RESUMO
IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.
Assuntos
Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Animais , Camundongos , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias de Mama Triplo Negativas/genética , Microambiente Tumoral , Neoplasias Pulmonares/genética , Interleucina-1alfa/genéticaRESUMO
The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.
Assuntos
Ciclofilina A , Linfócitos T , Camundongos , Animais , Humanos , Ciclofilina A/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Ativação Linfocitária , Timo/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.
Assuntos
Actinas , Cardiomiopatia Dilatada , Criança , Animais , Camundongos , Humanos , Actinas/genética , Actinas/metabolismo , Cardiomiopatia Dilatada/metabolismo , Citoesqueleto de Actina/metabolismo , Fenótipo , Morte Súbita Cardíaca/etiologia , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Células COS , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Chlorocebus aethiops , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Metilação , Complexo Repressor Polycomb 2/análise , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de ProteínasRESUMO
Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Adolescente , Autofagia/genética , Autofagia/fisiologia , Cardiomiopatia Dilatada/genética , Criança , Feminino , Genótipo , Humanos , Masculino , Mutação/genéticaRESUMO
NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Células Matadoras Naturais/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Sindecana-4/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Humanos , Neoplasias/imunologia , Ligação Proteica/imunologia , Receptores Imunológicos/imunologiaRESUMO
Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express â¼ 5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was â¼ 5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly(219)-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.
Assuntos
Ciclofilina A/metabolismo , Regulação da Expressão Gênica/fisiologia , Imunofilinas/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Humanos , Células Jurkat , Transdução de Sinais/fisiologiaRESUMO
Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII, the two alternative spliced forms of CRK, possess an N-terminal Src homology 2 domain, followed by a Src homology 3 (SH3) domain, whereas CrkII possesses in addition a C-terminal linker region plus a SH3 domain, which operate as regulatory moieties. In this study, we investigated the ability of immunophilins, which function as peptidyl-prolyl isomerases, to regulate Crk proteins in human T lymphocytes. We found that endogenous CrkII, but not CrkI, associates with the immunophilins, cyclophilin A, and 12-kDa FK506-binding protein, in resting human Jurkat T cells. In addition, cyclophilin A increased Crk SH3 domain-binding guanine-nucleotide releasing factor (C3G) binding to CrkII, whereas inhibitors of immunophilins, such as cyclosporine A (CsA) and FK506, inhibited CrkII, but not CrkI association with C3G. Expression in Jurkat T cells of phosphorylation indicator of Crk chimeric unit plasmid, a plasmid encoding the human CrkII1-236 sandwiched between cyan fluorescent protein and yellow fluorescent protein, demonstrated a basal level of fluorescence resonance energy transfer, which increased in response to cell treatment with CsA and FK506, reflecting increased trans-to-cis conversion of CrkII. Crk-C3G complexes are known to play an important role in integrin-mediated cell adhesion and migration. We found that overexpression of CrkI or CrkII increased adhesion and migration of Jurkat T cells. However, immunophilin inhibitors suppressed the ability of CrkII- but not CrkI-overexpressing cells to adhere to fibronectin-coated surfaces and migrate toward the stromal cell-derived factor 1α chemokine. The present data demonstrate that immunophilins regulate CrkII, but not CrkI activity in T cells and suggest that CsA and FK506 inhibit selected effector T cell functions via a CrkII-dependent mechanism.
Assuntos
Ciclofilina A/antagonistas & inibidores , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Linfócitos T/imunologia , Adesão Celular/imunologia , Movimento Celular/imunologia , Quimiocina CXCL12/imunologia , Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Plasmídeos/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/imunologia , Tacrolimo/farmacologia , Domínios de Homologia de src/genéticaRESUMO
Amyloid-ß peptides generated by proteolysis of the ß-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2 α (cPLA2 α) plays a role in elevated APP protein expression induced by aggregated amyloid-ß1-42 (Aß) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2 α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aß for 24 h and in cortical neuron of human amyloid-ß1-42 brain infused mice. Prevention of cPLA2 α up-regulation and its activity by oligonucleotide antisense against cPLA2 α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2 α in the signals leading to APP induction, increased cPLA2 α expression and activity induced by Aß was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2 ) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aß. Inhibition of elevated cPLA2 α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aß which coincided with cPLA2 α dependent activation of CREB in the cortex of Aß brain infused mice. Our results show that accumulation of Aß induced elevation of APP protein expression mediated by cPLA2 α, PGE2 release, and CREB activation via protein kinase A pathway.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Fosfolipases A2 do Grupo IV/metabolismo , Degeneração Neural/metabolismo , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Doença de Alzheimer/patologia , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.
Assuntos
Heparitina Sulfato/metabolismo , Células Matadoras Naturais/imunologia , Receptores KIR2DL4/metabolismo , Sulfotransferases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Linhagem Celular , Cricetulus , Endocitose , Células HEK293 , Heparina/metabolismo , Humanos , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Receptores KIR2DL4/genética , Receptores KIR2DL4/imunologia , Transdução de Sinais/imunologia , Sindecana-4/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.
Assuntos
Células Matadoras Naturais/química , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/química , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Linhagem Celular , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
Cellular senescence (CS) is recognized as one of the hallmarks of aging, and an important player in a variety of age-related pathologies. Accumulation of senescent cells can promote a pro-inflammatory and pro-cancerogenic microenvironment. Among potential senotherapeutics are extracellular vesicles (EVs) (40-1000â¯nm), including exosomes (40-150â¯nm), that play an important role in cell-cell communications. Here, we review the most recent studies on the impact of EVs derived from stem cells (MSCs, ESCs, iPSCs) as well as non-stem cells of various types on CS and discuss potential mechanisms responsible for the senotherapeutic effects of EVs. The analysis revealed that (i) EVs derived from stem cells, pluripotent (ESCs, iPSCs) or multipotent (MSCs of various origin), can mitigate the cellular senescence phenotype both in vitro and in vivo; (ii) this effect is presumably senomorphic; (iii) EVs display cross-species activity, without apparent immunogenic responses. In summary, stem cell-derived EVs appear to be promising senotherapeutics, with a feasible application in humans.
Assuntos
Senescência Celular , Vesículas Extracelulares , Senoterapia , Humanos , Vesículas Extracelulares/fisiologia , Senescência Celular/fisiologia , Animais , Senoterapia/farmacologia , Células-Tronco/fisiologia , Envelhecimento/fisiologiaRESUMO
Radiation therapy (RT) remains a common treatment for cancer patients worldwide, despite the development of targeted biological compounds and immunotherapeutic drugs. The challenge in RT lies in delivering a lethal dose to the cancerous site while sparing the surrounding healthy tissues. Low linear energy transfer (low-LET) and high linear energy transfer (high-LET) radiations have distinct effects on cells. High-LET radiation, such as alpha particles, induces clustered DNA double-strand breaks (DSBs), potentially inducing cell death more effectively. However, due to limited range, alpha-particle therapies have been restricted. In human cancer, mutations in TP53 (encoding for the p53 tumor suppressor) are the most common genetic alteration. It was previously reported that cells carrying wild-type (WT) p53 exhibit accelerated senescence and significant rates of apoptosis in response to RT, whereas cells harboring mutant p53 (mutp53) do not. This study investigated the combination of the alpha-emitting atoms RT based on internal Radium-224 (224Ra) sources and systemic APR-246 (a p53 reactivating compound) to treat tumors with mutant p53. Cellular models of colorectal cancer (CRC) or pancreatic ductal adenocarcinoma (PDAC) harboring mutant p53, were exposed to alpha particles, and tumor xenografts with mutant p53 were treated using 224Ra source and APR-246. Effects on cell survival and tumor growth, were assessed. The spread of alpha emitters in tumors was also evaluated as well as the spatial distribution of apoptosis within the treated tumors. We show that mutant p53 cancer cells exhibit radio-sensitivity to alpha particles in vitro and to alpha-particles-based RT in vivo. APR-246 treatment enhanced sensitivity to alpha radiation, leading to reduced tumor growth and increased rates of tumor eradication. Combining alpha-particles-based RT with p53 restoration via APR-246 triggered cell death, resulting in improved therapeutic outcomes. Further preclinical and clinical studies are needed to provide a promising approach for improving treatment outcomes in patients with mutant p53 tumors.
Assuntos
Partículas alfa , Radiossensibilizantes , Proteína Supressora de Tumor p53 , Partículas alfa/uso terapêutico , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Camundongos , Radiossensibilizantes/farmacologia , Mutação , Quinuclidinas/farmacologia , Linhagem Celular Tumoral , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias/radioterapia , Neoplasias/genética , Neoplasias/patologiaRESUMO
Robust elevation of the cytosolic calcium concentration is a crucial early step for T cell activation triggered by the T cell antigen receptor. Vav1 is a proto-oncogene expressed in hematopoietic cells that is indispensable for transducing the calcium-mobilizing signal. Following T cell receptor stimulation, Vav1 facilitates formation of signaling microclusters through multiple interactions with other proteins participating in the signaling cascade. Truncation of the N terminus of Vav1 produces its oncogenic version, which is unable to support normal calcium flux following T cell activation. We show here that truncation of the N-terminal region of Vav1 alters the fine structure of protein complexes in the signaling clusters, affecting the interaction of Vav1 with phospholipase Cγ1 (PLCγ1). This alteration is accompanied by a decrease in PLCγ1 phosphorylation and inhibition of inositol 1,4,5-trisphosphate production. We suggest that the structural integrity of the N-terminal region of Vav1 is important for the proper formation of the Vav1-associated signaling complexes. The oncogenic truncation of this region elicits conformational changes that interfere with the Vav1-mediated activation of PLCγ1 and that inhibit calcium mobilization.
Assuntos
Cálcio/metabolismo , Mutação , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linhagem Celular , Ativação Enzimática/genética , Humanos , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Ativação Linfocitária/genética , Fosfolipase C gama/genética , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-vav/genética , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1ß in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1ß, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1ß correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1ß can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1ß promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.
Assuntos
Quimiotaxia de Leucócito/imunologia , Mediadores da Inflamação/fisiologia , Interleucina-1alfa/fisiologia , Interleucina-1beta/fisiologia , Células Mieloides/imunologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito/genética , Células HEK293 , Humanos , Inflamação/classificação , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/classificação , Interleucina-1alfa/deficiência , Interleucina-1alfa/genética , Interleucina-1beta/deficiência , Interleucina-1beta/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
NK cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only natural cytotoxicity receptor that is expressed exclusively by primate NK cells, yet its cellular ligands remain largely unknown. Proliferating cell nuclear Ag (PCNA) is overexpressed in cancer cells. In this study, we show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through NKp44/ITIM. The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Antígeno Nuclear de Célula em Proliferação/imunologia , Evasão Tumoral/imunologia , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Sinapses Imunológicas/imunologia , Imunoprecipitação , Ligantes , Microscopia Confocal , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
IL-1alpha, like IL-1beta, possesses multiple inflammatory and immune properties. However, unlike IL-1beta, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1alpha translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1alpha is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1alpha concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1alpha is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking IL-1alpha failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (20-50 pg) of the IL-1alpha precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive IL-1alpha precursor was released into the supernatant. Thus, after an ischemic event, the IL-1alpha precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by IL-1alpha is tightly controlled.
Assuntos
Apoptose , Cromatina/metabolismo , Interleucina-1alfa/metabolismo , Animais , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interleucina-1alfa/genética , Interleucina-1alfa/farmacologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Necrose , Transporte Proteico , TransfecçãoRESUMO
Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at â¼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.
Assuntos
Transdução de Sinais , Linfócitos T , Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Domínios de Homologia de src , Fosforilação , Ubiquitinação , Proteínas Proto-Oncogênicas c-cbl/metabolismoRESUMO
Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.
Assuntos
Peptídeos , Peptidilprolil Isomerase , Animais , Camundongos , Humanos , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteína Quinase C-theta/genética , Camundongos Endogâmicos C57BL , Peptidilprolil Isomerase de Interação com NIMA/genética , Receptores de Antígenos de Linfócitos T , Prolina/química , Prolina/metabolismoRESUMO
BACKGROUND: The study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals. METHODS: Plasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy. RESULTS: The results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself. CONCLUSIONS: The dual fluorescent labelling is a valid method for detecting heavy metal accumulation in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.