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Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo. Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.
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Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.
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Endocannabinoides , Glicéridos , Humanos , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Lipoproteína Lipasa/metabolismo , Ácidos Araquidónicos/metabolismo , DolorRESUMEN
BACKGROUND: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) remains challenging. Thus, we investigated whether resistant cancer cells that expand shortly after EGFR-TKI treatment would eventually cause the resistant phenotype. METHODS: We generated two EGFR-mutant lung cancer cell lines resistant to gefitinib (PC9GR and HCC827GR). The parent cell lines were exposed to short-term treatment with gefitinib or paclitaxel and then were assessed for EGFR T790M mutation and C-MET expression. These experiments were repeated in vivo and in clinically relevant patient-derived cell (PDC) models. For validation in clinical cases, we measured these gene alterations in plasma circulating tumor DNA (ctDNA) before and 8 weeks after starting EGFR-TKIs in four patients with EGFR-mutant lung cancer. RESULTS: T790M mutation was only detected in the PC9GR cells, whereas C-MET amplification was detected in the HCC827GR cells. The T790M mutation level significantly increased in PC9 cells after short-term treatment with gefitinib but not in the paclitaxel. C-MET mRNA expression was only significantly increased in gefitinib-treated HCC827 cells. We confirmed that the C-MET copy number in HCC827 cells that survived after short-term gefitinib treatment was significantly higher than that in dead HCC827 cells. These findings were reproduced in the in vivo and PDC models. An early on-treatment increase in the plasma ctDNA level of these gene alterations was correlated with the corresponding resistance mechanism to EGFR-TKIs, a finding that was confirmed in post-treatment tumor tissues. CONCLUSIONS: Early on-treatment kinetics in resistance-related gene alterations may predict the final mechanism of EGFR-TKI resistance.
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Here, we apply quantitative chemical proteomics and untargeted lipidomics to assign a polyunsaturated fatty acid (PUFA)-specific triacylglycerol (TAG) lipase activity for diacylglycerol lipase-beta (DAGLß) in macrophages. We demonstrate that DAGLß but not DAGLα is expressed and active in bone marrow-derived macrophages (BMDMs) as determined by activity-based protein profiling analysis of SILAC BMDMs. Genetic disruption of DAGLß resulted in accumulation of cellular TAGs composed of PUFA but not saturated/low unsaturated fatty acid counterparts, which is recapitulated in wild-type macrophages treated with a DAGLß-selective inhibitor. Biochemical assays with synthetic substrates confirm PUFA-TAGs as authentic DAGLß substrates. In summary, our findings identify DAGLß as a PUFA-specific TAG lipase in primary macrophages.
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Ácidos Grasos Insaturados/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Macrófagos/metabolismo , Animales , Diferenciación Celular , Cromatografía Liquida , Ácidos Grasos Insaturados/química , Lipasa/química , Lipoproteína Lipasa/química , Espectrometría de Masas , Metabolómica , RatonesRESUMEN
Lipids exert key structural, metabolic, and signaling functions in cells. Lipid diversity found in cells and tissues is regulated principally by metabolic enzymes whose activity is modulated posttranslationally to shape head group and fatty acyl composition of membrane lipids. Methodologies capable of monitoring in vivo changes in the lipidome are needed to assign substrate specificity of metabolic enzymes, which represents a key step toward understanding structure-function of lipids in living systems. The resulting lipid annotations also serve as important biomarkers for understanding mode of action for pharmacological agents targeting metabolic enzymes in cells and animal models. In this chapter, we describe a general metabolomics workflow to complement (chemo)proteomic efforts to modulate lipid pathways for basic science and translational applications.
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Metabolismo de los Lípidos , Lipidómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Biocatálisis , Fraccionamiento Químico/métodos , Cromatografía Liquida/métodos , Humanos , Lípidos/análisis , Lípidos/aislamiento & purificaciónRESUMEN
A mixture of nanothin exfoliated (NTE) graphite and urea (CO(NH2)2) powder was treated with radio frequency (RF) thermal plasma to achieve in situ purification and nitrogen doping of NTE graphite using the high-temperature flame of the RF plasma. Reactive species such as NH3, NH2, and HCNO generated by the thermolysis of urea play an important role in the purification and nitrogen doping of NTE graphite. The nitrogen content of NTE graphite subjected to plasma treatment increased by 5 times compared with that of raw NTE graphite. Three types of nitrogen species, namely, quaternary N, pyridinic N, and pyrrolic N, were observed after N doping with plasma treatment. The sheet resistance of N-doped NTE graphite reduced to 12-21% compared to that of the untreated NTE graphite, with the corresponding resistivity being ~7 × 10-6 Ω m.
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Diacylglycerol lipase-ß (DAGLß) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLß regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLß in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLß inactivation. Collectively, we identify DAGLß pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.
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Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Lipoproteína Lipasa/metabolismo , Animales , Antígenos/metabolismo , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diglicéridos/metabolismo , Endocannabinoides/metabolismo , Femenino , Glicéridos/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The metabolic serine hydrolases hydrolyze ester, amide, or thioester bonds found in broad small molecule substrates using a conserved activated serine nucleophile. The mammalian central nervous system (CNS) express a diverse repertoire of serine hydrolases that act as (phospho)lipases or lipid amidases to regulate lipid metabolism and signaling vital for normal neurocognitive function and CNS integrity. Advances in genomic DNA sequencing have provided evidence for the role of these lipid-metabolizing serine hydrolases in neurologic, psychiatric, and neurodegenerative disorders. This review briefly summarizes recent progress in understanding the biochemical and (patho)physiological roles of these lipid-metabolizing serine hydrolases in the mammalian CNS with a focus on serine hydrolases involved in the endocannabinoid system. The development and application of specific inhibitors for an individual serine hydrolase, if available, are also described. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.
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Sistema Nervioso Central/metabolismo , Endocannabinoides/metabolismo , Hidrolasas/metabolismo , Metabolismo de los Lípidos/fisiología , Mamíferos/metabolismo , Serina/metabolismo , Animales , HumanosRESUMEN
Ritanserin was tested in the clinic as a serotonin receptor inverse agonist but recently emerged as a novel kinase inhibitor with potential applications in cancer. Here, we discovered that ritanserin induced apoptotic cell death of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells via a serotonin-independent mechanism. We used quantitative chemical proteomics to reveal a ritanserin-dependent kinase network that includes key mediators of lipid [diacylglycerol kinase α, phosphatidylinositol 4-kinase ß] and protein [feline encephalitis virus-related kinase, rapidly accelerated fibrosarcoma (RAF)] signaling, metabolism [eukaryotic elongation factor 2 kinase, eukaryotic translation initiation factor 2-α kinase 4], and DNA damage response [tousled-like kinase 2] to broadly kill lung tumor cell types. Whereas ritanserin exhibited polypharmacology in NSCLC proteomes, this compound showed unexpected specificity for c-RAF in the SCLC subtype, with negligible activity against other kinases mediating mitogen-activated protein kinase signaling. Here we show that ritanserin blocks c-RAF but not B-RAF activation of established oncogenic signaling pathways in live cells, providing evidence in support of c-RAF as a key target mediating its anticancer activity. Given the role of c-RAF activation in RAS-mutated cancers resistant to clinical B-RAF inhibitors, our findings may have implications in overcoming resistance mechanisms associated with c-RAF biology. The unique target landscape combined with acceptable safety profiles in humans provides new opportunities for repositioning ritanserin in cancer.
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Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteómica , Ritanserina/farmacología , Secuencia de Aminoácidos , Línea Celular Tumoral , Reposicionamiento de Medicamentos , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/efectos de los fármacos , Serotonina/metabolismoRESUMEN
Extracellular signal-regulated kinases (ERKs) mediate downstream signaling of RAS-RAF-MEK as key regulators of the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is a hallmark of cancer and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. However, the rapid rise of resistance to clinical RAF and MEK inhibitors has prompted interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. Current methods for evaluating activity of inhibitors against ERK isoforms are based primarily on analysis of recombinant proteins. Strategies to directly and independently profile native ERK1 and ERK2 activity would greatly complement current cell biological tools used to probe and target ERK function. Here, we present a quantitative chemoproteomic strategy that utilizes active-site directed probes to directly quantify native ERK activity in an isoform-specific fashion. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across a variety of cell types, tissues, and species. We used our chemoproteomic strategy to determine potency and selectivity of academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors. Correlation of potency estimates by chemoproteomics with anti-proliferative activity of VX-11e and Ulixertinib revealed that >90% inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of inhibitors. Our findings introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to advance drug discovery of oncogenic MAPK pathways.
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Diacylglycerol lipase-beta (DAGLß) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLß inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLß inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLß-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of â¼5 µg of liposomal KT109 was sufficient to achieve â¼80% inactivation of DAGLß in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLß inhibitors to macrophages.
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Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Lipoproteína Lipasa/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Dolor/tratamiento farmacológico , Fagocitosis/efectos de los fármacos , Triazoles/farmacología , Urea/análogos & derivados , Urea/farmacología , Animales , Antiinflamatorios/uso terapéutico , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/complicaciones , Inflamación/inmunología , Lipopolisacáridos/inmunología , Lipoproteína Lipasa/metabolismo , Liposomas , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Dolor/inmunología , Fagocitosis/inmunología , Resultado del Tratamiento , Triazoles/uso terapéutico , Urea/uso terapéuticoRESUMEN
The electrophilic natural product parthenolide has generated significant interest as a model for potential chemotherapeutics. Similar to other α,ß-unsaturated carbonyl electrophiles, parthenolide induces the heat shock response in leukemia cells, potentially through covalent adduction of heat shock proteins. Other thiol-reactive electrophiles have also been shown to induce the heat shock response as well as to covalently adduct members of the heat shock protein family, such as heat shock protein 72 (Hsp72). To identify sites of modification of Hsp72 by parthenolide, we used high-resolution tandem mass spectrometry to detect 10 lysine, histidine, and cysteine residues of recombinant Hsp72 as modified in vitro by 10 and 100 µM parthenolide. To further ascertain that modification of Hsp72 by parthenolide occurs inside cells and not simply as an in vitro artifact, an alkyne-labeled derivative of parthenolide was synthesized to enable enrichment and detection of protein targets of parthenolide using copper-catalyzed [3 + 2] azide-alkyne cycloaddition. The alkyne-labeled parthenolide derivative displays an half maximal inhibitory concentration (IC50) in undifferentiated acute monocytic leukemia cells (THP-1) of 13.1 ± 1.1 µM, whereas parthenolide has an IC50 of 4.7 ± 1.1 µM. Concentration dependence of protein modification by the alkyne-parthenolide derivative was demonstrated, as well as in vitro adduction of Hsp72. Following treatment of THP-1 cells in culture by the alkyne-parthenolide, adducted proteins were isolated with neutravidin resin and detected by immunoblotting in the enriched protein fraction. Hsp70 proteins were detected in the enriched proteins, indicating that Hsp70 proteins were adducted intracellularly by the alkyne-parthenolide derivative.