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1.
Nat Commun ; 12(1): 5337, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504101

RESUMO

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.


Assuntos
Proteínas 14-3-3/genética , Proteínas Fetais/genética , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/genética , Ubiquitina/genética , Proteínas 14-3-3/metabolismo , Células A549 , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Fetais/antagonistas & inibidores , Proteínas Fetais/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Biochim Biophys Acta Rev Cancer ; 1876(2): 188619, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34454048

RESUMO

Phosphoinositide metabolism is crucial intracellular signaling system that regulates a plethora of biological functions including mitogenesis, cell proliferation and division. Phospholipase C gamma 1 (PLCγ1) which belongs to phosphoinositide-specific phospholipase C (PLC) family, is activated by many extracellular stimuli including hormones, neurotransmitters, growth factors and modulates several cellular and physiological functions necessary for tumorigenesis such as cell survival, migration, invasion and angiogenesis by generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) via hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2). Cancer remains as a leading cause of global mortality and aberrant expression and regulation of PLCγ1 is linked to a plethora of deadly human cancers including carcinomas of the breast, lung, pancreas, stomach, prostate and ovary. Although PLCγ1 cross-talks with many onco-drivers and signaling circuits including PI3K, AKT, HIF1-α and RAF/MEK/ERK cascade, its precise role in carcinogenesis is not completely understood. This review comprehensively discussed the status quo of this ubiquitously expressed phospholipase as a tumor driver and highlighted its significance as a novel therapeutic target in cancer. Furthermore, we have highlighted the significance of somatic driver mutations in PLCG1 gene and molecular roles of PLCγ1 in several major human cancers, a knowledgebase that can be utilized to develop novel, isoform-specific small molecule inhibitors of PLCγ1.


Assuntos
Neoplasias/enzimologia , Neoplasias/patologia , Fosfolipase C gama/metabolismo , Carcinogênese , Proliferação de Células/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Transdução de Sinais
3.
Eur J Immunol ; 51(9): 2251-2265, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34323286

RESUMO

Bruton's tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B- cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B-cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF-κB and Akt/S6 signaling was decreased in Btk-deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk-effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho-CD79a and phospho-PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfócitos B/imunologia , Antígenos CD79/metabolismo , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Antineoplásicos/farmacologia , Linfócitos B/citologia , Benzamidas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunoglobulina M/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Transdução de Sinais/imunologia , Quinase Syk/metabolismo
4.
Molecules ; 26(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062884

RESUMO

Osteoporosis is a systemic metabolic bone disorder that is caused by an imbalance in the functions of osteoclasts and osteoblasts and is characterized by excessive bone resorption by osteoclasts. Targeting osteoclast differentiation and bone resorption is considered a good fundamental solution for overcoming bone diseases. ß-boswellic acid (ßBA) is a natural compound found in Boswellia serrata, which is an active ingredient with anti-inflammatory, anti-rheumatic, and anti-cancer effects. Here, we explored the anti-resorptive effect of ßBA on osteoclastogenesis. ßBA significantly inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by receptor activator of nuclear factor-B ligand (RANKL) and suppressed bone resorption without any cytotoxicity. Interestingly, ßBA significantly inhibited the phosphorylation of IκB, Btk, and PLCγ2 and the degradation of IκB. Additionally, ßBA strongly inhibited the mRNA and protein expression of c-Fos and NFATc1 induced by RANKL and subsequently attenuated the expression of osteoclast marker genes, such as OC-STAMP, DC-STAMP, ß3-integrin, MMP9, ATP6v0d2, and CtsK. These results suggest that ßBA is a potential therapeutic candidate for the treatment of excessive osteoclast-induced bone diseases such as osteoporosis.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Reabsorção Óssea , Regulação da Expressão Gênica , Osteoclastos/metabolismo , Fosfolipase C gama/metabolismo , Ligante RANK , Triterpenos/farmacologia , Animais , Boswellia , Diferenciação Celular , Técnicas de Cocultura , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Fosforilação , Transdução de Sinais
5.
Front Immunol ; 12: 667430, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093563

RESUMO

Background: Autoinflammatory phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) is a rare autoinflammatory disease caused by gain-of-function mutations in the PLCG2 gene. Here we report a rare case of APLAID patient carrying a novel heterozygous missense PLCG2 I169V mutation with gangrenous pyoderma and concomitant high serum immunoglobulin (Ig) E level. Methods: The patient was diagnosed as APLAID and has been treated in our department. His phenotype and genotype were carefully documented and studied. We also conducted a comprehensive literature review on APLAID. Results: A 23-year-old Chinese Han man presented with recurrent fever for 18 years and vesiculopustular rashes for 9 years, along with chronic bronchitis, leukocytosis, increased C-reactive protein, immunodeficiency and high serum IgE. Skin biopsy showed chronic inflammatory cells infiltration. A paternal heterozygous missense variant in exon 6 of the PLCG2 gene p. I169V was identified. His vesiculopustular and IgE level responded to medium dose corticosteroids. After withdrawal of steroids, he developed severe arthritis and a large deteriorating ulceration resembling pyoderma gangrenosum on the left knee. Large dose corticosteroids were suboptimal. Then he received adalimumab with satisfactory response for arthritis and skin lesion. But he got an immunodeficiency-associated lymphoproliferative disorder 2 months later. Through literature review, there were a total of 10 APLAID patients reported by six English-language publications. Vesiculopustular rashes, sinopulmonary infection and immunodeficiency were the most frequent symptoms of APLAID patients. Glucocorticoids, intravenous immunoglobulin and biologics were clinically used to treat APLAID but none of these patients had a complete recovery. Conclusions: The rarity and diversity of APLAID make it difficult to be diagnosed. Our study reported the first case of APLAID with gangrenous pyoderma and concomitant high IgE carrying a novel PLCG2 mutation, which may expand the clinical phenotype and genotype of APLAID.


Assuntos
Síndromes de Imunodeficiência/genética , Fosfolipase C gama/genética , Pioderma Gangrenoso/genética , Éxons , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/enzimologia , Masculino , Mutação de Sentido Incorreto , Fenótipo , Fosfolipase C gama/metabolismo , Pioderma Gangrenoso/complicações , Adulto Jovem
6.
PLoS Pathog ; 17(6): e1009635, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34143834

RESUMO

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman's Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y481EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y481EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y481EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.


Assuntos
Infecções por Herpesviridae/metabolismo , Fosfolipase C gama/metabolismo , Proteínas não Estruturais Virais/metabolismo , Quinases da Família src/metabolismo , Células HEK293 , Herpesvirus Humano 8/fisiologia , Humanos , Fosforilação , Ativação Viral/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia
7.
Cells ; 10(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073225

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton's tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.


Assuntos
Tirosina Quinase da Agamaglobulinemia/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Proteínas Tirosina Quinases/efeitos dos fármacos , Tirosina Quinase da Agamaglobulinemia/metabolismo , Artrite Reumatoide/metabolismo , Linfócitos B/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo
8.
Front Immunol ; 12: 689472, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34177947

RESUMO

Since the first clinical report in 2013, inhibitors of the intracellular kinase BTK (BTKi) have profoundly altered the treatment paradigm of B cell malignancies, replacing chemotherapy with targeted agents in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström's macroglobulinemia. There are over 20 BTKi, both irreversible and reversible, in clinical development. While loss-of-function (LoF) mutations in the BTK gene cause the immunodeficiency X-linked agammaglobulinemia, neither inherited, nor somatic BTK driver mutations are known. Instead, BTKi-sensitive malignancies are addicted to BTK. BTK is activated by upstream surface receptors, especially the B cell receptor (BCR) but also by chemokine receptors, and adhesion molecules regulating B cell homing. Consequently, BTKi therapy abrogates BCR-driven proliferation and the tissue homing capacity of the malignant cells, which are being redistributed into peripheral blood. BTKi resistance can develop over time, especially in MCL and high-risk CLL patients. Frequently, resistance mutations affect the BTKi binding-site, cysteine 481, thereby reducing drug binding. Less common are gain-of-function (GoF) mutations in downstream signaling components, including phospholipase Cγ2 (PLCγ2). In a subset of patients, mechanisms outside of the BCR pathway, related e.g. to resistance to apoptosis were described. BCR signaling depends on many proteins including SYK, BTK, PI3K; still based on the resistance pattern, BTKi therapy only selects GoF alterations in the NF-κB arm, whereas an inhibitor of the p110δ subunit of PI3K instead selects resistance mutations in the RAS-MAP kinase pathway. BTK and PLCγ2 resistance mutations highlight BTK's non-redundant role in BCR-mediated NF-κB activation. Of note, mutations affecting BTK tend to generate clone sizes larger than alterations in PLCγ2. This infers that BTK signaling may go beyond the PLCγ2-regulated NF-κB and NFAT arms. Collectively, when comparing the primary and acquired mutation spectrum in BTKi-sensitive malignancies with the phenotype of the corresponding germline alterations, we find that certain observations do not readily fit with the existing models of BCR signaling.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfócitos B/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hematológicas/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Regulação Neoplásica da Expressão Gênica , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Transdução de Sinais/genética , Proteínas ras/genética , Proteínas ras/metabolismo
9.
Allergol Immunopathol (Madr) ; 49(3): 42-49, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33938187

RESUMO

BACKGROUND: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). MATERIALS AND METHODS: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. RESULTS: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. CONCLUSIONS: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases.


Assuntos
Degranulação Celular/efeitos dos fármacos , Dissacarídeos/farmacologia , Eicosanoides/biossíntese , Mediadores da Inflamação/metabolismo , Inula/química , Mastócitos/efeitos dos fármacos , Quercetina/análogos & derivados , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Leucotrieno C4/biossíntese , Mastócitos/metabolismo , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fosfolipase C gama/metabolismo , Fosfolipases A2/metabolismo , Fosforilação/efeitos dos fármacos , Prostaglandina D2/biossíntese , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33929486

RESUMO

The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here, we report that phospholipase Cγ1 (PLCγ1) promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly cross-links LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK activation and SLP76 phosphorylation. Intriguingly, a nonmonotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.


Assuntos
Ativação Linfocitária/imunologia , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Animais , Bovinos , Humanos , Células Jurkat , Fosfolipase C gama/genética , Fosforilação , Ligação Proteica , Linfócitos T/imunologia
11.
Arthritis Rheumatol ; 73(9): 1614-1625, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33645887

RESUMO

OBJECTIVE: Gain-of-function mutations and genome-wide association studies have linked phospholipase Cγ2 (PLCγ2) to various inflammatory diseases, including arthritis in humans and mice. PLCγ2-deficient (Plcg2-/- ) mice are also protected against experimental arthritis. This study was undertaken to test how PLCγ2 triggers autoantibody-induced arthritis in mice. METHODS: PLCγ2 was deleted from various mouse cellular lineages. Deletion efficacy and specificity were tested by immunoblotting and intracellular flow cytometry. Autoantibody-induced arthritis was triggered by K/BxN serum transfer. The role of neutrophil PLCγ2 was further investigated by analysis of the inflammatory exudate, competitive in vivo migration assays, and in vitro functional studies. RESULTS: PLCγ2 deficiency in the entire hematopoietic compartment completely blocked autoantibody-induced arthritis. Arthritis development was abrogated by deletion of PLCγ2 from myeloid cells or neutrophils but not from mast cells or platelets. Neutrophil infiltration was reduced in neutrophil-specific PLCγ2-deficient (Plcg2Δ PMN ) mice. However, this was not due to an intrinsic migration defect since Plcg2Δ PMN neutrophils accumulated normally when wild-type cells were also present in mixed bone marrow chimeras. Instead, the Plcg2Δ PMN mutation blocked the accumulation of interleukin-1ß, macrophage inflammatory protein 2 (MIP-2), and leukotriene B4 (LTB4 ) in synovial tissues and reduced the secondary infiltration of macrophages. These findings were supported by in vitro studies showing normal chemotactic migration but defective immune complex-induced respiratory burst and MIP-2 or LTB4 release in PLCγ2-deficient neutrophils. CONCLUSION: Neutrophil PLCγ2 is critical for arthritis development, supposedly through the generation of the inflammatory microenvironment. PLCγ2-expressing neutrophils exert complex indirect effects on other inflammatory cells. PLCγ2-targeted therapies may provide particular benefit in inflammatory diseases with a major neutrophil component.


Assuntos
Artrite Experimental/metabolismo , Autoanticorpos/metabolismo , Neutrófilos/metabolismo , Fosfolipase C gama/metabolismo , Animais , Plaquetas/metabolismo , Inflamação/metabolismo , Mastócitos/metabolismo , Camundongos , Fosfolipase C gama/genética
12.
Medicine (Baltimore) ; 100(11): e25008, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33725976

RESUMO

ABSTRACT: The tumor microenvironment (TME) plays an important role in the occurrence and development of soft tissue sarcoma (STS). A number of studies have shown that to inhibit tumor growth, the TME can be remodeled into an environment unsuitable for tumor proliferation. However, a lack of understanding exists regarding the dynamic regulation of TME.In this study, we used CIBERSORT and ESTIMATE calculation methods from the Cancer Genome Atlas (TCGA) database to calculate the proportion of tumor infiltrating immune cells (TICs) and the number of immune and stromal components in 263 STS samples. Differential expression genes (DEGs) shared by Immune Score and Stromal Score were obtained via difference analysis. Univariate Cox regression analysis and construction of protein-protein interaction (PPI) networks were applied to the DEGs.Through intersection analysis of univariate COX and PPI, PLCG2 was determined as the indicator. Further analysis showed that PLCG2 expression was positively correlated with the survival of STS patients. Gene set enrichment analysis (GSEA) showed that genes in the highly expressed PLCG2 group were enriched in immune-related activities. In the low-expression PLCG2 group, genes were enriched in the E2F, G2M, and MYC pathways. Difference analysis and correlation analysis showed that CD8+ T cells, gamma delta T cells, monocytes, and M1 macrophages were positively correlated with PLCG2 expression, indicating that PLCG2 may represent the immune status of TME.Therefore, the level of PLCG2 may aid in determining the prognosis of STS patients, especially the status of TME. These data provide additional insights into the remodeling of TME.


Assuntos
Fosfolipase C gama/metabolismo , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Microambiente Tumoral/genética , Biomarcadores Tumorais , Bases de Dados Genéticas , Feminino , Humanos , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/metabolismo , Ativação de Macrófagos/genética , Masculino , Modelos de Riscos Proporcionais , Mapas de Interação de Proteínas , Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidade , Células Estromais/metabolismo , Transcriptoma
13.
Elife ; 102021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33755016

RESUMO

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.


Assuntos
Receptores ErbB/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteômica/métodos , Catálise , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ocludina/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacologia , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Domínios de Homologia de src
14.
Oncol Rep ; 45(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760219

RESUMO

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B­lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen­activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor­κB (NF­κB) activation, suggesting an anti­metastatic effect of BTK inhibitors on MCF­7 cells that leads to the downregulation of matrix metalloproteinase (MMP)­9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti­metastatic activity of BTK inhibitors was examined in MCF­7 cells focusing on MMP­9 expression in 12­O­tetradecanoylphorbol­13­acetate (TPA)­stimulated MCF­7 cells. The expression and activity of MMP­9 in MCF­7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX­774 (10 µM)] significantly attenuated TPA­induced cell invasion and migration in MCF­7 cells and inhibited the activation of the phospholipase Cγ2/PKCß signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP­9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA­induced MMP­9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF­κB/activator protein­1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP­9 expression in MCF­7 cells.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Neoplasias da Mama/patologia , Metaloproteinase 9 da Matriz/genética , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Fosfolipase C gama/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Acetato de Tetradecanoilforbol/toxicidade , Fator de Transcrição AP-1/metabolismo
15.
Cancer Cell ; 39(4): 509-528.e20, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33577785

RESUMO

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteogenômica , Neoplasias Encefálicas/patologia , Biologia Computacional/métodos , Glioblastoma/patologia , Humanos , Metabolômica/métodos , Mutação/genética , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteogenômica/métodos , Proteômica/métodos
16.
Methods Mol Biol ; 2251: 225-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481244

RESUMO

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.


Assuntos
Ensaios Enzimáticos/métodos , Fosfatidilinositol 4,5-Difosfato/análogos & derivados , Fosfolipases Tipo C/análise , Fluoresceína-5-Isotiocianato/química , Hidrólise , Isoenzimas/análise , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase C gama/análise , Fosfolipase C gama/metabolismo , Ligação Proteica/fisiologia , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo
17.
Int Immunopharmacol ; 90: 107039, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33127334

RESUMO

Patients with sepsis and sepsis-related complications have a high mortality. Endothelial cell dysfunction plays a central role in sepsis pathophysiological process. In sepsis patients, endothelial cell apoptosis is associated with intracellular calcium overload. Multiple functions in the apoptotic process have been found to be regulated by calcium signaling. Our previous work had proved that LPS-induced cell injury was associated with store-operated calcium (SOC) entry mediated by stromal interaction molecule-1 (STIM 1) in Human umbilical vein endothelial cells (HUVEC), but the underlying molecular mechanism has not been adequately defined. Here we report that the LPS-induced cell injury is related to the calcium overload in HUVEC. SOC entry mediated by calcium release-activated calcium modulator (Orai) 1 and transient receptor potential canonical (TRPC) 1 was associated with LPS-induced calcium overload and cell apoptosis. Bruton's tyrosine kinase (Btk)/Phospholipase C(PLC) γ/inositol 1,4,5-triphosphate receptor (IP3R) played a major role in regulating calcium overload in LPS-induced HUVEC. Knockdown of Btk markedly inhibited the expressions of Orai 1 and its downstream molecule IP3R but not that of TRPC1 in LPS-induced HUVEC. In mice, knockdown of Btk and Orai 1 inhibited LPS-induced calcium overload, pulmonary vascular endothelial cell (VEC) injury and acute lung injury. These findings demonstrated that Btk acts as a regulator of calcium-dependent signaling, especially in the Orai 1-mediated SOC entry of the LPS-induced VEC.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Tirosina Quinase da Agamaglobulinemia/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Pulmão/irrigação sanguínea , Proteína ORAI1/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteína ORAI1/genética , Fosfolipase C gama/metabolismo
18.
Biochem Biophys Res Commun ; 534: 1020-1025, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131771

RESUMO

Significant cellular morphology changes in renal tubules were observed in diabetes patients and animal models. However, the interaction between insulin and tubular epithelial cells microvillar structure remains obscure. To understand microvillar dynamics, we used Scanning Ion Conductance Microscope to visualize microvillar in the living cell. Here, we found two layers of microvilli on the tubular epithelial cell surface: short compact microvilli and netlike long microvilli. Insulin treatment could increase microvilli length and density. This process was mediated by the PI3K/PLCγ signaling pathway, other than the PI3K/Arp2/3 signal pathway. In conclusion, our findings present a novel insulin signaling transduction mechanism, which contributes to understanding renal tubular epithelial cell microvilli dynamic regulation.


Assuntos
Células Epiteliais/metabolismo , Insulina/metabolismo , Túbulos Renais/metabolismo , Microvilosidades/metabolismo , Fosfolipase C gama/metabolismo , Transdução de Sinais , Animais , Anuros , Linhagem Celular , Células Epiteliais/citologia , Humanos , Túbulos Renais/citologia
19.
Insect Sci ; 28(2): 430-444, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32108427

RESUMO

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis (Bt) cotton in China. Additionally, 20-hydroxyecdysone (20E) has important functions in many biological processes, including insect reproduction. Phospholipase C (PLC), which is an essential enzyme for phosphoinositide metabolism, is involved in 20E signal transduction, but its function in 20E-mediated reproduction in A. lucorum remains unclear. In this study, 20E increased AlPLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis. The 20E treatment also induced the considerable accumulation of two second messengers, inositol triphosphate and diacylglycerol. The expression levels of genes encoding vitellogenin (AlVg) and soluble trehalase (AlTre-1) were similar to those of AlPLCγ, and were upregulated in response to 20E. The silencing of AlPLCγ resulted in downregulated expression of AlTre-1 and AlVg. However, the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression. Moreover, the silencing of AlVg did not alter AlTre-1 expression. Furthermore, an examination of the insect specimens indicated that AlPLCγ is required for female adult reproduction, and that downregulated expression of this gene is associated with decreases in fecundity, adult longevity, and egg hatching rate as well as delayed oocyte maturation. We propose that 20E regulates AlTre-1 expression via AlPLCγ and affects Vg expression as well as ovary development to facilitate the reproductive activities of A. lucorum females.


Assuntos
Heterópteros/fisiologia , Proteínas de Insetos/genética , Fosfolipase C gama/genética , Trealase/metabolismo , Sequência de Aminoácidos , Animais , Ecdisterona/administração & dosagem , Feminino , Fertilidade/efeitos dos fármacos , Heterópteros/genética , Heterópteros/crescimento & desenvolvimento , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Fosfolipase C gama/química , Fosfolipase C gama/metabolismo , Filogenia
20.
Sci Rep ; 10(1): 20940, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262354

RESUMO

Eucalyptus oil has been used since ancient times for its bactericidal, anti-inflammatory, analgesic and sedative effects. In recent years, the action of Eucalyptus oil has been scientifically proven, and there have been reports that Eucalyptus oil suppresses the production of chemokines, cytokines and lipid mediators in basophils, alveolar macrophages and monocytes. Based on this information, we aimed to verify whether Eucalyptus oil can be used for allergic dermatitis, the incidence of which has been increasing among human skin diseases. This effect was verified using a mouse IgE-mediated local allergic model. In conclusion, topical application of Eucalyptus oil suppressed oedema and vascular permeability enhancement due to IgE-mediated allergic on the skin. In addition, we also verified the degranuration of mast cells, which is a part of its action, and examined whether 1,8-cineole, which is the main component of Eucalyptus oil, suppresses the phosphorylation of PLCγ and p38 directly or indirectly. 1,8-cineole was found to suppress degranulation of mast cells.


Assuntos
Degranulação Celular , Regulação para Baixo , Óleo de Eucalipto/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Imunoglobulina E/metabolismo , Mastócitos/fisiologia , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Células da Medula Óssea/efeitos dos fármacos , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Eucaliptol/farmacologia , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Espaço Intracelular/metabolismo , Mastócitos/efeitos dos fármacos , Camundongos , Modelos Biológicos , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src
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