RESUMO
BACKGROUND AND AIM: Diacylglycerol kinase (DGK) isoforms catalyze an enzymatic reaction that removes diacylglycerol (DAG) and thereby terminates protein kinase C signaling by converting DAG to phosphatidic acid. DGKδ (type II isozyme) downregulation causes insulin resistance, metabolic inflexibility, and obesity. Here we determined whether DGKδ overexpression prevents these metabolic impairments. METHODS: We generated a transgenic mouse model overexpressing human DGKδ2 under the myosin light chain promoter (DGKδ TG). We performed deep metabolic phenotyping of DGKδ TG mice and wild-type littermates fed chow or high-fat diet (HFD). Mice were also provided free access to running wheels to examine the effects of DGKδ overexpression on exercise-induced metabolic outcomes. RESULTS: DGKδ TG mice were leaner than wild-type littermates, with improved glucose tolerance and increased skeletal muscle glycogen content. DGKδ TG mice were protected against HFD-induced glucose intolerance and obesity. DGKδ TG mice had reduced epididymal fat and enhanced lipolysis. Strikingly, DGKδ overexpression recapitulated the beneficial effects of exercise on metabolic outcomes. DGKδ overexpression and exercise had a synergistic effect on body weight reduction. Microarray analysis of skeletal muscle revealed common gene ontology signatures of exercise and DGKδ overexpression that were related to lipid storage, extracellular matrix, and glycerophospholipids biosynthesis pathways. CONCLUSION: Overexpression of DGKδ induces adaptive changes in both skeletal muscle and adipose tissue, resulting in protection against HFD-induced obesity. DGKδ overexpression recapitulates exercise-induced adaptations on energy homeostasis and skeletal muscle gene expression profiles.
Assuntos
Diacilglicerol Quinase , Dieta Hiperlipídica , Camundongos Transgênicos , Obesidade , Animais , Diacilglicerol Quinase/metabolismo , Diacilglicerol Quinase/genética , Obesidade/metabolismo , Obesidade/genética , Camundongos , Dieta Hiperlipídica/efeitos adversos , Masculino , Glucose/metabolismo , Condicionamento Físico Animal/fisiologia , Músculo Esquelético/metabolismo , Humanos , Intolerância à Glucose/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/prevenção & controle , Camundongos Endogâmicos C57BL , Resistência à Insulina/genéticaRESUMO
Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol (DAG) kinase (DGK) 5, an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants.
Assuntos
Ácidos Fosfatídicos , Plantas , Estresse Fisiológico , Ácidos Fosfatídicos/metabolismo , Plantas/metabolismo , Diacilglicerol Quinase/metabolismoRESUMO
Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.
Assuntos
Asma , Diacilglicerol Quinase , Transdução de Sinais , Asma/metabolismo , Asma/patologia , Asma/fisiopatologia , Asma/enzimologia , Humanos , Diacilglicerol Quinase/metabolismo , Animais , Diglicerídeos/metabolismo , Proteína Quinase C/metabolismoRESUMO
BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. METHODS: Immunohistochemistry (IHC) with anti-DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (-, 1+, 2+, 3+) and divided into two groups (DGKα- and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki-67 expression was evaluated as a cell proliferation marker. The number of Ki-67-positive cells was counted, and the relationship with DGKα expression was examined. RESULTS: DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα- group (-: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence-free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki-67-positive cells increased in association with the staining levels of DGKα. CONCLUSION: Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.
Assuntos
Neoplasias dos Ductos Biliares , Biomarcadores Tumorais , Proliferação de Células , Colangiocarcinoma , Diacilglicerol Quinase , Antígeno Ki-67 , Humanos , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/mortalidade , Diacilglicerol Quinase/metabolismo , Diacilglicerol Quinase/genética , Masculino , Feminino , Prognóstico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/mortalidade , Idoso , Antígeno Ki-67/metabolismo , Adulto , Imuno-Histoquímica , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/metabolismoRESUMO
Reactive oxygen species (ROS) and phosphatidic acid (PA) are important second messengers in plant immunity. PA binding to RBOHD, an NADPH oxidase responsible for ROS production, enhances RBOHD stability and promotes ROS production. Distinct phosphorylation of the lipid kinase DGK5 optimizes the PA burst in regulating ROS production.
Assuntos
Homeostase , Ácidos Fosfatídicos , Imunidade Vegetal , Espécies Reativas de Oxigênio , Ácidos Fosfatídicos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Imunidade Vegetal/fisiologia , NADPH Oxidases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/imunologia , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Quinase/metabolismo , FosforilaçãoRESUMO
BACKGROUND: Glycoursodeoxycholic acid (GUDCA) has been acknowledged for its ability to regulate lipid homeostasis and provide benefits for various metabolic disorders. However, the impact of GUDCA on arterial thrombotic events remains unexplored. The objective of this study is to examine the effects of GUDCA on thrombogenesis and elucidate its underlying mechanisms. METHODS: Plasma samples from patients with arterial thrombotic events and diet-induced obese mice were collected to determine the GUDCA concentrations using mass spectrometry. Multiple in vivo murine thrombosis models and in vitro platelet functional assays were conducted to comprehensively evaluate the antithrombotic effects of GUDCA. Moreover, lipidomic analysis was performed to identify the alterations of intraplatelet lipid components following GUDCA treatment. RESULTS: Plasma GUDCA level was significantly decreased in patients with arterial thrombotic events and negatively correlated with thrombotic propensity in diet-induced obese mice. GUDCA exhibited prominent suppressing effects on platelet reactivity as evidenced by the attenuation of platelet activation, secretion, aggregation, spreading, and retraction (P<0.05). In vivo, GUDCA administration robustly alleviated thrombogenesis (P<0.05) without affecting hemostasis. Mechanistically, GUDCA inhibited DGK (diacylglycerol kinase) activity, leading to the downregulation of the phosphatidic acid-mediated signaling pathway. Conversely, phosphatidic acid supplementation was sufficient to abolish the antithrombotic effects of GUDCA. More importantly, long-term oral administration of GUDCA normalized the enhanced DGK activity, thereby remarkably alleviating the platelet hyperreactivity as well as the heightened thrombotic tendency in diet-induced obese mice (P<0.05). CONCLUSIONS: Our study implicated that GUDCA reduces platelet hyperreactivity and improves thrombotic propensity by inhibiting DGKs activity, which is a potentially effective prophylactic approach and promising therapeutic agent for arterial thrombotic events.
Assuntos
Plaquetas , Diacilglicerol Quinase , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Trombose , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/metabolismo , Trombose/prevenção & controle , Trombose/sangue , Trombose/enzimologia , Trombose/tratamento farmacológico , Humanos , Masculino , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/metabolismo , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Feminino , Agregação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pessoa de Meia-Idade , Fibrinolíticos/farmacologia , Estudos de Casos e Controles , Camundongos Obesos , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Obesidade/sangue , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
Phosphatidic acid (PA) as a universal second messenger is transiently and rapidly produced upon immune activation in plants. A recent study by Kong et al. elucidated a mechanism for maintaining PA homeostasis via two uncoupled phosphorylation events of DIACYLGLYCEROL KINASE 5 (DGK5) at different phosphorylation sites by two distinct kinases.
Assuntos
Diacilglicerol Quinase , Homeostase , Ácidos Fosfatídicos , Imunidade Vegetal , Fosforilação , Diacilglicerol Quinase/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Phosphatidic acid (PA) and reactive oxygen species (ROS) are cellular messengers that relay signals to regulate diverse biological processes. In recent issues of Cell Host & Microbe and Cell, Qi et al. and Kong et al., respectively, investigate diacylglycerol kinase 5-mediated PA in regulating ROS signaling and plant immunity.
Assuntos
Diacilglicerol Quinase , Transdução de Sinais , Espécies Reativas de Oxigênio , Diacilglicerol Quinase/metabolismo , Ácidos FosfatídicosRESUMO
Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Diacilglicerol Quinase , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Quinase/metabolismo , NADPH Oxidases/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosforilação , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.
Assuntos
Diacilglicerol Quinase , Diglicerídeos , Animais , Camundongos , Fosforilação , Diglicerídeos/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismoRESUMO
Lipid phosphorylation by diacylglycerol kinase (DGK) that produces phosphatidic acid (PA) plays important roles in various biological processes, including stress responses, but the underlying mechanisms remain elusive. Here, we show that DGK5 and its lipid product PA suppress ABA biosynthesis by interacting with ABA-DEFICIENT 2 (ABA2), a key ABA biosynthesis enzyme, to negatively modulate plant response to abiotic stress tested in Arabidopsis thaliana. Loss of DGK5 function rendered plants less damaged, whereas overexpression (OE) of DGK5 enhanced plant damage to water and salt stress. The dgk5 mutant plants exhibited decreased total cellular and nuclear levels of PA with increased levels of diacylglycerol, whereas DGK5-OE plants displayed the opposite effect. Interestingly, we found that both DGK5 and PA bind to the ABA-synthesizing enzyme ABA2 and suppress its enzymatic activity. Consistently, the dgk5 mutant plants exhibited increased levels of ABA, while DGK5-OE plants showed reduced ABA levels. In addition, we showed that both DGK5 and ABA2 are detected in and outside the nuclei, and loss of DGK5 function decreased the nuclear association of ABA2. We found that both DGK5 activity and PA promote nuclear association of ABA2. Taken together, these results indicate that both DGK5 and PA interact with ABA2 to inhibit its enzymatic activity and promote its nuclear sequestration, thereby suppressing ABA production in response to abiotic stress. Our study reveals a sophisticated mechanism by which DGK5 and PA regulate plant stress responses.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Quinase/metabolismo , Ácido Abscísico/metabolismo , Fosforilação , Arabidopsis/metabolismo , Lipídeos , Regulação da Expressão Gênica de PlantasRESUMO
Adipose tissue (AT) comprises distinct fat depots such as white AT and brown AT. White and brown adipocytes exhibit different morphological and physiological properties. White adipocytes containing large single lipid droplet (LD) provide energy on demand whereas brown adipocytes loaded with multilocular LDs consume energy to generate heat or dissipate excess energy. Recent studies have shown that multilocular brown-like cells emerge in white AT under certain conditions. These cells termed beige adipocytes participate in energy expenditure and heat generation. In the process of lipolysis, TG is broken down into free fatty acid and diacylglycerol (DG). In this regard, DG also serves as a signaling molecule activating some proteins such as protein kinase C. Therefore, DG kinase (DGK), an enzyme which phosphorylates DG into phosphatidic acid (PA), plays a pivotal role in integrating energy homeostasis and intracellular signaling. Recently, we described that DGKε-KO mice exhibit increased adiposity in visceral white AT accompanied with impaired glucose tolerance early (40 days) in the course of high fat diet (HFD) feeding, although these mice exhibit "browning or beiging" in visceral white AT associated with improved glucose tolerance after longer term HFD feeding (180 days). This study was conducted to understand the overall features of adipose tissues and investigate changes in subcutaneous (inguinal) white AT and interscapular brown AT of DGKε-KO mice during the course of HFD feeding. Results demonstrated that fat accumulation is promoted in all fat depots under 40 days of HFD feeding conditions. Remarkably, "whitening" of brown adipocytes was identified in DGKε-deficient brown AT during the course of HFD feeding, suggesting brown adipocyte dysfunction. In addition, insulin levels were considerably elevated in DGKε-KO mice under 180 days of HFD feeding conditions. Collectively, these findings suggest that brown adipocytes are dysfunctional in DGKε-KO mice, which promotes browning or beiging in visceral white AT. Beige adipocytes may take over energy disposal and contribute to improving glucose tolerance with the aid of high levels of insulin in DGKε-KO mice upon excess feeding.
Assuntos
Tecido Adiposo Marrom , Insulinas , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Dieta Hiperlipídica , Glucose/metabolismo , Insulinas/metabolismoRESUMO
Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.
Assuntos
Cisteína , Diacilglicerol Quinase , Camundongos , Humanos , Animais , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Transdução de Sinais , Citosol/metabolismo , Metabolismo dos LipídeosRESUMO
Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.
Assuntos
Diacilglicerol Quinase , Neoplasias , Animais , Camundongos , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Linfócitos T/patologia , Neoplasias/patologia , ImunoterapiaRESUMO
Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG) to phosphatidic acid (PtdOH). A common approach to examine the activity of these enzymes relys on a radiometric assay (Epand and Topham, 2007; Tu-Sekine and Raben, 2017). This assay quantifies the DGK-catalyzed incorporation of 32P into DAG from AT32P to generate 32PtdOH and is perhaps been the most widely used assay. While sensitive, its drawbacks are the expense and the potential negative impacts on health and the environment. In this report, we describe a new assay which utilizes fluorescent labeled NBD-DAG (1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-Glycero-3-diacylglycerol) to quantify the DGK-θ-catalyzed conversion of NBD-DAG to NBD-PtdOH. Furthermore, we show the assay is sufficiently sensitive as the measured specific activity was similar to that previously determined with AT32P (Tu-Sekine and Raben, 2012) and was able to detect the activation of DGK-θ by synaptotagmin-1 (Barber et al., 2022). Overall, this assay is inexpensive, sensitive, and reproducible making it an attractive alternative to currently established assays.
Assuntos
Diacilglicerol Quinase , Diglicerídeos , Humanos , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The enhancement of T cell and NK cell function is an immunotherapeutic strategy for combating cancer. Antibodies that block inhibitory receptors, such as PD-1 and CTLA4, augment T cell function and have been successful in curing patients with some types of cancer. As an alternative approach to targeting specific inhibitory receptors by antibodies, small molecule drugs that inhibit negative regulators of T cell activation have been sought. One potential pharmacological target is diacylglycerol (DAG) kinase (DGK)ζ, which is an enzyme that acts as a negative regulator of DAG by phosphorylating DAG and converting it into phosphatidic acid. DAG-mediated signaling is critical for T cell activation through its T cell receptor and NK cell activation downstream of a variety of activating receptors. Thus, DGKζ-deficient T cells and NK cells display increased function upon activating receptor engagement. Moreover, treatment with the DGKζ-selective inhibitor ASP1570 augments T cell function. In this study, we sought to test whether the acute inhibition of DGKζ by ASP1570 augments NK cell function. We find that ASP1570 enhances DAG-mediated signaling in immunoreceptor-stimulated NK cells. Accordingly, ASP1570 treatment enhanced IFNγ production and degranulation of immunoreceptor-activated NK cells in vitro and NK cell-mediated tumor clearance in vivo. Thus, ASP1570 enhances both T and NK cell function, which could possibly induce more durable anti-tumor responses for immunotherapy.
Assuntos
Diacilglicerol Quinase , Neoplasias , Humanos , Diacilglicerol Quinase/metabolismo , Linfócitos T , Transdução de Sinais , Células Matadoras Naturais/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Allergic airway diseases are caused by inappropriate immune responses directed against inhaled environmental antigens. We previously reported that the inhibition of diacylglycerol (DAG) kinaseζ (DGKζ),an enzyme that terminates DAG-mediated signaling,protects against T cell-mediated allergic airway inflammation by blocking Th2 cell differentiation.In this study, we tested whether DGKζ deficiency also affects allergic airway disease mediated by type 2 innate lymphoid cells (ILC2)s. DGKζ-deficient mice displayed diminished ILC2 function and reduced papain-induced airway inflammation compared to wildtype mice. Unexpectedly, however, mice with hematopoietic cell-specific deletion ofDGKζ displayed intact airway inflammation upon papain challenge. Rather, bone marrow chimera studies revealed thatDGKζ deficiency in the non-hematopoietic compartment was responsible for the reduction in papain-induced airway inflammation. These data suggest that DGK might represent a novel therapeutic target not only for T cell-dependent but also ILC2-dependent allergic airway inflammation by affecting non-hematopoietic cells.
Assuntos
Hipersensibilidade , Imunidade Inata , Animais , Camundongos , Papaína , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Linfócitos , InflamaçãoRESUMO
Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Camundongos , Animais , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Proteômica , Diacilglicerol Quinase/metabolismo , Linfócitos T , LipídeosRESUMO
The sleep state is widely observed in animals. The molecular mechanisms underlying sleep regulation, however, remain largely unclear. In the nematode Caenorhabditis elegans, developmentally timed sleep (DTS) and stress-induced sleep (SIS) are 2 types of quiescent behaviors that fulfill the definition of sleep and share conserved sleep-regulating molecules with mammals. To identify novel sleep-regulating molecules, we conducted an unbiased forward genetic screen based on DTS phenotypes. We isolated 2 mutants, rem8 and rem10, that exhibited significantly disrupted DTS and SIS. The causal gene of the abnormal sleep phenotypes in both mutants was mapped to dgk-1, which encodes diacylglycerol kinase. Perhaps due to the diminished SIS, dgk-1 mutant worms exhibited decreased survival following exposure to a noxious stimulus. Pan-neuronal and/or cholinergic expression of dgk-1 partly rescued the dgk-1 mutant defects in DTS, SIS, and post-stress survival. Moreover, we revealed that pkc-1/nPKC participates in sleep regulation and counteracts the effect of dgk-1; the reduced DTS, SIS, and post-stress survival rate were partly suppressed in the pkc-1; dgk-1 double mutant compared with the dgk-1 single mutant. Excessive sleep observed in the pkc-1 mutant was also suppressed in the pkc-1; dgk-1 double mutant, implying that dgk-1 has a complicated mode of action. Our findings indicate that neuronal DGK-1 is essential for normal sleep and that the counterbalance between DGK-1 and PKC-1 is crucial for regulating sleep and mitigating post-stress damage.
Assuntos
Caenorhabditis elegans , Diacilglicerol Quinase , Animais , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Neurônios/metabolismo , Fosforilação , Sono/genética , MamíferosRESUMO
BACKGROUND: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). METHODS: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. RESULTS: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. CONCLUSIONS: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.