RESUMEN
BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.
Asunto(s)
Neoplasias , Nucleósido-Difosfato Quinasa , Animales , Membranas Intracelulares , Ratones , Mitocondrias , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Nucleósido Difosfato Quinasa D/metabolismo , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismoRESUMEN
Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K1+2+3, k1+2+3+4) and the serine protease (SP) region (K5-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.
Asunto(s)
Trampas Extracelulares/enzimología , Fibrinolisina/metabolismo , Fibrinólisis/fisiología , Elastasa Pancreática/metabolismo , Plasminógeno/metabolismo , Choque Séptico/sangre , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Coagulación Intravascular Diseminada/sangre , Humanos , Persona de Mediana Edad , Elastasa Pancreática/genéticaRESUMEN
Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease.
Asunto(s)
Anexina A1/metabolismo , Apoptosis/inmunología , Autoinmunidad , Granulomatosis con Poliangitis/inmunología , Neutrófilos/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anexina A1/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Biomarcadores/metabolismo , Calreticulina/inmunología , Calreticulina/metabolismo , Femenino , Granulomatosis con Poliangitis/sangre , Granulomatosis con Poliangitis/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Mieloblastina/inmunología , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Proteínas de Transferencia de Fosfolípidos/inmunología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteómica , Transducción de Señal/inmunología , Adulto JovenRESUMEN
BACKGROUND: α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. METHODS: We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. RESULTS: Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. CONCLUSIONS: Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. GENERAL SIGNIFICANCE: Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.
Asunto(s)
Cuerpos de Lewy/química , Neuroblastoma/patología , alfa-Sinucleína/fisiología , Línea Celular Tumoral , Humanos , Procesamiento Proteico-Postraduccional , Proteoma , Electroforesis Bidimensional Diferencial en Gel , alfa-Sinucleína/químicaRESUMEN
Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH-SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or -1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH-SMC (fold change 1.5≤ or -1.5≥, p < 0.05). HUASMC expressed increased amount of α-smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH-SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH-SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH-SMC. There was a trend toward reduced proliferation of PAH-SMC with paxillin-si-RNA and increased proliferation with ELAVL1-siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH-SMC proliferation.
Asunto(s)
Hipertensión Pulmonar/patología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Mapas de Interacción de Proteínas , Proteoma/análisis , Arteria Pulmonar/patología , Proliferación Celular , Células Cultivadas , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Paxillin/genética , Paxillin/metabolismo , Proteoma/genética , Proteoma/metabolismo , Arteria Pulmonar/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
UNLABELLED: Hepatitis E virus (HEV) causes acute enterically transmitted hepatitis. In industrialized countries, it is a zoonotic disease, with swine being the major reservoir of human HEV contamination. The occurrence and severity of the disease are variable, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In the absence of a robust cell culture system or small-animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with three different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, two-dimensional difference in gel electrophoresis, and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the three HEV strains replicate similarly in swine and that they modulate several cellular pathways, suggesting that HEV impairs several cellular processes, which can account for the various types of disease expression. Several proteins, such as heterogeneous nuclear ribonucleoprotein K, apolipoprotein E, and prohibitin, known to be involved in other viral life cycles, were upregulated in HEV-infected livers. Some differences were observed between the three strains, suggesting that HEV's genetic variability may induce variations in pathogenesis. This comparative analysis of the liver proteome modulated during infection with three different strains of HEV genotype 3 provides an important basis for further investigations on the factors involved in HEV replication and the mechanism of HEV pathogenesis. IMPORTANCE: Hepatitis E virus (HEV) is responsible for acute hepatitis, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, with swine being the principal reservoir for human contamination. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol and lipid metabolism or cell survival were modulated during HEV infection in the swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.
Asunto(s)
Virus de la Hepatitis E/fisiología , Hepatitis E/patología , Hepatitis E/virología , Interacciones Huésped-Patógeno , Proteoma/análisis , Animales , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Variación Genética , Virus de la Hepatitis E/patogenicidad , Hígado/patología , Espectrometría de Masas , Proteómica/métodos , Porcinos , VirulenciaAsunto(s)
Cryptomeria , Alérgenos , Antígenos de Plantas , Giberelinas , Humanos , Proteínas de Plantas , PolenRESUMEN
BACKGROUND: The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins. STUDY DESIGN AND METHODS: Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method. RESULTS: Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1. CONCLUSION: The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile.
Asunto(s)
Conservación de la Sangre/métodos , Furocumarinas/farmacología , Plasma/química , Factores de Coagulación Sanguínea/análisis , Proteínas Sanguíneas/química , Proteínas Sanguíneas/efectos de los fármacos , Humanos , Proteómica/métodos , Rayos UltravioletaRESUMEN
Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an â¼30 and â¼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/genética , VIH-1/patogenicidad , Proteínas Nucleares/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/deficiencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Western Blotting , Electroforesis/métodos , Silenciador del Gen , Células HEK293 , VIH-1/metabolismo , Humanos , Proteómica , ARN Interferente Pequeño , Tetraspanina 28/metabolismo , Ultracentrifugación , Virión/metabolismo , alfa-Glucosidasas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague-Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and two groups were refed for one week with a standard diet supplemented with nonessential amino acids group or Cit (CIT group). Results of the proteomic approach were validated using targeted Western blot analysis and assessment of gene expression of the related genes. Maximal activities of the key enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituents and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs. AL and R, p < 0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs. AL, p < 0.05). However, maximal activities of key enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs. AL and R, p < 0.05). In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia toward anaerobia.
Asunto(s)
Citrulina/farmacología , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteoma/efectos de los fármacos , Animales , Dieta , Metabolismo Energético , Masculino , Proteínas Musculares/análisis , Proteínas Musculares/química , Músculo Esquelético/química , Proteoma/análisis , Proteoma/química , Proteoma/metabolismo , Proteómica , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re-induction remain largely unknown. Here, we used a DIGE-based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re-induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re-induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by-product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re-induced BBB functions than in cells with limited BBB functions.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Electroforesis en Gel Bidimensional/métodos , Células Endoteliales/metabolismo , Neuroglía/metabolismo , Animales , Arginina/análogos & derivados , Barrera Hematoencefálica/citología , Bovinos , Medios de Cultivo , Immunoblotting , Fenotipo , Ratas , Reproducibilidad de los ResultadosRESUMEN
Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma-derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neoangiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neovascularización Patológica/metabolismo , Células-Madre Neurales/metabolismo , Proteómica , Adulto , Animales , Movimiento Celular , Medios de Cultivo Condicionados , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Glioblastoma/patología , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Neovascularización Patológica/patología , Células-Madre Neurales/patología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy-coat PCs (PCT-PCs) compared to those from conventional PCs. STUDY DESIGN AND METHODS: Four PCT-PCs and four conventional PCs were stored for 6.5 days and PLT function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored PLTs were taken, washed, and suspended in Tyrode's buffer before testing. RESULTS: PLT counts and morphology were conserved although a slight increase in the PLT volume was observed after PCT. Glycoprotein (GP) IIbIIIa, IaIIa, and VI expression remained stable while GPIbα declined similarly in both types of PCs. A steep decrease (50%) in GPV occurred on Day 1.5 in PCT-PCs and Day 2.5 in control PCs. For both PCT- and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. PCT- and control PCs were fully responsive to aggregation agonists up to Day 4.5 and exhibited similar perfusion functionality. Mitochondrial membrane potential and annexin A5 binding of PCT-PCs and control PCs were comparable. Two-dimensional differential in-gel electrophoresis and mass spectrometry profiles for 1882 protein spots revealed only three proteins selectively changed in PCT-PCs compared to control-PCs. CONCLUSION: Washed treated and untreated PCs have similar functional, morphologic, and proteomic characteristics provided that PLTs are suspended in an appropriate medium during testing.
Asunto(s)
Plaquetas/citología , Conservación de la Sangre/métodos , Seguridad de la Sangre/métodos , Patógenos Transmitidos por la Sangre/efectos de la radiación , Furocumarinas/farmacología , Rayos Ultravioleta , Anexina A5/metabolismo , Almacenamiento de Sangre/métodos , Capa Leucocitaria de la Sangre/citología , Capa Leucocitaria de la Sangre/microbiología , Plaquetas/metabolismo , Plaquetas/microbiología , Criopreservación/métodos , Humanos , Potencial de la Membrana Mitocondrial , Selectina-P/metabolismo , Fármacos Fotosensibilizantes/farmacología , Adhesividad Plaquetaria , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transfusión de Plaquetas , ProteómicaRESUMEN
Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Leishmania donovani/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Leishmania donovani/crecimiento & desarrollo , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosforilación , Unión Proteica , Proteoma/química , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity® analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.
Asunto(s)
Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Microvasos/citología , Proteoma/metabolismo , Dermis/irrigación sanguínea , Electroforesis en Gel Bidimensional , Células Endoteliales/efectos de los fármacos , Perfilación de la Expresión Génica , Salud , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Pulmón/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Donantes de Tejidos , Tretinoina/farmacologíaRESUMEN
Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K.
Asunto(s)
IMP Deshidrogenasa/metabolismo , Microdominios de Membrana/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 2/metabolismo , Células HEK293 , Humanos , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/inmunología , Inmunidad Innata/fisiología , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunologíaRESUMEN
Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood-brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm-2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.
Asunto(s)
Células Endoteliales , Proteómica , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Estrés MecánicoRESUMEN
Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation.
Asunto(s)
Neoplasias Encefálicas/genética , Isocitrato Deshidrogenasa/genética , Mutación , Oligodendroglioma/genética , Proteoma/genética , Proteómica , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas , Persona de Mediana Edad , Oligodendroglioma/enzimología , Oligodendroglioma/metabolismo , Oligodendroglioma/patología , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Electroforesis Bidimensional Diferencial en Gel/métodosRESUMEN
The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos). Using 2D-DIGE and mass spectrometry techniques, we identified 41 proteins significantly overexpressed between F/P-Eos and C-Eos. Among them, 17.8% belonged to the oxidoreductase family. We further observed a down-expression of peroxiredoxin-2 (PRX-2) and an overexpression of src-homology-2 domain containing tyrosine phosphatase (SHP-1), enzymes regulating PDGFR downstream pathways, and especially intracellular reactive oxygen species (ROS) production. This profile, confirmed in immunoblot analysis, appears specific to F/P-Eos compared to controls and patients with idiopathic HES. In this clonal disorder possibly involving a pluripotent hematopoietic stem cell, we postulate that the well documented relationships between PDGFRA downstream signals and intracellular ROS levels might influence the phenotype of this leukemia.
Asunto(s)
Eosinófilos , Síndrome Hipereosinofílico/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteoma/análisis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Adulto , Anciano , Animales , Línea Celular , Bases de Datos de Proteínas , Eosinófilos/química , Eosinófilos/metabolismo , Femenino , Humanos , Síndrome Hipereosinofílico/genética , Síndrome Hipereosinofílico/fisiopatología , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Electroforesis Bidimensional Diferencial en Gel/métodos , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.