RESUMEN
BACKGROUND: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function. RESULTS: In this study, we identify in T lymphocytes several adaptor proteins as potential substrates targeted by LYP, including FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2. We also show that LYP co-localizes with SLP76 in microclusters, upon TCR engagement. CONCLUSIONS: These data indicate that LYP may modulate T cell activation by dephosphorylating several adaptor proteins, such as FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2 upon TCR engagement.
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Proteínas Adaptadoras Transductoras de Señales , Fosfoproteínas , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Linfocitos T , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Jurkat , Activación de Linfocitos , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genética , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/metabolismoRESUMEN
Ectophosphatases catalyse the hydrolysis of phosphorylated molecules, such as phospho-amino acids, in the extracellular environment. Nevertheless, the hydrolysis of nucleotides in the extracellular environment is typically catalysed by ectonucleotidases. Studies have shown that acid ectophosphatase, or transmembrane-prostatic acid phosphatase (TM-PAP), a membrane-bound splice variant of prostatic acid phosphatase, has ecto-5'-nucleotidase activity. Furthermore, it was demonstrated that ectophosphatase cannot hydrolyse ATP, ADP, or AMP in triple-negative breast cancer cells. In contrast to previous findings in MDA-MB-231 cells, the ectophosphatase studied in the present work displayed a remarkable capacity to hydrolyse AMP in luminal A breast cancer cells (MCF-7). We showed that AMP dose-dependently inhibited p-nitrophenylphosphate (p-NPP) hydrolysis. The p-NPP and AMP hydrolysis showed similar biochemical behaviours, such as increased hydrolysis under acidic conditions and comparable inhibition by NiCl2, ammonium molybdate, and sodium orthovanadate. In addition, this ectophosphatase with ectonucleotidase activity was essential for the release of adenosine and inorganic phosphate from phosphorylated molecules available in the extracellular microenvironment. This is the first study to show that prostatic acid phosphatase on the membrane surface of breast cancer cells (MCF-7) is correlated with cell adhesion and migration.
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Fosfatasa Ácida , Neoplasias de la Mama , Humanos , Células MCF-7 , Femenino , Hidrólisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/enzimología , Fosfatasa Ácida/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina Monofosfato/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Nitrofenoles/farmacología , Nitrofenoles/metabolismo , Línea Celular Tumoral , Compuestos OrganofosforadosRESUMEN
In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.
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Neoplasias Colorrectales , Proteínas Tirosina Fosfatasas , Neoplasias Colorrectales/patología , Humanos , Indoles , Masculino , Mitocondrias/metabolismo , Peso Molecular , Proteínas Tirosina Fosfatasas/metabolismo , TirosinaRESUMEN
Protein phosphorylation is a major post-translational modification involved in cell signalling that regulates many physiological and pathological processes. Despite their biological importance, protein phosphatases are less studied than protein kinases. Importantly, the activity of Cys-based protein tyrosine phosphatases (PTPs) can be regulated by reversible oxidation. The initial two-electron oxidation product of the active site Cys is a sulfenic acid (Cys-SOH) that can then undergo distinct outcomes, such as the disulfide bond or a sulfenyl amide formation. Here, we review the biochemical and structural features of PTPs to find patterns that might specify their oxidation products, aiming to get insights into redox regulatory mechanisms. Initially, the structure and biochemistry of PTP1B is presented. Then, we describe structural aspects that are relevant for substrate recognition and catalysis. Notably, all PTPs contain critical Cys residues for the catalysis of dephosphorylation that is prone to oxidative inactivation, which are frequently found oxidized in cells under physiological conditions, such as upon growth factor stimuli. However, direct oxidations of Cys residues in PTPs by H2 O2 are rather slow. Therefore, we discuss possible mechanisms that may account for this apparent contradiction between biological and chemical redox aspects of PTPs. Furthermore, we performed a systematic analysis of the distance between active site cysteine and its backdoor cysteine with the attempt to analyse the preference between disulfide bond formation or sulfenyl amide interaction upon oxidation. In summary, PTPs have been showing many possibilities to auto-protect from irreversible oxidation, which is important for cell signalling regulation.
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Cisteína , Ácidos Sulfénicos , Amidas/química , Cisteína/química , Disulfuros/metabolismo , Oxidación-Reducción , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Ácidos Sulfénicos/química , Ácidos Sulfénicos/metabolismoRESUMEN
Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.
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Proteínas de la Membrana Bacteriana Externa/química , Cisteína/química , Óxido Nítrico/química , Proteínas Tirosina Fosfatasas/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biotina/metabolismo , Catálisis , Cristalografía por Rayos X/métodos , Cisteína/metabolismo , Humanos , Espectrometría de Masas/métodos , Estructura Molecular , Óxido Nítrico/metabolismo , Oxidación-Reducción , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Yersinia enterocolitica/metabolismoRESUMEN
The main post-translational reversible modulation of proteins is phosphorylation and dephosphorylation, catalyzed by protein kinases (PKs) and protein phosphatases (PPs) which is crucial for homeostasis. Imbalance in this crosstalk can be related to diseases, including cancer. Plenty of evidence indicates that protein tyrosine phosphatases (PTPs) can act as tumor suppressors and tumor promoters. In gastric cancer (GC), there is a lack of understanding of the molecular aspects behind the tumoral onset and progression. Here we describe several members of the PTP family related to gastric carcinogenesis. We discuss the associated molecular mechanisms which support the down or up modulation of different PTPs. We emphasize the Helicobacter pylori (H. pylori) virulence which is in part associated with the activation of PTP receptors. We also explore the involvement of intracellular redox state in response to H. pylori infection. In addition, some PTP members are under influence by genetic mutations, epigenetics mechanisms, and miRNA modulation. The understanding of multiple aspects of PTPs in GC may provide new targets and perspectives on drug development.
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Proteínas Tirosina Fosfatasas/metabolismo , Neoplasias Gástricas/metabolismo , Helicobacter pylori/enzimología , Humanos , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias Gástricas/diagnósticoRESUMEN
BACKGROUND: PTP4A3 is a subclass of a protein tyrosine phosphatase super family and is expressed in a range of epithelial neoplasms. We evaluated PTP4A3 expression and its association with clinicopathological parameters in different types of functioning pituitary adenomas. METHODS: A total of 34 functioning pituitary adenomas samples were evaluated in this observational study. PTP4A3 expression was examined by immunohistochemical staining, and, possible correlations between PTP4A3 and some clinicopathological variables were investigated. RESULTS: PTP4A3 was expressed in 19 out of 34 tumours (55%), at the cytoplasmic level of tumorous cells. Moreover, there was significant association (p=0.042) between PTP4A3 expression and tumorous size. CONCLUSIONS: PTP4A3 was expressed in more than half of the tumours analysed, with there being a significant association with the tumorous size of functioning adenomas. This allows to speculate that PTP4A3 may regulate tumour growth, although further investigations are necessary to determine if this phosphatase can serve as a biomarker or used as a therapeutic target in pituitary macroadenomas.
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Adenoma/diagnóstico , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisarias/diagnóstico por imagen , Proteínas Tirosina Fosfatasas/metabolismo , Adenoma/metabolismo , Adenoma/patología , Adulto , Citoplasma/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Hipófisis/metabolismo , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Proteínas Tirosina Fosfatasas/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
Identification of allosteric inhibitors of PTPs has attracted great interest as a new strategy to overcome the challenge of discover potent and selective molecules for therapeutic intervention. YopH is a virulence factor of the genus Yersinia, validated as an antimicrobial target. The finding of a second substrate binding site in YopH has revealed a putative allosteric site that could be further exploited. Novel chalcone compounds that inhibit PTPs activity were designed and synthesized. Compound 3j was the most potent inhibitor, interestingly, with different mechanisms of inhibition for the panel of enzymes evaluated. Further, our results showed that compound 3j is an irreversible non-competitive inhibitor of YopH that binds to a site different than the catalytic site, but close to the well-known second binding site of YopH.
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Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Chalcona/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Factores de Virulencia/antagonistas & inhibidores , Sitio Alostérico/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Chalcona/síntesis química , Chalcona/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , Proteínas Tirosina Fosfatasas/metabolismo , Relación Estructura-Actividad , Factores de Virulencia/metabolismoRESUMEN
Colorectal Cancer (CRC) therapy confronts challenges as chemoresistance and side effects. Therefore, drugs with antitumor properties that downmodulate aggressiveness mediators are required. Studies have shown the relevance of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP), Protein Tyrosine Phosphatase 1B (PTP1B), and Transforming Growth Factor ß (TGFß) in mediating proliferation, chemoresistance, and metastasis. In this study, we aimed to investigate the responsiveness of colorectal cancer lines (HT29 and HCT116) towards Vemurafenib and whether this treatment could modulate these aggressiveness mediators. Cytotoxicity Assays (MTT and Trypan Exclusion Test) were performed to evaluate the viability of HT29 and HCT116 cells treated with Vemurafenib. Western blotting was performed to analyze the amount and/or the activity of mediators (LMWPTP, PTP1B, TGFß, SMAD3), and the immunoprecipitation was performed to evaluate LMWPTP activity. This study brought up novel aspects of Vemurafenib action in colorectal cancer, which can decrease the activity of protein tyrosine phosphatases (LMWPTP and PTP1B) and the TGFß pathway, making them important in the CRC aggressiveness. By downmodulating colorectal cancer hallmarks, Vemurafenib appears as an interesting candidate for CRC therapeutic protocols.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Vemurafenib/farmacología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Células HCT116 , Células HT29 , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína smad3/antagonistas & inhibidores , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
In the last decade, several reports highlight the importance of the low molecular weight protein tyrosine phosphatase (LMWPTP) in cancer aggressiveness and resistance. Specifically, in chronic myeloid leukemia, we have reported that high expression of the LMWPTP maintains Src and Bcr-Abl kinases in an activated status and the glucose metabolism is directed to lactate production and, in turn, favor the pentoses pathway (one of the key process for antioxidant and protective responses). In this present study, we investigated the possible correlation between the LMWPTP and autophagy. In resistant chronic myeloid leukemia cells, the antioxidant response is supported by the glycolytic metabolism and antioxidant enzymes such as SOD and catalase, both favored by the LMWPTP. Therefore, when the cells were challenged by hydrogen peroxide treatment, the LMWPTP level goes down as well as SOD, and in turn, autophagy process was stimulated. The findings presented here reveal a novel aspect by which LMWPTP cooperates for the resistance of CML towards stressor stimuli.
Asunto(s)
Antioxidantes/metabolismo , Autofagia , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patologíaRESUMEN
Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/química , Humanos , Modelos Moleculares , Fosforilación , Proteínas Tirosina Fosfatasas/químicaRESUMEN
Vasoinhibin belongs to a family of angiogenesis inhibitors generated when the fourth α-helix (H4) of the hormone prolactin (PRL) is removed by specific proteolytic cleavage. The antiangiogenic properties are absent in uncleaved PRL, indicating that conformational changes create a new bioactive domain. However, the solution structure of vasoinhibin and the location of its bioactive domain are unknown. Molecular dynamic simulation (MD) showed that the loss of H4 exposes the hydrophobic nucleus of PRL and leads to the compression of the molecule into a three-helix bundle that buries the hydrophobic nucleus again. Compression occurs by the movement of loop 1 (L1) and its interaction with α-helix 1 (H1) generating a new L1 conformation with electrostatic and hydrophobic surfaces distinct from those of PRL, that may correspond to a bioactive domain. Consistent with this model, a recombinant protein containing the first 79 amino acids comprising H1 and L1 of human PRL inhibited the proliferation and migration of endothelial cells and upregulated the vasoinhibin target genes, IL1A and ICAM1. This bioactivity was comparable to that of a conventional vasoinhibin having the 123 residues encompassing H1, L1, Η2, L2, and Η3 of human PRL. These findings extend the vasoinhibin family to smaller proteins and provide important structural information, which will aid in antiangiogenic drug development.
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Inhibidores de la Angiogénesis/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Endotelio Vascular/citología , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Proteínas Tirosina Fosfatasas/metabolismo , Inhibidores de la Angiogénesis/química , Movimiento Celular , Proliferación Celular , Células Cultivadas , Endotelio Vascular/fisiología , Humanos , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas Tirosina Fosfatasas/químicaRESUMEN
KEY MESSAGE: The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Ácido Glutámico/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Raíces de Plantas/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/biosíntesis , Proliferación Celular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Proteínas de Transporte de Membrana/metabolismo , Meristema/efectos de los fármacos , Meristema/enzimología , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Mutación/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Proteínas Tirosina Fosfatasas/biosíntesis , Factores de Transcripción/metabolismoRESUMEN
Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.
Asunto(s)
Virus del Orf/enzimología , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Cristalografía por Rayos X , Disulfuros/metabolismo , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Especificidad por SustratoRESUMEN
In chemoresistant leukemia cells (Lucena-1), the low molecular weight protein tyrosine phosphatase (LMWPTP) is about 20-fold more active than in their susceptible counterpart (K562). We found this phosphatase ensures the activated statuses of Src and Bcr-Abl. Since, phosphorylation and dephosphorylation of proteins represent a key post-translational regulation of several enzymes, we also explored the kinome. We hereby show that LMWPTP superactivation, together with kinome reprogramming, cooperate towards glucose addiction. Resistant leukemia cells present lower levels of oxidative metabolism, in part due to downexpression of the following mitochondrial proteins: pyruvate dehydrogenase subunit alpha 1, succinate dehydrogenase, and voltage-dependent anion channel. Those cells displayed higher expression levels of glucose transporter 1 and higher production of lactate. In addition, Lucena-1 siRNA LMWPTP cells showed lower expression levels of glucose transporter 1 and lower activity of lactate dehydrogenase. On the other hand, K562 cells overexpressing LMWPTP presented higher expression/activity of both proteins. In this study, we show that LMWPTP is a pivotal mediator of metabolic reprogramming that confers survival advantages to leukemia cells against death stimuli. J. Cell. Biochem. 118: 3846-3854, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Resistencia a Antineoplásicos , Glucólisis , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Enfermedad Aguda , Humanos , Células K562 , Leucemia/patología , FosforilaciónRESUMEN
The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens.IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia, and EspC, an autotransporter protein secreted by enteropathogenic E. coli, require a functional T3SS for host cell translocation. Both proteins have an intermediate extracellular step before their T3SS-dependent translocation. Here, we show an alternative delivery mechanism for these extracellularly secreted virulence factors that are then incorporated into the T3SS to enter the cells; this novel mechanism coexists with but diverges from the canonical injection model that involves the passage of the protein inside the injectisome.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Yersinia/metabolismo , Transporte de ProteínasRESUMEN
In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)-induced increase in intracellular calcium concentration ([Ca2+ ]i ). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P-induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P-induced acrosome reaction and in the P-induced calcium influx. Motile spermatozoa were capacitated for 18 h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca2+ ]i in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)-FITC, and [Ca2+ ]i was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P-induced acrosome reaction (p < 0.001). However, only the highest concentrations of PKA inhibitors reduced the P-induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P-induced acrosome reaction or the P-induced calcium influx, except for the PP2B inhibitors that significantly reduced the P-induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P-induced acrosome reaction and reduced the amplitude of the P-induced calcium transient (p < 0.001) as well as the amplitude of the plateau phase (p < 0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some point downstream of calcium entry and that Tyr phosphatases have a role on the acrosome reaction upstream of calcium entry.
Asunto(s)
Reacción Acrosómica/fisiología , Calcio/metabolismo , Progesterona/farmacología , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Espermatozoides/metabolismo , Reacción Acrosómica/efectos de los fármacos , Humanos , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/fisiología , Espermatozoides/efectos de los fármacosRESUMEN
Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis.
Asunto(s)
Caspasa 3/metabolismo , Dieta de Carga de Carbohidratos/efectos adversos , Dieta con Restricción de Proteínas/efectos adversos , Regulación hacia Abajo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Pie , Péptidos y Proteínas de Señalización Intracelular , Masculino , Desarrollo de Músculos , Músculo Esquelético/enzimología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteolisis , Distribución Aleatoria , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , UbiquitinaciónRESUMEN
In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.
Asunto(s)
Polisacáridos Bacterianos/biosíntesis , Proteínas Tirosina Fosfatasas/metabolismo , Sinorhizobium meliloti/enzimología , Sinorhizobium meliloti/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Medicago sativa/microbiología , Nodulación de la Raíz de la Planta , Raíces de Plantas/microbiologíaRESUMEN
BACKGROUND: Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. OBJECTIVE: To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. DESIGN, SETTING, AND PARTICIPANTS: Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. RESULTS AND LIMITATIONS: LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. CONCLUSIONS: Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. PATIENT SUMMARY: These findings implicate low-molecular-weight protein tyrosine phosphatase as a novel oncogene in prostate cancer and could offer the possibility of using this protein as biomarker or target for treatment of this disease.