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1.
Molecules ; 26(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809496

RESUMO

BACKGROUND: Carnosine is a dipeptide molecule (ß-alanyl-l-histidine) with anti-inflammatory, antioxidant, anti-glycation, and chelating properties. It is used in exercise physiology as a food supplement to increase performance; however, in vitro evidence suggests that carnosine may exhibit anti-cancer properties. METHODS: In this study, we investigated the effect of carnosine on breast, ovarian, colon, and leukemic cancer cell proliferation. We further examined U937 promonocytic, human myeloid leukemia cell phenotype, gene expression, and cytokine secretion to determine if these are linked to carnosine's anti-proliferative properties. RESULTS: Carnosine (1) inhibits breast, ovarian, colon, and leukemic cancer cell proliferation; (2) upregulates expression of pro-inflammatory molecules; (3) modulates cytokine secretion; and (4) alters U937 differentiation and phenotype. CONCLUSION: These effects may have implications for a role for carnosine in anti-cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Carnosina/farmacologia , Dipeptídeos/farmacologia , Neoplasias/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Neoplasias/genética , Células U937
2.
Nat Commun ; 12(1): 2211, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850121

RESUMO

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.


Assuntos
Morte Celular/fisiologia , Necroptose/fisiologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Sítios de Ligação , Membrana Celular , Cristalografia por Raios X , Células HT29 , Humanos , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Recombinantes , Alinhamento de Sequência , Células U937
3.
Nat Commun ; 12(1): 2346, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879767

RESUMO

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antígeno B7-H1/química , Antígeno CTLA-4/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Neoplasias/terapia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/fisiologia , Células U937 , Ubiquitinação/efeitos dos fármacos
4.
Cytokine ; 142: 155496, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33773396

RESUMO

Efforts to understand host factors critical for COVID-19 pathogenesis have identified high mobility group box 1 (HMGB1) to be crucial for regulating susceptibility to SARS-CoV-2. COVID-19 disease severity is correlated with heightened inflammatory responses, and HMGB1 is an important extracellular mediator in inflammation processes.In this study, we evaluated the effect of HMGB1 inhibitor Glycyrrhizin on the cellular perturbations in lung cells expressing SARS-CoV-2 viral proteins. Pyroptosis in lung cells transfected with SARS-CoV-2 S-RBD and Orf3a, was accompanied by elevation of IL-1ß and extracellular HMGB1 levels. Glycyrrhizin mitigated viral proteins-induced lung cell pyroptosis and activation of macrophages. Heightened release of proinflammatory cytokines IL-1ß, IL-6 and IL-8, as well as ferritin from macrophages cultured in conditioned media from lung cells expressing SARS-CoV-2 S-RBD and Orf3a was attenuated by glycyrrhizin. Importantly, Glycyrrhizin inhibited SARS-CoV-2 replication in Vero E6 cells without exhibiting cytotoxicity at high doses. The dual ability of Glycyrrhizin to concomitantly halt virus replication and dampen proinflammatory mediators might constitute a viable therapeutic option in patients with SARS-CoV-2 infection.


Assuntos
/metabolismo , Ácido Glicirrízico/farmacologia , Proteína HMGB1/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/efeitos dos fármacos , Células A549 , /genética , Proteína HMGB1/genética , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Células U937 , /genética
5.
Life Sci ; 273: 119304, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662432

RESUMO

AIMS: Necroptosis, an inflammatory form of regulated necrosis mediated by receptor-interacting kinase 1 (RIP1), RIP3, and pseudokinase mixed lineage kinase domain-like protein (MLKL) is extensively implicated in liver inflammatory disease. Thus identification small-molecule inhibitor of necroptosis has emerged as a potential therapeutic strategy to prevent liver damage. In this study, we identified 5-((7-chloro-6-fluoro-1 h-indol-3-yl) methyl)-3-methylimidazolidine-2,4-dione (F-nec) as a novel potent necroptosis inhibitor. MAIN METHODS: To find out the potent chemical inhibitors of necroptosis, human monocytic U937 cells were treated with a combination of tumor necrosis factor alpha (TNFα) and a pan-caspase inhibitor z-VAD-fmk. LPS and D-galactosamine (LPS/GalN) were further employed to simulate acute liver failure to explore therapeutic potency of F-nec in vivo. In addition, a specific inhibitor of c-Jun NH (2)-terminal kinases (JNK) SP600125 and its activator anisomycin are used to elucidate its mechanisms in acute liver failure therapy. Necroptosis pathway related proteins were tested by western blot. KEY FINDINGS: In this study, we identified F-nec as a novel potent RIP1 inhibitor which efficiently blocked TNFα-induced necroptosis in human and mice cells. Furthermore, pre-treatment of F-nec could prevent hepatic necrosis by reducing RIP1-mediated necroptosis also effectively ameliorated LPS/GalN induced acute liver failure by attenuating cell death signaling-stimulated JNK pathway activation and then suppressing JNK-triggered inflammation. SIGNIFICANCE: Altogether, this study demonstrates that F-nec is a potent inhibitor of RIP1 and highlights its great potential for use in the treatment of RIP1-driven inflammatory liver diseases.


Assuntos
Proteínas Ativadoras de GTPase/antagonistas & inibidores , Galactosamina/toxicidade , Indóis/química , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/tratamento farmacológico , Necroptose , Substâncias Protetoras/farmacologia , Animais , Humanos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células U937
6.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672787

RESUMO

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge.


Assuntos
Proteínas de Membrana/deficiência , Monoacilglicerol Lipases/deficiência , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/patologia , Ganho de Peso , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Células Cultivadas , Ácidos Graxos/metabolismo , Humanos , Inflamação/patologia , Metabolismo dos Lipídeos , Fígado/patologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoacilglicerol Lipases/metabolismo , Ácido Oleico , Fenótipo , Células U937
7.
Life Sci ; 276: 119402, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33785335

RESUMO

In our previous study, we observed that donor pulmonary intravascular nonclassical monocytes play a major role in early PGF, but the specific mechanism remained unclear. In this study, we investigated the mechanistic role of monocytes in inducing pyroptosis of human pulmonary microvascular endothelial cells (HPMECs) during IRI. A murine hilar ligation model of IRI was utilized whereby left lungs underwent 1 h of ischemia and 23 h of reperfusion. Monocyte depletion by intraperitoneal clodronate-liposome treatment on pulmonary edema and pyroptosis activation were determined. In vitro experiments, we performed the co-culture experiments under hypoxia-reoxygenation (H/R) conditions to mimic the IRI environment. We monitored the expression of NLRP3, caspase-1 and IL-1ß in co-cultures of monocytes (U937 cells) and HPMECs under H/R conditions. NLRP3, IL-1ß and IL-1R siRNA knockdown, caspase-1 and NF-κB pathway inhibitors were employed to elucidate the mechanism modulating HPMEC pyroptosis during H/R. Treatment of mice with clodronate-liposome attenuated IR-induced pulmonary edema, cytokine production and pyroptosis activation. In vitro, NLRP3 knockdown in monocytes reduced caspase-1 and IL-1ß secretion in co-cultures of monocytes and HPMECs. Reduced HPMEC pyroptosis was also observed either containing HPMECs with genetically engineered IL-1R knockdown or in co-culture treated with a Triplotide inhibitor that disrupts NF-κB signaling. Monocytes play a vital role in the development of transplant-associated ischemia-reperfusion injury. A potential role is that monocytes secrete IL-1ß to induce HPMEC pyroptosis via the IL-1R/NF-κB/NLRP3 pathway.


Assuntos
Endotélio Vascular/patologia , Inflamação/patologia , Pneumopatias/complicações , Monócitos/patologia , Piroptose , Traumatismo por Reperfusão/complicações , Doenças Vasculares/patologia , Animais , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamassomos , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Células U937 , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
8.
Nat Commun ; 12(1): 1628, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712615

RESUMO

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Tolerância a Medicamentos/genética , Tolerância a Medicamentos/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Combinação de Medicamentos , Descoberta de Drogas , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Mutação , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Células U937
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(1): 17-25, 2021 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-33554791

RESUMO

OBJECTIVE: To investigate the antileukemia activity of phosphatidylinositol-3 kinase (PI3K) inhibitor ZSTK474 on human leukemia cell line U937. METHODS: MTT, soft agar assay, flow cytometric analysis and western blot were used to detect the effect of ZSTK474 on U937 cell proliferation, tumorigenicity, cell cycle, cell apoptosis and phosphorylation levels of the key factor of PI3K/AKT pathway. Chou-Talalay method was used to evaluate the combination of ZSTK474 with Cytarabine or Homoharringtonine. RESULTS: PI3K inhibitor ZSTK474 could inhibit the proliferation and tumorigenicity of U937 cell, induce G1 cell cycle arrest and promote cell apoptosis, and enhance intracellular ROS production and decrease MMP, downregulate Cyclin D1, p-Rb, BCL-2 and upregulate p27, caspase-9, caspase-3, PARP and BAX. Furthermore, the phosphorylation of PDK1, GSK-3ß, AKT and mTOR could be downregulated by ZSTK474. The combination of ZSTK474 with Homoharringtonine was synergistic. CONCLUSION: ZSTK474 can inhibit the pathway of PI3K/AKT, ZSTK474 alone or in combination with Homoharringtonine shows potential antileukemia activity on U937 cells.


Assuntos
Fosfatidilinositol 3-Quinases , Triazinas , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas Proto-Oncogênicas c-akt , Células U937
10.
J Med Chem ; 64(3): 1510-1523, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33522230

RESUMO

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity in vitro and in vivo in animal models of traumatic brain injury, acute myocardial infarction, and drug-induced liver toxicity. Consequently, this study may pave the way for the development of novel therapies for the treatment, inhibition, or prevention of a large number of hitherto untreatable diseases.


Assuntos
Necroptose/efeitos dos fármacos , Necrose/prevenção & controle , Elastase Pancreática/antagonistas & inibidores , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Animais , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Morte Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , RNA Interferente Pequeno , Células U937
11.
Molecules ; 26(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430125

RESUMO

To prevent accumulation of misfolded proteins in the endoplasmic reticulum, chaperones perform quality control on newly translated proteins and redirect misfolded proteins to the cytosol for degradation by the ubiquitin-proteasome system. This pathway is called ER-associated protein degradation (ERAD). The human cytomegalovirus protein US2 induces accelerated ERAD of HLA class I molecules to prevent immune recognition of infected cells by CD8+ T cells. Using US2-mediated HLA-I degradation as a model for ERAD, we performed a genome-wide CRISPR/Cas9 library screen to identify novel cellular factors associated with ERAD. Besides the identification of known players such as TRC8, p97, and UBE2G2, the ubiquitin-fold modifier1 (UFM1) pathway was found to affect degradation of HLA-I. UFMylation is a post-translational modification resembling ubiquitination. Whereas we observe ubiquitination of HLA-I, no UFMylation was detected on HLA-I or several other proteins involved in degradation of HLA-I, suggesting that the UFM1 pathway impacts ERAD in a different manner than ubiquitin. Interference with the UFM1 pathway seems to specifically inhibit the ER-to-cytosol dislocation of HLA-I. In the absence of detectable UFMylation of HLA-I, UFM1 may contribute to US2-mediated HLA-I degradation by misdirecting protein sorting indirectly. Mass spectrometry analysis of US2-expressing cells showed that ribosomal proteins are a major class of proteins undergoing extensive UFMylation; the role of these changes in protein degradation may be indirect and remains to be established.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Citomegalovirus/metabolismo , Degradação Associada com o Retículo Endoplasmático , Antígenos HLA/metabolismo , Proteínas/metabolismo , Proteólise , Proteínas do Envelope Viral/metabolismo , Linfócitos T CD8-Positivos/virologia , Citomegalovirus/genética , Antígenos HLA/genética , Humanos , Proteínas/genética , Células U937
12.
Nat Methods ; 18(1): 84-91, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398190

RESUMO

Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug.


Assuntos
Benzamidas/farmacologia , Membrana Celular/metabolismo , Dioxóis/farmacologia , Proteínas de Membrana/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Membrana Celular/efeitos dos fármacos , Humanos , Células K562 , Ligantes , Proteínas de Membrana/análise , Proteínas de Membrana/efeitos dos fármacos , Ligação Proteica , Proteoma/análise , Proteoma/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Temperatura , Células U937
13.
Mol Immunol ; 131: 171-179, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33461764

RESUMO

Alzheimer's disease (AD) is characterized by the accumulation in the brain of extracellular amyloid ß (Aß) plaques as well as intraneuronal inclusions (neurofibrillary tangles) consisting of total tau and phosphorylated tau. Also present are dystrophic neurites, loss of synapses, neuronal death, and gliosis. AD genetic studies have highlighted the importance of inflammation in this disease by identifying several risk associated immune response genes, including TREM2. TREM2 has been strongly implicated in basic microglia function including, phagocytosis, apoptosis, and the inflammatory response to Aß in mouse brain and primary cells. These studies show that microglia are key players in the response to Aß and in the accumulation of AD pathology. However, details are still missing about which apoptotic or inflammatory factors rely on TREM2 in their response to Aß, especially in human cell lines. Given these previous findings our hypothesis is that TREM2 influences the response to Aß toxicity by enhancing phagocytosis and inhibiting both the BCL-2 family of apoptotic proteins and pro-inflammatory cytokines. Aß42 treatment of the human microglial cell line, HMC3 cells, was performed and TREM2 was overexpressed or silenced and the phagocytosis, apoptosis and inflammatory response were evaluated. Results indicate that a robust phagocytic response to Aß after 24 h requires TREM2 in HMC3 cells. Also, TREM2 inhibits Aß induced apoptosis by activating the Mcl-1/Bim complex. TREM2 is involved in activation of IP-10, MIP-1a, and IL-8, while it inhibits FGF-2, VEGF and GRO. Taken together, TREM2 plays a role in enhancing the microglial functional response to Aß toxicity in HMC3 cells. This novel information suggests that therapeutic strategies that seek to activate TREM2 may not only enhance phagocytosis and inhibit apoptosis, but may also inhibit beneficial inflammatory factors, emphasizing the need to define TREM2-related inflammatory activity in not only mouse models of AD, but also in human AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Apoptose/fisiologia , Inflamação/metabolismo , Glicoproteínas de Membrana/metabolismo , Fagócitos/metabolismo , Receptores Imunológicos/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Microglia/metabolismo , Fagocitose/fisiologia , Placa Amiloide/metabolismo , Células THP-1 , Células U937
14.
Methods Mol Biol ; 2201: 163-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975797

RESUMO

Opioids play a pivotal role in pain transmission but are also able to modulate immune cell functions. In the last decades a connection between opioids and integrins-adhesion molecules involved, among many other processes, in leukocyte recruitment at inflamed site-has been established. To study immune cell integrin-mediated adhesion, cell adhesion assay is a simple, reproducible, and valuable tool capable of unraveling concentration-dependent effects of a test candidate on integrin-mediated cell adhesion.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/efeitos dos fármacos , Imuno-Histoquímica/métodos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Inflamação/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Células Jurkat , Leucócitos/metabolismo , Células U937
15.
Methods Mol Biol ; 2217: 47-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215376

RESUMO

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.


Assuntos
Adesão Celular/efeitos dos fármacos , Imunoprecipitação/métodos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linhagem Celular , Reagentes para Ligações Cruzadas/química , Expressão Gênica , Humanos , Células Jurkat , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Cultura Primária de Células , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Succinimidas/química , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Tetraspanina 28/genética , Tetraspanina 29/genética , Tetraspanina 30/genética , Células U937
16.
Proc Natl Acad Sci U S A ; 117(52): 33272-33281, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318170

RESUMO

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.


Assuntos
Necroptose , Proteínas Quinases/metabolismo , Humanos , Concentração Inibidora 50 , Células Jurkat , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Multimerização Proteica , Células U937 , Xantina/farmacologia
17.
Int J Mol Sci ; 21(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371413

RESUMO

Guaiane-type sesquiterpene lactones are naturally occurring compounds which have attracted attention due to their array of biological activities. In this study, chlorinated guaianolides 1-8, isolated from plants of the genus Centaurea, were evaluated against the human leukemia cell lines HL-60, U-937, a specific U-937 cell line that overexpresses the anti-apoptotic Bcl-2 protein and the human melanoma cell line SK-MEL-1. This established the relevant structure-growth inhibition relationships. Chlorohyssopifolins A (1), C (3) and D (4) and linichlorin A (6) were the most potent compounds in terms of inducing growth inhibition in the four cell lines. IC50 values were below 10 µM in all cases. Chlorohyssopifolins A (1) and D (4) and linichlorin A (6) were potent apoptotic inducers in human U-937 leukemia cells, as determined by fluorescent microscopy and flow cytometry, and their mechanism of action was associated with cytochrome c release, caspase activation and poly(ADP-ribose)polymerase cleavage. Overall this study shows that guaianolides induce cytotoxicity against human tumor cells and provides important insights into the cell death pathways that are involved.


Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Lactonas/farmacologia , Leucemia/patologia , Sesquiterpenos de Guaiano/química , Apoptose , Citocromos c/metabolismo , Humanos , Leucemia/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/metabolismo , Células U937
18.
PLoS Biol ; 18(9): e3000813, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991574

RESUMO

Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ácidos Graxos Voláteis/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/prevenção & controle , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Feminino , Microbioma Gastrointestinal/imunologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Células U937
19.
Proc Natl Acad Sci U S A ; 117(37): 22984-22991, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868431

RESUMO

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.


Assuntos
Aciltransferases/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Lipídeo A/imunologia , Yersinia pestis/imunologia , Animais , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/imunologia , Células THP-1/imunologia , Células U937 , Yersinia pseudotuberculosis/imunologia
20.
Nat Cell Biol ; 22(9): 1042-1048, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32868903

RESUMO

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.


Assuntos
Ferroptose/fisiologia , Osmose/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Ferro/metabolismo , Células MCF-7 , Necrose/metabolismo , Necrose/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Células U937
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