S-homocysteinylation of the L55P-TTR mutant impairs protein stability, favouring the appearance of toxic monomers. Interestingly, S-homocysteinylation affected only mutant, not wt-TTR. Moreover, we also show that assemblies of S-homocysteinylated L55P-TTR impair cardiomyocytes functional parameters. GENERAL SIGNIFICANCE: Our study offers new insights on the negative impact of S-homocysteinylation on L55P-TTR stability, whose aggregation is considered the causative agent of a form of early-onset familial amyloid polyneuropathy and cardiomyopathy. Our results suggest that high homocysteine levels are a further risk factor for TTR cardiomyopathy in patients harbouring the L55P-TTR mutation.
Assuntos
Neuropatias Amiloides Familiares/genética , Cardiomiopatias/genética , Homocisteína/genética , Pré-Albumina/química , Neuropatias Amiloides Familiares/patologia , Cardiomiopatias/patologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Homocisteína/química , Humanos , Metionina/química , Mutação/genética , Miócitos Cardíacos , Pré-Albumina/genética , Pré-Albumina/ultraestrutura , Conformação Proteica , Estabilidade Proteica , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Relação Estrutura-AtividadeRESUMO
Cellular prion protein (PrP(c)) is a ubiquitous glycoprotein, whose physiological role is poorly characterized. It has been suggested that PrP(c) participates in neuritogenesis, neuroprotection, copper metabolism, and signal transduction. In this study we detailed the intracellular events induced by PrP(c) antibody-mediated cross-linking in PC12 cells. We found a Fyn-dependent activation of the Ras-Raf pathway, which leads to a rapid and transient phosphorylation of extracellular regulated kinases. In addition, this activation cascade relies on the engagement of integrins, and involves focal adhesion kinase activation. We demonstrated the tyrosine phosphorylation of caveolin-1 as a consequence of PrP(c) stimulation, and showed that phosphocaveolin-1 scaffolds and coordinates protein complexes involved in PrP(c)-dependent signaling. Moreover, we found that caveolin-1 phosphorylation, is a mechanism for recruiting the C-terminal Src kinase and inactivating Fyn, so as to terminate cell signaling. Furthermore our data support a significant role for PrP(c) as a response mediator in neuritogenesis and cell differentiation.
Assuntos
Caveolina 1/metabolismo , Diferenciação Celular/fisiologia , Neuritos/metabolismo , Neurogênese/fisiologia , Proteínas PrPC/metabolismo , Transdução de Sinais/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Proteína Tirosina Quinase CSK , Caveolina 1/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Neuritos/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Células PC12 , Fosforilação/efeitos dos fármacos , Proteínas PrPC/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo , Proteínas ras/metabolismo , Quinases da Família srcRESUMO
We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.
Assuntos
Aminoácidos Aromáticos/genética , Mutagênese Sítio-Dirigida , Proteínas Tirosina Fosfatases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Alanina/metabolismo , Substituição de Aminoácidos , Aminoácidos Aromáticos/química , Sítios de Ligação , Catálise , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética , Triptofano/química , Triptofano/genética , Tirosina/química , Tirosina/genéticaRESUMO
BACKGROUND: Low Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature. METHODS: We investigated, by proteomic analysis, the protein phosphorylation pattern of A375 human melanoma cells silenced for LMW-PTP. Two-dimensional electrophoresis (2-DE) analysis, followed by western blot was performed using anti-phosphotyrosine antibodies, in order to identify differentially phosphorylated proteins. RESULTS: Proteomic analysis pointed out that most of the identified proteins belong to the glycolytic metabolism, such as α-enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, suggesting an involvement of LMW-PTP in glucose metabolism. Assessment of lactate production and oxygen consumption demonstrated that LMW-PTP silencing enhances glycolytic flux and slow down the oxidative metabolism. In particular, LMW-PTP expression affects PKM2 tyrosine-phosphorylation and nuclear localization, modulating its activity. CONCLUSION: All these findings propose that tumor cells are subjected to metabolic reprogramming after LMW-PTP silencing, enhancing glycolytic flux, probably to compensate the inhibition of mitochondrial metabolism. GENERAL SIGNIFICANCE: Our results highlight the involvement of LMW-PTP in regulating glucose metabolism in A375 melanoma cells.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Neoplasias/metabolismo , Fluorescência , Humanos , Peso Molecular , Neoplasias/patologiaRESUMO
Tumor resistance to apoptosis is one the main causes of anticancer treatment failure. Previous studies showed that LMW-PTP overexpression enhances resistance of cancer cells to traditional anticancer drugs. Today, the role of LMW-PTP in inducing resistance to apoptosis in melanoma cells remains to be elucidated. Experimental setting include MTT assay, Annexin V/Pi method, and colony assay to assess whether silencing of LMW-PTP improves the sensitivity of A375 to dacarbazine, 5-FU, and radiotherapy. Pharmacological targeting of LMW-PTP was obtained using Morin, a LMW-PTP inhibitor. The ability of Morin to improve the effectiveness of anticancer drugs and radiotherapy was also studied. Moreover, PC3 cells were used as an alternative cellular model to confirm the data obtained with melanoma cells. We found that LMW-PTP silencing improves the effectiveness of dacarbazine, 5-FU, and radiotherapy. Identical results were obtained in vivo when Morin was used to target LMW-PTP. We demonstrated that Morin synergizes with dacarbazine, improving its cytotoxic activity. However, we showed that the combined treatment, Morin-anticancer drug, does not affect the viability of noncancerous cells. Knockdown of LMW-PTP sensitizes also PC3 cells to docetaxel and radiotherapy. In conclusion, we showed that LMW-PTP targeting improves effectiveness of anticancer drugs used for treatment of melanoma. Moreover, our results suggest that Morin could be used as adjuvant to improve the outcome of patients affected by metastatic melanoma.