RESUMO
Protein arginine N-methyltransferases (PRMTs) selectively replace N-H for N-CH(3) at substrate protein guanidines, a post-translational modification important for a range of biological processes, such as epigenetic regulation, signal transduction and cancer progression. Selective chemical probes are required to establish the dynamic function of individual PRMTs. Herein, model inhibitors designed to occupy PRMT binding sites for an arginine substrate and S-adenosylmethionine (AdoMet) co-factor are described. Expedient access to such compounds by modular synthesis is detailed. Remarkably, biological evaluation revealed some compounds to be potent inhibitors of PRMT1, but inactive against CARM1. Docking studies show how prototype compounds may occupy the binding sites for a co-factor and arginine substrate. Overlay of PRMT1 and CARM1 binding sites suggest a difference in a single amino acid that may be responsible for the observed selectivity.
Assuntos
Arginina/metabolismo , Inibidores Enzimáticos/síntese química , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , S-Adenosilmetionina/metabolismo , Arginina/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Escherichia coli , Humanos , Metilação , Modelos Moleculares , Peso Molecular , Plasmídeos , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/genética , S-Adenosilmetionina/antagonistas & inibidores , Especificidade por Substrato , Transformação BacterianaRESUMO
SHIP-1 negatively regulates the PI3K pathway in hematopoietic cells and has an emerging role in T lymphocyte biology. PI3K and SHIP can regulate cell migration in leukocytes, particularly in neutrophils, although their role in T cell migration has been less clear. Therefore, we sought to explore the role of SHIP-1 in human CD4(+) T lymphocyte cell migration responses to chemoattractants using a lentiviral-mediated expression system and a short hairpin RNA approach. Silencing of SHIP-1 leads to increased basal phosphorylation of protein kinase B/Akt and its substrate GSK3ß, as well as an increase in basal levels of polymerized actin, suggesting that SHIP-1 might regulate changes in the cytoskeleton. Accordingly, silencing of SHIP-1 led to loss of microvilli and ezrin/radixin/moesin phosphorylation, which could not be rescued by the PI3K inhibitor Ly294002. There were striking morphological changes, including a loss of microvilli projections, which mirrored changes in wild type cells after stimulation with the chemokine CXCL11. There was no defect in directional T cell migration toward CXCL11 in the SHIP-1-silenced cells but, importantly, there was a defect in the overall basal motility of SHIP-1 knockdown cells. Taken together, these results implicate SHIP-1 as a key regulator of basal PI3K signaling in human CD4(+) T lymphocytes with important phosphatase-independent actions, which together are key for maintaining normal morphology and basal motility.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Movimento Celular , Monoéster Fosfórico Hidrolases/metabolismo , Actinas/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/ultraestrutura , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL11/farmacologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citometria de Fluxo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Inositol Polifosfato 5-Fosfatases , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Varredura , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
Prototype inhibitors of protein arginine methyltransferases (PRMTs) have been constructed by attaching guanidine functionality via a variable linker to non-reactive amine analogues of the cellular co-factor (S)-adenosyl methionine (AdoMet). Potent inhibition of PRMT1 (IC(50) of approximately 3-6 microM) combined with weak inhibition of the lysine methyltransferase SET7 (approximately 50% of activity at 100 microM) was observed for two such compounds.
Assuntos
Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Concentração Inibidora 50 , Metilação , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Especificidade por SubstratoRESUMO
The phosphoinositide 3-kinase signaling pathway regulates a range of T lymphocyte cellular functions including growth, proliferation, cytokine secretion and survival. Aberrant regulation of phosphoinositide 3-kinase-dependent signaling in T lymphocytes has been implicated in inflammatory and autoimmune diseases. In common with much of the immune system, several mechanisms exist to ensure the pathway is tightly regulated to elicit appropriate responses. One level of control involves the Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1) that modulates phosphoinositide 3-kinase signaling by degrading the key signaling lipid PI(3,4,5)P(3) to PI(3,4)P(2), but also serves as a key scaffolding molecule in the formation of multi-protein complexes. Here we discuss the role of SHIP-1 in regulating T lymphocyte and immune function, as well as its potential as a therapeutic target.
Assuntos
Doenças Autoimunes/enzimologia , Proliferação de Células , Monoéster Fosfórico Hidrolases/metabolismo , Linfócitos T/enzimologia , Animais , Doenças Autoimunes/imunologia , Sobrevivência Celular/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inositol Polifosfato 5-Fosfatases , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/imunologia , Linfócitos T/imunologia , Domínios de Homologia de src/imunologiaRESUMO
To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Assuntos
Engenharia Genética/métodos , Imunoterapia Adotiva/métodos , Linfócitos T/transplante , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Linfócitos T/enzimologia , Animais , Movimento Celular , Desenho de Fármacos , Marcação de Genes , Humanos , Inositol Polifosfato 5-Fosfatases , Ativação Linfocitária , Modelos Biológicos , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kgamma provides a key migratory signal for T lymphocyte migration. Since PI3Kgamma can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kgamma was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kgamma), class II (PI3KC2alpha and PI3KC2beta) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kgamma. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.
Assuntos
Técnicas de Cultura de Células , Quimiotaxia de Leucócito , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Transfecção/métodos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Fatores de TempoRESUMO
Stimulation of resting CD4 T cells with anti-CD3/CD28-coated beads leads to rapid polarization of lipid rafts (LRs). It has been postulated that a major role of costimulation is to facilitate LR aggregation. CD86 is up-regulated or expressed aberrantly on immune cells in a wide array of autoimmune and infectious diseases. Using an Ig fusion with the extracellular domain of CD86 (CD86Ig) bound to a magnetic bead or K562 cells expressing CD86, we demonstrated that ligation of CD28 by its natural ligand, but not by Ab, induced polarization of LRs at the cell-bead interface of fresh human CD4 T cells in the absence of TCR ligation. This correlated with activation of Vav-1, increase of the intracellular calcium concentration, and nuclear translocation of NF-kappaB p65, but did not result in T cell proliferation or cytokine production. These studies show, for the first time, that LR polarization can occur in the absence of TCR triggering, driven solely by the CD28/CD86 interaction. This result has implications for mechanisms of T cell activation. Abnormalities in this process may alter T and B cell tolerance and susceptibility to infection.
Assuntos
Antígeno B7-2/fisiologia , Antígenos CD28/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Microdomínios da Membrana/fisiologia , Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Cálcio/metabolismo , Células Cultivadas , Humanos , Ativação Linfocitária , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T , Fator de Transcrição RelA/metabolismoRESUMO
CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.
Assuntos
Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/fisiologia , Antígenos CD , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Antígeno CTLA-4 , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Ácido Okadáico/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Receptor de Morte Celular Programada 1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.
Assuntos
Apoptose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Antígenos CD , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose , Antígenos CD28/genética , Antígenos CD28/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Receptor de Morte Celular Programada 1 , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/metabolismoRESUMO
Ligation of either CD28 or inducible costimulatory protein (ICOS) produces a second signal required for optimal T cell activation and proliferation. One prominent difference between ICOS- and CD28-costimulated T cells is the quantity of IL-2 produced. To understand why CD28 but not ICOS elicits major increases in IL-2 expression, we compared the abilities of these molecules to activate the signal transduction cascades implicated in the regulation of IL-2. Major differences were found in the regulation of phosphatidylinositol 3-kinase activity (PI3K) and c-jun N-terminal kinase. ICOS costimulation led to greatly augmented levels of PI3K activity compared with CD28 costimulation, whereas only CD28 costimulation activated c-jun N-terminal kinase. To examine how these differences in signal transduction affected IL-2 production, we transduced primary human CD4 T cells with a lentiviral vector that expressed the murine CD28 extracellular domain with a variety of human CD28 and ICOS cytoplasmic domain swap constructs. These domains were able to operate as discrete signaling units, suggesting that they can function independently. Our results show that even though the ICOS Src homology (SH) 2 binding domain strongly activated PI3K, it was unable to substitute for the CD28 SH2 binding domain to induce high levels of IL-2 and Bcl-x(L). Moreover, the CD28 SH2 binding domain alone was sufficient to mediate optimal levels of Bcl-x(L) induction, whereas the entire CD28 cytoplasmic tail was required for high levels of IL-2 expression. Thus, differences within their respective SH2 binding domains explain, at least in part, the distinct regulation of IL-2 and Bcl-x(L) expression following ICOS- or CD28-mediated costimulation.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos CD28/fisiologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-2/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Domínios de Homologia de src/fisiologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Citoplasma/genética , Citoplasma/fisiologia , Ativação Enzimática/imunologia , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Interleucina-2/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Imunológicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína bcl-X , Domínios de Homologia de src/genéticaRESUMO
Lymphocyte activation initiates a program of cell growth, proliferation, and differentiation that increases metabolic demand. Although T cells increase glucose uptake and glycolysis during an immune response, the signaling pathways that regulate these increases remain largely unknown. Here we show that CD28 costimulation, acting through phosphatidylinositol 3'-kinase (PI3K) and Akt, is required for T cells to increase their glycolytic rate in response to activation. Furthermore, CD28 controls a primary response pathway, inducing a level of glucose uptake and glycolysis in excess of that needed to maintain cellular ATP/ADP levels or macromolecular synthesis. These data suggest that CD28 costimulation functions to increase glycolytic flux, allowing T cells to anticipate energetic and biosynthetic needs associated with a sustained response.