RESUMO
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22 , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Humanos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Mutação com Ganho de Função , Linfócitos T/metabolismo , Linfócitos T/imunologia , Células Jurkat , Células HEK293RESUMO
Chronic lymphocytic leukaemia (CLL) is characterised by malignant mature-like B cells. Supportive to CLL cell survival is chronic B-cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T-helper (Th) cells in a CD40L-dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non-malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient-derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin-1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin-1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin-1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin-1 and CD43-mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B , Ligante de CD40 , Proliferação de Células , Galectina 1 , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos T Auxiliares-IndutoresRESUMO
Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation.
Assuntos
Autoimunidade/imunologia , Imunidade/imunologia , Interferon Tipo I/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Receptores Toll-Like/imunologia , Animais , Artrite/genética , Artrite/imunologia , Autoimunidade/genética , Linhagem Celular , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Sulfato de Dextrana/imunologia , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/genética , Immunoblotting , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Ubiquitinação/imunologiaRESUMO
Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.
Assuntos
Membrana Celular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Homologia de SequênciaRESUMO
Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.
Assuntos
Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Gordura Subcutânea/patologia , Células 3T3-L1 , Adipogenia/genética , Animais , Diferenciação Celular/genética , Respiração Celular , Tamanho Celular , Transporte de Elétrons , Deleção de Genes , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise , Hipertrofia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaboloma , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Biológicos , PPAR gama/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Transdução de Sinais/fisiologia , Sinoviócitos/metabolismo , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Artrite Reumatoide/metabolismo , Proteínas de Ciclo Celular/fisiologia , Humanos , Camundongos , Proteínas de Sinalização YAPRESUMO
Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Bibliotecas de Moléculas Pequenas , Animais , Sítios de Ligação , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Camundongos Obesos , Modelos Biológicos , Estrutura Molecular , Peso Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.
Assuntos
Antígenos CD2/genética , Cromossomos de Mamíferos/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Alelos , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Antígenos CD2/imunologia , Cromossomos de Mamíferos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica/imunologia , Loci Gênicos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). OBJECTIVE: We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. METHODS: Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA. RESULTS: T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive B cells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. CONCLUSION: Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive B cells in contributing to autoimmunity if not properly controlled.
Assuntos
Linfócitos B/imunologia , Antígenos Comuns de Leucócito/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Adulto , Cálcio/metabolismo , Células Cultivadas , Anergia Clonal , Humanos , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Quinase Syk/genética , Regulação para Cima , Quinases da Família src/genéticaRESUMO
OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.
Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/fisiologia , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Artrite Reumatoide/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/transplante , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos Nus , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/transplante , Regulação para CimaRESUMO
The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function in the heart and in cardiac diseases remains unknown. To investigate the in vivo role of LMPTP in cardiac function, we generated mice with genetic inactivation of the Acp1 locus and studied their response to long-term pressure overload. Acp1(-/-) mice develop normally and ageing mice do not show pathology in major tissues under basal conditions. However, Acp1(-/-) mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the postnatal stage, and increased in the adult mouse failing heart. We also show that LMPTP expression increases in end-stage heart failure in humans. Consistent with their protected phenotype, Acp1(-/-) mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of fibrotic genes. Transcriptional profiling and analysis of molecular signalling show that the resistance of Acp1(-/-) mice to pathological cardiac stress correlates with marginal re-expression of fetal cardiac genes, increased insulin receptor beta phosphorylation, as well as PKA and ephrin receptor expression, and inactivation of the CaMKIIδ pathway. Our data show that ablation of Lmptp inhibits pathological cardiac remodelling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of human heart failure.
Assuntos
Insuficiência Cardíaca/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Cardiomiopatia de Takotsubo/metabolismo , Animais , Modelos Animais de Doenças , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RatosRESUMO
Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.
Assuntos
Alanina/análogos & derivados , Bioensaio/métodos , Cumarínicos/química , Corantes Fluorescentes/química , Organofosfatos/química , Peptídeos/química , Proteínas Tirosina Fosfatases/química , Alanina/química , Eletroforese em Gel de Poliacrilamida , Humanos , Peptídeos/genética , Proteínas Tirosina Fosfatases/genética , Especificidade por SubstratoRESUMO
Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.
Assuntos
Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/metabolismo , Antraz/tratamento farmacológico , Antraz/metabolismo , Bacillus anthracis , Citoproteção/efeitos dos fármacos , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo/métodos , Humanos , Células Jurkat , Oligopeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
OBJECTIVE: The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS. METHODS: A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides. RESULTS: PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION: These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.
Assuntos
Artrite Reumatoide/enzimologia , Fibroblastos/enzimologia , Osteoartrite/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Membrana Sinovial/enzimologia , Artrite Reumatoide/genética , Linhagem Celular , Movimento Celular , Fibroblastos/patologia , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Oligonucleotídeos Antissenso/farmacologia , Osteoartrite/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais , Membrana Sinovial/patologia , Regulação para CimaRESUMO
PTPN22 encodes a tyrosine phosphatase that inhibits Src-family kinases responsible for Ag receptor signaling in lymphocytes and is strongly linked with susceptibility to a number of autoimmune diseases. As strength of TCR signal is critical to the thymic selection of regulatory T cells (Tregs), we examined the effect of murine PTPN22 deficiency on Treg development and function. In the thymus, numbers of pre-Tregs and Tregs increased inversely with the level of PTPN22. This increase in Tregs persisted in the periphery and could play a key part in the reduced severity observed in the PTPN22-deficient mice of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This could explain the lack of association of certain autoimmune conditions with PTPN22 risk alleles.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/fisiologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Timo/enzimologia , Timo/imunologia , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 22/deficiência , Linfócitos T Reguladores/patologia , Timo/patologia , Regulação para Cima/imunologiaRESUMO
OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.
Assuntos
Artrite Reumatoide , Microdissecção e Captura a Laser , Análise de Sequência de RNA , Membrana Sinovial , Humanos , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Membrana Sinovial/patologia , Membrana Sinovial/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Transcriptoma , Idoso , Osteoartrite/genética , Osteoartrite/patologia , Análise de Componente Principal , Perfilação da Expressão GênicaRESUMO
Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Peso Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Tirosina , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000-member library of drugs and drug-like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac-ARLIEDNE-pCAP-TAREG-NH2, peptide 1) and a small-molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin-3,5-digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC50 of 50 nM and significant activity in T-cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Fosfotirosina/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Células Cultivadas , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Células Jurkat , Modelos Moleculares , Estrutura Molecular , Peptídeos/análise , Peptídeos/química , Fosfotirosina/análogos & derivados , Fosfotirosina/química , Proteínas Tirosina Fosfatases/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Protein phosphatases act as key regulators of multiple important cellular processes and are attractive therapeutic targets for various diseases. Although extensive effort has been dedicated to phosphatase-targeted drug discovery, early expeditions for competitive phosphatase inhibitors were plagued by druggability issues, leading to the stigmatization of phosphatases as difficult targets. Despite challenges, persistent efforts have led to the identification of several drug-like, non-competitive modulators of some of these enzymes - including SH2 domain-containing protein tyrosine phosphatase 2, protein tyrosine phosphatase 1B, vascular endothelial protein tyrosine phosphatase and protein phosphatase 1 - reigniting interest in therapeutic targeting of phosphatases. Here, we discuss recent progress in phosphatase drug discovery, with emphasis on the development of selective modulators that exhibit biological activity. The roles and regulation of protein phosphatases in immune cells and their potential as powerful targets for immuno-oncology and autoimmunity indications are assessed.
Assuntos
Doenças Autoimunes , Neoplasias , Humanos , Proteínas Tirosina Fosfatases/metabolismo , Fosfoproteínas Fosfatases , Doenças Autoimunes/tratamento farmacológico , Neoplasias/tratamento farmacológico , ImunoterapiaRESUMO
More than half of the known protein tyrosine phosphatases (PTPs) in the human genome are expressed in T cells, and significant progress has been made in elucidating the biology of these enzymes in T-cell development and function. Here we provide a systematic review of the current understanding of the roles of PTPs in T-cell activation, providing insight into their mechanisms of action and regulation in T-cell receptor signalling, the phenotypes of their genetically modified mice, and their possible involvement in T-cell-mediated autoimmune disease. Our projection is that the interest in PTPs as mediators of T-cell homeostasis will continue to rise with further functional analysis of these proteins, and PTPs will be increasingly considered as targets of immunomodulatory therapies.