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BACKGROUND: Tyrosine metabolism (TM) plays an important role in the progression of cancer, but its role in lung adenocarcinoma (LUAD) is still unclear. This study aims to construct TM-related prognostic features for LUAD. METHODS: Transcriptomes and clinical data of LUAD were collected from public databases. A TM-related risk score (TMRS) model was constructed using 42 TM-related genes (TMRGs). The prognostic value of the model was comprehensively analyzed through survival analysis, enrichment analysis, immune assessment, and drug sensitivity prediction. The expression of key genes was also verified in LUAD cell lines and patient PBMCs. RESULTS: A 14-gene prognostic model (TMRS) was constructed. TMRS was an independent prognostic factor for LUAD. The low TMRS group has a more active immune microenvironment and may be more sensitive to immunotherapy. Patients with high TMRS may be more sensitive to various chemotherapy drugs. The model genes were specifically expressed in different cell types, suggesting that they may be involved in metabolic reprogramming and tumor progression. CONCLUSION: This study establishes a foundation for personalized risk assessment and treatment decisions in LUAD, highlighting the prognostic significance of TM.
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Adenocarcinoma del Pulmón , Biomarcadores de Tumor , Neoplasias Pulmonares , Tirosina , Humanos , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico , Pronóstico , Tirosina/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Masculino , Regulación Neoplásica de la Expresión Génica , Transcriptoma , Línea Celular Tumoral , Relevancia ClínicaRESUMEN
Tyrosinase is an enzyme that metabolizes L-tyrosine and is found in various organisms. Its overactivity can lead to health issues in humans, such as hyperpigmentation, and can adversely affect human skin, leading to skin cancers. This has heightened the significance of tyrosinase inhibitors in pharmaceuticals and cosmetics, particularly skin-whitening formulations. In this study, we developed a high-throughput screening assay for identifying tyrosinase inhibitors. This assay leverages the strong chemiluminescence signal emitted by L-tyrosine upon nanosecond UV irradiation in the presence of L-012 chemiluminescence dye, which is based on the formation of reactive oxygen species (ROS). We measured the decrease in chemiluminescence signal induced by tyrosinase enzyme, which converts chemiluminescent L-tyrosine into non-chemiluminescent L-DOPA. The addition of tyrosinase inhibitors prevents this conversion, leading to recovery in chemiluminescence of L-tyrosine. However, the reliability of the assay can be compromised by the ROS-scavenging activity and phenolic nature of certain enzyme inhibitors. To mitigate potential false results caused by some inhibitors, tyrosinase was immobilized on the microplate surface, and the inhibitors were incubated with the fixed enzyme, then, the enzyme activity was assessed after washing away the inhibitors. The proposed assay successfully facilitated high-throughput screening (less than 1 min per sample) of numerous tyrosinase inhibitor candidates from various pharmacological classes. The percentage inhibition of tyrosinase activity determined by our assay was statistically compared with results from a previously reported assay, revealing comparable outcomes and confirming the reliability of our approach. In addition, we evaluated the environmental impact and applicability of the assay using two recent metrics, yielding promising results.
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Inhibidores Enzimáticos , Ensayos Analíticos de Alto Rendimiento , Mediciones Luminiscentes , Monofenol Monooxigenasa , Rayos Ultravioleta , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/análisis , Mediciones Luminiscentes/métodos , Luminiscencia , Tirosina/metabolismo , Tirosina/química , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In this study, nanoscale Fe-N co-doped L/D chiral carbon nanotubes (Fe-N-L/D-CCNTs) were combined with molecular-level L/D histidine adsorbed metal-organic framework (L/D-His-ZIF-8) to construct a highly efficient nano-molecular dual-dimensional chiral recognition biosensor (Fe-N-L/D-CCNTs@L/D-His-ZIF-8), which was used for detecting chiral amino acids (AAs) and providing mechanistic insights. In this biosensor, Fe-N-L/D-CCNTs served as the substrate, and L/D-His-ZIF-8 acted as the chiral recognition molecule. The synergetic effect of both components enhanced chiral recognition performance for tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys) enantiomers. Fe-N-L/D-CCNTs@L/D-His-ZIF-8 identified Tyr enantiomers with detection limits of as low as 10.5 nM and 14.4 nM, with ID-Tyr/IL-Tyr and IL-Tyr/ID-Tyr ratios up to 2.78 and 2.26, respectively. This performance, which was also observed in Trp and Cys detections, confirmed the excellent chiral recognition performance of Fe-N-L/D-CCNTs@L/D-His-ZIF-8. In addition, density functional theory (DFT) was performed to elucidate the mechanism of the chiral biosensor recognition for L/D-amino acids (AAs). The biosensor realized efficient analysis and detection of actual samples, exhibiting good selectivity, reproducibility, and stability. The findings of this study broaden the scope of chiral amino acid identification, provide an experimental and theoretical basis for the design of novel multi-dimensional chiral recognition biosensors, and carry significant implications in drug screening, clinical diagnosis, and life science.
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Aminoácidos , Técnicas Biosensibles , Estructuras Metalorgánicas , Nanotubos de Carbono , Técnicas Biosensibles/métodos , Nanotubos de Carbono/química , Aminoácidos/química , Aminoácidos/aislamiento & purificación , Estructuras Metalorgánicas/química , Estereoisomerismo , Límite de Detección , Tirosina/química , Histidina/química , Hierro/química , Triptófano/química , Cisteína/químicaRESUMEN
Cytochrome P450 enzymes (also designated with the prefix CYP) catalyze oxygenation reactions on a wide array of organic substrates. The reaction cycle involves two high-potential intermediates that are oxidized one and two electrons above the resting enzyme. We have suggested that hole transfer along chains of tryptophan, tyrosine, and cysteine residues can prevent enzyme damage by these intermediates when reaction with substrate fails. This work is an examination of the distributions of tryptophan, tyrosine, and cysteine residues closest to the heme in the structures of 188 P450 enzymes. Tyrosine residues tend to be closest in bacterial enzymes, whereas tryptophan residues dominate in eukaryotic enzymes. A tryptophan residue hydrogen-bonded to the D-propionate of the heme is a common structural motif in eukaryotes. A tryptophan in a similar location in the bacterial CYP102A1 enzyme has been shown in prior work to extend the lifetime of the enzyme during turnover. The eukaryotic tryptophan residues might perform a similar protective role.
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Aminoácidos , Sistema Enzimático del Citocromo P-450 , Hemo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/química , Hemo/metabolismo , Oxidación-Reducción , Triptófano/química , Tirosina/química , Cisteína/química , Aminoácidos/químicaRESUMEN
Protein phosphorylation is critical to selective gene expression; the proteins that regulate transcription are often phosphorylated at multiple sites. Serine and threonine phosphorylation in transcription factors such as NF-κB has been studied, and specific serines are involved in transcriptional activation. Tyrosine phosphorylation of NF-κB p50 subunit can also facilitate the NF-κB-mediated expression of CD40 protein. This study seeks to determine whether tyrosine phosphorylations at positions not normally phosphorylated in vivo could nonetheless affect protein expression mediated by NF-κB. The alterations studied included p50 analogs having pTyr at positions 59 and 61, which do not contain Tyr naturally, and an analog containing a metabolically stable tyrosine methylene phosphonate at position 60. Additionally, to explore the structural basis for enhanced NF-κB binding to CD40 promoter DNA by tyrosine phosphorylation, an analog of NF-κB p50 containing pTyr at both positions 60 and 82 is prepared. The results reflected changes in the ability of the modified NF-κBs containing an altered p50 subunit to bind to CD40 promoter DNA in vitro, and to direct the synthesis of CD40 in cellulo.
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Antígenos CD40 , Subunidad p50 de NF-kappa B , Tirosina , Fosforilación , Tirosina/metabolismo , Tirosina/química , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/genética , Humanos , Antígenos CD40/genética , Antígenos CD40/metabolismo , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: To analyze the uptake patterns of dual-nuclide PET using 2-[ 18 F]-fluoro-2-deoxy- D -glucose (FDG) and O-(2-[ 18 F]-fluoroethyl)- L -tyrosine (FET) in intracranial lesions. METHODS: This study was conducted from July 2023 to September 2024. Patients with intracranial lesions underwent FDG PET and FET-PET scans on separate days before surgery. Four uptake patterns were observed: type A, positive uptake of both FET and FDG; type B, positive uptake of FET with negative uptake of FDG; type C, negative uptake of FET with positive uptake of FDG; and type D, negative uptake of both FET and FDG. Correlations between standardized uptake values (SUVs) of FET/FDG and tumor proliferation marker Ki-67/tumor protein p53 (P53) were analyzed using Pearson correlation analysis. RESULTS: Among 28 patients, type A was observed in 17, type B in nine, and type D in two patients. Dual-nuclide PET-based diagnoses were completely consistent with the pathology in nine, partially consistent in 17, and inconsistent in only two patients. Except for mature teratomas, all intracranial tumors demonstrated positive FET uptake. Correlations were observed between FDG and Ki-67: SUV max ( P â =â 0.011) and SUV mean ( P â =â 0.012). In addition, correlations were observed between the FDG/FET ratio and Ki-67: SUV max ( P â =â 0.029) and SUV mean ( P â =â 0.021). CONCLUSION: Dual-nuclide PET is valuable in diagnosing intracranial lesions. Type A is the most common uptake pattern. The SUV of FDG is positively correlated with the Ki-67 level. Compared with single-modality PET, dual-nuclide PET uptake patterns provide diagnostic insights into intracranial lesions.
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Neoplasias Encefálicas , Fluorodesoxiglucosa F18 , Tomografía de Emisión de Positrones , Tirosina , Humanos , Masculino , Femenino , Persona de Mediana Edad , Adulto , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/metabolismo , Fluorodesoxiglucosa F18/metabolismo , Anciano , Tirosina/análogos & derivados , Tirosina/metabolismo , Adulto Joven , Transporte Biológico , Adolescente , Niño , Antígeno Ki-67/metabolismoRESUMEN
Ulcerative colitis (UC) is a chronic inflammatory disease of the colon, characterized by recurrent flare-ups and limited effectiveness of available drug therapies. Asiatic acid (AA), a triterpenoid compound extracted from Centella asiatica, has shown promising pharmacological activities and therapeutic potential in several inflammation-related diseases. However, AA's efficacy in treating UC and its precise mechanisms of action remain to be fully explored. This study aimed to provide a comprehensive assessment of AA's therapeutic effects on UC and to investigate its underlying mechanisms, with a focus on gut microbiota interactions. In our study, a dextran sulfate sodium-induced UC mouse model was used to evaluate AA's therapeutic potential and explore its impact on gut microbiota composition and function. We further used an antibiotic cocktail and fecal microbiota transplantation assays to substantiate the role of gut microbiota in AA's mechanisms of action. A metabolomic analysis was also conducted to identify key metabolic pathways and gut microbiota-derived metabolites involved in AA's effects. Our findings demonstrated that AA significantly alleviates symptoms of UC, including reducing weight loss, slowing disease progression, mitigating colonic inflammation, and restoring immune balance. Mechanistically, the beneficial effects of AA were strongly linked to alterations in the gut microbiota and its metabolites, particularly γ-glutamyltyrosine. This metabolite was found to regulate the cyclic adenosine monophosphate/protein kinase A/nuclear factor kappa-B signaling pathway, which plays a crucial role in inflammatory responses. Overall, these findings strongly suggest that AA holds promise as a therapeutic agent for UC by modulating the gut microbiota and influencing critical inflammatory pathways.
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Colitis Ulcerosa , Microbioma Gastrointestinal , Triterpenos Pentacíclicos , Tirosina , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , FN-kappa B/metabolismo , Triterpenos Pentacíclicos/farmacología , Ratones , Masculino , Transducción de Señal/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Sulfato de Dextran , Trasplante de Microbiota Fecal , Ratones Endogámicos C57BL , Tirosina/análogos & derivados , Tirosina/metabolismo , Colon/efectos de los fármacosRESUMEN
Biomolecular condensates often form through the self-assembly of disordered proteins with low-complexity sequences. In these polypeptides, the aromatic amino acids phenylalanine and tyrosine act as key 'sticker' residues, driving the cohesion of dense phases. Recent studies on condensates suggest a hierarchy in sticker strength, with tyrosine being more adhesive than phenylalanine. This hierarchy aligns with experimental data on amino acids solubilities and potentials of mean force derived from atomistic simulations. However, it contradicts conventional chemical intuition based on hydrophobicity scales and pairwise contact statistics from experimental structures of proteins, which suggest that phenylalanine should be the stronger sticker. In this work, we use molecular dynamics simulations and quantum chemistry calculations to resolve this apparent discrepancy. Using simple model peptides and side-chain analogues, we demonstrate that the experimentally observed hierarchy arises from the lower free energy of transfer of tyrosine into the condensate, mediated by both stronger protein-protein interactions and solvation effects in the condensate environment. Notably, as the dielectric constant of the media surrounding the stickers approaches that of an apolar solvent, the trend reverses, and phenylalanine becomes the stronger sticker. These findings highlight the role of the chemical environment in modulating protein-protein interactions, providing a clear explanation for the crossover in sticker strength between tyrosine and phenylalanine in different media.
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Aminoácidos Aromáticos , Condensados Biomoleculares , Fenilalanina , Tirosina , Fenilalanina/química , Fenilalanina/metabolismo , Tirosina/química , Tirosina/metabolismo , Simulación de Dinámica Molecular , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Aminoácidos Aromáticos/químicaRESUMEN
INTRODUCTION: Protein tyrosine sulfation is of growing scientific interest due to its biological and clinical significance, yet it remains an underexplored post-translational modification (PTM). Catalyzed by Golgi-localized TPST1 and TPST2, tyrosine sulfation modulates protein-protein interactions and receptor-ligand binding in inflammation, hemostasis, immunity, and viral entry. Despite functional relevance, this modification is underrepresented in databases such as UniProt (accessed July 2025), in large part due to a lack of robust analytical strategies. Advances in mass spectrometry (MS)-based analyses have recently improved sensitivity of detection, expanding the known tyrosine 'sulfome.' Systematic profiling of sulfated residues can now be undertaken, expanding knowledge of their regulatory roles in both health and disease, and for pioneering new sulfation-targeted therapeutics. AREAS COVERED: We review known biological roles of protein sulfation by TPSTs and approaches for characterization of sulfation of tyrosine and other residues such as cysteine. More broadly, we consider how these strategies might be useful in a clinical context. EXPERT OPINION: High throughput MS-based proteomics has proven invaluable for the discovery of PTMs, advancing understanding of their roles in human health and disease. With recent advances in strategies for the characterization of protein sulfation, the field is now ready for exploration in a clinical context.
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Espectrometría de Masas , Proteómica , Tirosina , Humanos , Proteómica/métodos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Tirosina/metabolismoRESUMEN
This study aimed to prospectively examine the systemic effects of curative radiotherapy on dynamic thiol/disulfide homeostasis and nitrosative stress markers in patients diagnosed with lung cancer. Forty-one patients diagnosed with lung cancer and 41 healthy controls were enrolled in the study. Serum nitric oxide, 3-nitrotyrosine, total thiol, native thiol, and disulfide levels were measured from blood specimens taken from patients before radiotherapy and 24 h after radiotherapy. Nitric oxide levels were analyzed by chemiluminescence technique, 3-nitrotyrosine levels by ELISA, and thiol/disulfide homeostasis parameters by spectrophotometric methods. Serum nitric oxide (NO) and 3-nitrotyrosine levels of patients were significantly elevated in both pre-radiotherapy and post-radiotherapy periods compared to the healthy control group (p < 0.001). NO levels tended to increase after RT, but this difference did not reach statistical significance. Total thiol and native thiol levels in the pre-radiotherapy and post-radiotherapy periods were decreased when compared to the controls (p < 0.001). Post-radiotherapy disulfide levels were markedly higher than the pre-radiotherapy (p < 0.05) and the controls (p < 0.05). Similarly, while disulfide/total thiol and disulfide/native thiol ratios were increased in the post-radiotherapy period, a marked decline was observed in the native thiol/total thiol ratio in the post-radiotherapy period (p < 0.05). Since chemotherapy is also known to affect redox homeostasis, the combined treatment (chemoradiotherapy) is likely responsible for the observed biochemical changes seen in our study. Our suggest that radiotherapy further exacerbates the already imbalanced redox homeostasis in lung cancer patients by enhancing NO production and promoting thiol oxidation. Concurrent chemoradiotherapy appears to exacerbate the imbalance in redox homeostasis in lung cancer patients.
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Disulfuros , Neoplasias Pulmonares , Estrés Nitrosativo , Compuestos de Sulfhidrilo , Humanos , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Compuestos de Sulfhidrilo/sangre , Disulfuros/sangre , Femenino , Masculino , Persona de Mediana Edad , Óxido Nítrico/sangre , Anciano , Estrés Nitrosativo/efectos de la radiación , Tirosina/análogos & derivados , Tirosina/sangre , Adulto , Estudios de Casos y Controles , Estudios ProspectivosAsunto(s)
Humanos , Inmunohistoquímica , Receptor ErbB-2 , Tirosina , Proteínas , Diagnóstico , Metástasis de la NeoplasiaRESUMEN
Peptide-based coacervates are biocompatible and tunable but inherently unstable due to weak, reversible noncovalent interactions, making them vulnerable to stress from pH and temperature. This is especially difficult for coacervates used in commercial products with low-temperature storage or lyophilization. We engineered lyophilization-tolerant nanocoacervates by incorporating aromatic tyrosine residues into oligopeptide [(YR)2R10 + (YR)2D10] and reinforcing them with tannic acid (TA). This approach leverages π-π stacking, cation-π interactions, hydrogen bonds, and electrostatic interactions to create a stabilized network. The coacervates maintain their structure, count (107 particles/mL), size (<500 nm), and morphology after exposure to ionic strength of 10-100 mM, pH 3-10, elevated temperatures of 25-80 °C, biological media, and, crucially, freeze drying/rehydration. The study confirms that all interactions are essential for recovery from lyophilization. This is the first peptide-based coacervate system with lyophilization tolerance, supporting scalable applications in drug delivery, biostabilization, and regenerative medicine without cold-chain storage.
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Oligopéptidos , Péptidos , Polifenoles , Tirosina , Liofilización , Tirosina/química , Taninos/química , Polifenoles/química , Péptidos/química , Oligopéptidos/química , Concentración de Iones de Hidrógeno , Enlace de Hidrógeno , TemperaturaRESUMEN
Celiac disease (CD) is a chronic autoimmune disorder characterized by gluten-induced intestinal inflammation, epithelial barrier dysfunction, and malabsorption of nutrients, including amino acids (AAs). While essential AAs have been extensively studied, the roles of conditionally essential amino acids (CEAAs), cysteine (Cys), and tyrosine (Tyr) in CD pathogenesis remain comparatively underexplored. This review synthesizes current evidence on the contributions of these AAs to intestinal barrier integrity, immune regulation, oxidative stress mitigation, and gut microbiota modulation in CD. Key findings indicate that asparagine (Asn), glycine (Gly), Cys, glutamate (Glu), aspartate (Asp), and glutamine (Gln) contribute to the reinforcement of intestinal barrier integrity. In parallel, Cys and Gly, along with Gln and Asp, play important roles in suppressing oxidative stress, while Gln and Asp have been shown to positively influence the composition and function of the gut microbiota. Serine (Ser) may contribute to gliadin-induced epithelial damage but could also have potential protective effects during treatment. Arginine (Arg), through its metabolism via the inducible nitric oxide synthase (iNOS) and arginase pathways, contributes to immune regulation. This review underscores the therapeutic potential of AAs as adjunctive targets to gluten-free diets (GFD), offering avenues for nutritional interventions, enzyme therapies, and biomarker development. A deeper understanding of AA-mediated mechanisms may pave the way for personalized management strategies to improve clinical outcomes in CD.
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Aminoácidos Esenciales , Enfermedad Celíaca , Cisteína , Tirosina , Humanos , Enfermedad Celíaca/metabolismo , Enfermedad Celíaca/patología , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/microbiología , Cisteína/metabolismo , Tirosina/metabolismo , Microbioma Gastrointestinal , Animales , Aminoácidos Esenciales/metabolismo , Inflamación/metabolismo , Inflamación/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Estrés Oxidativo , Dieta Sin GlutenRESUMEN
Toll-like receptors (TLRs) are transmembrane proteins that recognize microbial components or cellular danger signals and activate intracellular signaling pathways, leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein that binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed that TLR9 was phosphorylated sequentially on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling. IMPORTANCE: Toll-like receptors (TLRs) are critical components of cellular innate immune responses to microbial infection or tissue damage. TLRs are transmembrane proteins that require activation to mount a successful host response; TLR mutations are associated with human diseases. TLR ligands are also used as vaccine adjuvants to amplify the inflammatory responses of the host, activation of TLRs, and their regulation are essential. Here, we report the molecular mechanisms of TLR9 activation by tyrosine phosphorylation of its cytoplasmic domain. TLR9 interacts with EGFR and Syk, the tyrosine kinases, which phosphorylate two specific tyrosine residues on the TLR9 cytoplasmic domain. Mutation of these tyrosine residues or deficiency of these tyrosine kinases leads to impaired TLR9 signaling. Therefore, our results elucidate the early events of TLR9 signaling with implications in inflammatory diseases.
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Receptores ErbB , Transducción de Señal , Quinasa Syk , Receptor Toll-Like 9 , Tirosina , Quinasa Syk/metabolismo , Quinasa Syk/genética , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/genética , Fosforilación , Receptores ErbB/metabolismo , Receptores ErbB/genética , Animales , Tirosina/metabolismo , Humanos , Ratones , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , LigandosRESUMEN
Rh(III)-catalyzed [4 + 2] annulation of C3Ar-formyl tyrosine ester with internal alkynes furnished di-substituted pyranone-mounted tyrosine-based unnatural amino acids in high yields. Selected compounds exhibited intrinsic viscosity-responsive fluorescence and favorable biocompatibility in microglial and neuroblastoma cells. Of these, one of the products showed efficient cellular uptake, preserved mitochondrial health, reduced ROS levels, and suppressed nitrite production in LPS-activated microglia, underscoring its potential as a bioactive fluorescent probe for modulating neuroinflammatory responses.
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Antiinflamatorios , Pironas , Rodio , Tirosina , Rodio/química , Catálisis , Tirosina/química , Tirosina/farmacología , Tirosina/síntesis química , Tirosina/análogos & derivados , Microglía/efectos de los fármacos , Microglía/metabolismo , Pironas/química , Pironas/farmacología , Animales , Humanos , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/síntesis química , Lipopolisacáridos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Estructura Molecular , Ratones , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tubulin detyrosination is an important α-tubulin specific posttranslational modification which has been implicated in various disorders including neurodegeneration and cancer. As such, the enzymes involved in the generation of this modification emerged as promising therapeutic targets. Previous studies have identified the members of the vasohibin family, VASH1 and VASH2, as the first class of enzymes involved in the generation of detyrosination. Recently, we have discovered Tubulin MetalloCarboxyPeptidase 1 (TMCP1) as the second class of enzymes catalyzing this modification. Here we describe the development of a highly sensitive FRET-based enzymatic assay to study and monitor the activity of TMCP1 and VASH2. The originality of this assay lies in the use of 3-nitrotyrosine as a quencher, which not only restores fluorescence upon cleavage but also closely mimics the natural tyrosine substrate, ensuring optimal enzyme recognition. The selected fluorogenic substrate, named FS2, exhibited strong quenching efficiency and a high signal-to-noise ratio, allowing for real-time kinetic monitoring of TMCP1 and VASH2 activity. Enzyme kinetics, competition assays, and metal ion dependency studies confirmed the assay's specificity, robustness, and physiological relevance. This optimized assay provides a powerful and reliable tool for the future identification and characterization of inhibitors of α-tubulin detyrosination.
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Carboxipeptidasas , Moléculas de Adhesión Celular , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Tubulina (Proteína) , Tirosina , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Tirosina/análogos & derivados , Tirosina/metabolismo , Tirosina/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Carboxipeptidasas/metabolismo , Carboxipeptidasas/química , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/química , Pruebas de Enzimas/métodos , CinéticaRESUMEN
Caspase-8 expression is upregulated in many tumors where, despite its canonical apoptotic pathway, it sustains cancer progression promoting cell migration, NF-kB activation and inflammation. Here, we provide the first evidence for a novel role of Caspase-8 in promoting the metabolic rewiring of cancer cells. By performing transcriptomic, proteomic and phosphoproteomic analyses on glioblastoma cellular models, we identify Caspase-8 as an unexpected modulator of NRF2. Here we show that Caspase-8 expression and phosphorylation affect NRF2 activity and mitochondrial homeostasis. Mechanistically, we demonstrate that Src-dependent phosphorylation of Caspase-8 on Tyrosine 380 (Y380), frequently reported in cancers including glioblastoma, sustains mTORC1 activation, thus promoting energy metabolism. mTORC1 activity results in p62 phosphorylation allowing its dependent sequestration of KEAP1 protein and constitutive NRF2 signaling activation, as a consequence. Overall, this work depicted a novel unexpected role for Caspase-8 in the modulation of cancer cell metabolism, bridging together Src, mTORC1 and NRF2 signaling.
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Caspasa 8 , Glioblastoma , Factor 2 Relacionado con NF-E2 , Familia-src Quinasas , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Caspasa 8/metabolismo , Caspasa 8/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/genética , Transducción de Señal , Fosforilación , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Tirosina/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mitocondrias/metabolismoRESUMEN
OBJECTIVE: This study investigates the role of peroxisome proliferator-activated receptor alpha (PPARα) in regulating macrophage polarization and inflammatory signaling under stimulation by periodontal pathogens. METHODOLOGY: THP-1-derived macrophages were stimulated with Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in the presence or absence of PPARα agonists fenofibrate and WY14643, or the antagonist GW6471. Protein expression levels of TNF-α, IL-10, and phosphorylated NF-κB were assessed by Western blot. Immunofluorescence staining was used to evaluate IL-10, NF-κB, and CD36 expression. Flow cytometry quantified changes in macrophage polarization markers, including CD14+CD86+ (M1) and CD68+CD206+/CD163+ (M2) populations. THP-1 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter plasmid were treated with Pg-LPS (1 µg/mL) ± fenofibrate (50 µM) to assess NF-κB/AP-1 activity. PPARα reporter cells were treated with increasing concentrations of GW590735 or WY14643 and exposed to TNF-α, LPS, or GW6471+LPS to evaluate PPARα transcriptional activity. RESULTS: PPARα activation by fenofibrate reduced TNF-α expression in Pg-LPS-stimulated macrophages and attenuated NF-κB signaling via both TLR2 and TLR4 pathways. Fenofibrate significantly increased IL-10 and CD36 expression, inhibited Pg-LPS-induced NF-κB nuclear translocation, and promoted a phenotypic shift from pro-inflammatory M1 to anti-inflammatory M2 macrophages. Moreover, inflammatory stimuli such as TNF-α and LPS suppressed PPARα activity, which could be restored by potent PPARα agonists. CONCLUSION: These findings suggest that PPARα activation modulates macrophage polarization and suppresses inflammatory signaling in response to periodontal bacterial antigens.
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Macrófagos , PPAR alfa , Periodontitis , PPAR alfa/antagonistas & inhibidores , PPAR alfa/agonistas , PPAR alfa/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Lipopolisacáridos/farmacología , Porphyromonas gingivalis , Factor de Necrosis Tumoral alfa/análisis , Transducción de Señal/efectos de los fármacos , Interleucina-10/análisis , FN-kappa B/análisis , Periodontitis/metabolismo , Periodontitis/microbiología , Pirimidinas/farmacología , Citometría de Flujo , Fenofibrato/farmacología , Western Blotting , Células THP-1 , Técnica del Anticuerpo Fluorescente , Oxazoles , Tirosina/análogos & derivadosRESUMEN
Immunoglobulin (Ig) and Proline-rich Receptor-1 (IGPR-1, also called TMIGD2) is closely related to immune checkpoint CD28/PDL1 family receptors. It controls important cellular processes, including immune cell regulation, cell-cell adhesion, mechanosensing, and autophagy, and its activity is associated with multiple human malignancies. However, the role and signaling mechanism of IGPR-1 remains largely undefined. Here, we report that IGPR-1's pro-tumor signaling in melanoma cells is mediated by phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) tyrosine (Y222). IGPR-1 is phosphorylated at Y222 in human melanoma tissues and cell lines. Phosphorylation of Y222 is context-dependent and catalyzed by EGFR and Src kinase. Pharmacological inhibition of EGFR using Erlotinib, as well as EGFR knockdown via shRNA, suppressed phosphorylation of Y222. In contrast, stimulation with EGF enhanced Y222 phosphorylation in vivo, and recombinant active EGFR directly phosphorylated Y222 in an in vitro kinase assay. In vivo coimmunoprecipitation and in vitro GST pull-down assays demonstrated that phospho-Y222 facilitates the binding of IGPR-1 with the SH2 domain-containing protein SHC1. IGPR-1 stimulates multiple key downstream signal transduction pathways relevant to tumorigenesis, including AKT, mTOR, and MAPK pathways. Mutation of Y222 blocked IGPR-1-mediated activation of AKT and MAPK, leading to the inhibition of tumor spheroid growth. This study uncovered new findings that have potential diagnostic and therapeutic implications in melanoma.
Asunto(s)
Motivo de Activación del Inmunorreceptor Basado en Tirosina , Melanoma , Proteínas Proto-Oncogénicas c-akt , Humanos , Fosforilación , Melanoma/metabolismo , Melanoma/patología , Melanoma/genética , Transducción de Señal , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Receptores ErbB/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Tirosina/metabolismoRESUMEN
Tyrosinemia type II (Richner-Hanhart syndrome) is a rare disorder caused by mutations in the TAT gene, leading to elevated blood tyrosine and impaired metabolism. It presents with oculocutaneous symptoms, retinal tyrosine crystals, and neurological issues. Elevated tyrosine disrupts brain metabolism, neurotransmitters, and neurotrophic factors, causing neuroinflammation and affecting brain function. The exact mechanism of neurological damage is unclear, and the impact of dietary intervention on cognition is uncertain. While rodent models are commonly used, zebrafish are emerging as a cost-effective, genetically similar alternative for studying tyrosinemia type II. Thus, this study aims to determine whether acute exposure of zebrafish to elevated tyrosine concentrations can reproduce early central nervous system alterations associated with tyrosinemia type II. Zebrafish were exposed via immersion to 1 mM or 2 mM tyrosine for 1-24 h, with a total of 180 animals used across assays. Behavioral analysis was conducted using the novel tank test, and cholinergic and oxidative stress markers were assessed. Brain tyrosine levels were measured centrally. Exposure to 1 mM tyrosine for 24 h resulted in the highest brain accumulation, suggesting a non-linear dose-response. Behavioral testing revealed decreased locomotor activity and exploratory behavior, and ChAT activity was reduced in both exposure groups. No significant changes were observed in oxidative stress or protein damage. These findings indicate that acute tyrosine exposure induces early behavioral and cholinergic alterations without detectable oxidative stress, supporting the use of zebrafish as a preliminary model to study early neurochemical disturbances such in tyrosinemia type II. Further studies should explore different life stages, sex-specific responses, chronic exposure, and precise tyrosine kinetics, including potential non-linear effects due to the LAT1 transporter, to clarify mechanisms underlying neurotoxicity and improve translational relevance.