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Boletus aereus Fr. ex Bull. stands out as a delectable edible mushroom with high nutritional and medicinal values, featuring polysaccharides as its primary nutrient composition. In our continuous exploration of its beneficial substances, a novel polysaccharide (BAPN-1) with a molecular weight of 2279 kDa was prepared. It was identified as a glucan with a backbone composed of the residues â4)-α-Glcp-(1â and â4,6)-α-Glcp-(1â connected in a proportion of 5:1 and a ß-Glcp-(1â side residue attached at C6 of the â4,6)-α-Glcp-(1â residue. Biologically, BAPN-1 exhibited broad-spectrum antiproliferative activities against various NHL cells, including HuT-78, OCI-LY1, OCI-LY18, Jurkat, RL, and Karpas-299, with IC50 values of 0.73, 1.21, 3.18, 1.52, 3.34, and 4.25 mg/mL, respectively. Additionally, BAPN-1 significantly induced cell cycle arrest in the G2/M phase and caused apoptosis of NHL cells. Mechanistically, bulk RNA sequencing and Western blot analysis revealed that BAPN-1 could upregulate cyclin B1 and enhance cleaved caspase-9 expression through the inhibition of FGFR3 and RAF-MEK-ERK signaling pathways. This work supports the improved utilization of B. aereus in high-value health products.
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Antineoplásicos , Apoptose , Proliferação de Células , Linfoma não Hodgkin , Polissacarídeos , Humanos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Linfoma não Hodgkin/tratamento farmacológico , Polissacarídeos/química , Polissacarídeos/farmacologia , Polissacarídeos/isolamento & purificação , Peso Molecular , Basidiomycota/químicaRESUMO
BACKGROUND: Colorectal cancer (CRC) has a high incidence and mortality. Recent studies have shown that indole derivatives involved in gut microbiota metabolism can impact the tumorigenesis, progression, and metastasis of CRC. AIM: To investigate the effect of indole-3-acetaldehyde (IAAD) on CRC. METHODS: The effect of IAAD was evaluated in a syngeneic mouse model of CRC and CRC cell lines (HCT116 and DLD-1). Cell proliferation was assessed by Ki-67 fluorescence staining and cytotoxicity tests. Cell apoptosis was analysed by flow cytometry after staining with Annexin V-fluorescein isothiocyanate and propidium iodide. Invasiveness was investigated using the transwell assay. Western blotting and real-time fluorescence quantitative polymerase chain reaction were performed to evaluate the expression of epithelial-mesenchymal transition related genes and aryl hydrocarbon receptor (AhR) downstream genes. The PharmMapper, SEA, and SWISS databases were used to screen for potential target proteins of IAAD, and the core proteins were identified through the String database. RESULTS: IAAD reduced tumorigenesis in a syngeneic mouse model. In CRC cell lines HCT116 and DLD1, IAAD exhibited cytotoxicity starting at 24 h of treatment, while it reduced Ki67 expression in the nucleus. The results of flow cytometry showed that IAAD induced apoptosis in HCT116 cells but had no effect on DLD1 cells, which may be related to the activation of AhR. IAAD can also increase the invasiveness and epithelial-mesenchymal transition of HCT116 and DLD1 cells. At low concentrations (< 12.5 µmol/L), IAAD only exhibited cytotoxic effects without promoting cell invasion. In addition, predictions based on online databases, protein-protein interaction analysis, and molecular docking showed that IAAD can bind to matrix metalloproteinase-9 (MMP9), angiotensin converting enzyme (ACE), poly(ADP-ribose) polymerase-1 (PARP1), matrix metalloproteinase-2 (MMP2), and myeloperoxidase (MPO). CONCLUSION: Indole-3-aldehyde can induce cell apoptosis and inhibit cell proliferation to prevent the occurrence of CRC; however, at high concentrations (≥ 25 µmol/L), it can also promote epithelial-mesenchymal transition and invasion in CRC cells. IAAD activates AhR and directly binds MMP9, ACE, PARP1, MMP2, and MPO, which partly reveals why it has a bidirectional effect.
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The intestinal mucosal barrier serves as a vital guardian for gut health, maintaining a delicate equilibrium between gut microbiota and host immune homeostasis. Recent studies have found the intricate roles of Gasdermin D (GSDMD), a key executioner of pyroptosis downstream of the inflammasome, within the intestine, including controlling colitis in intestinal macrophage and the regulatory function in goblet cell mucus secretion. Thus, the exact role and nature of GSDMD's regulatory function in maintaining intestinal immune homeostasis and defending against pathogens remain elucidation. Here, we uncover that GSDMD plays a key role in defending against intestinal Citrobacter rodentium infection, with high expression in intestinal epithelial and lamina propria myeloid cells. Our results show that GSDMD specifically acts in intestinal epithelial cells to fight the infection, independently of its effects on antimicrobial peptides or mucin secretion. Instead, the resistance is mediated through GSDMD's N-terminal fragments, highlighting its importance in intestinal immunity. However, the specific underlying mechanism of GSDMD N-terminal activity in protection against intestinal bacterial infections still needs further study to clarify in the future.
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OSCA/TMEM63 is a newly identified family of mechanically activated (MA) ion channels in plants and animals, respectively, which convert physical forces into electrical signals or trigger intracellular cascades and are essential for eukaryotic physiology. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. However, the molecular architecture of the mammalian TMEM63 proteins remains unclear. Here we elucidate the structure of human TMEM63A in the presence of calcium by single particle cryo-EM, revealing a distinct monomeric architecture containing eleven transmembrane helices. It has structural similarity to the single subunit of the Arabidopsis thaliana OSCA proteins. We locate the ion permeation pathway within the monomeric configuration and observe a nonprotein density resembling lipid. These results lay a foundation for understanding the structural organization of OSCA/TMEM63A family proteins.
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Cálcio , Microscopia Crioeletrônica , Humanos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Cálcio/química , Canais Iônicos/química , Canais Iônicos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Chronic liver disease (CLD) imposes a heavy burden on millions of people worldwide. Despite substantial research on the pathogenesis of CLD disorders, no optimal treatment is currently available for some diseases, such as liver cancer. Exosomes, which are extracellular vesicles, are composed of various cellular components. Exosomes have unique functions in maintaining cellular homeostasis and regulating cell communication, which are associated with the occurrence of disease. Furthermore, they have application potential in diagnosis and treatment by carrying diverse curative payloads. Hepatic macrophages, which are key innate immune cells, show extraordinary heterogeneity and polarization. Hence, macrophage-derived exosomes may play a pivotal role in the initiation and progression of various liver diseases. This review focuses on the effects of macrophage-derived exosomes on liver disease etiology and their therapeutic potential, which will provide new insights into alleviating the global pressure of CLD.
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The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.
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Estimulantes do Sistema Nervoso Central , Cocaína , Metanfetamina , Animais , Suínos , Proteínas da Membrana Plasmática de Transporte de Serotonina , Microscopia Crioeletrônica , Detergentes , Serotonina , Cocaína/farmacologia , Metanfetamina/farmacologiaRESUMO
Aims: To evaluate the effects of different durations of continuous aerobic exercise on prediabetic patients. Materials and methods: The research encompassed randomized controlled trials that examined how various durations of aerobic exercise training affected outcomes related to Body Mass Index (BMI), Fasting blood glucose (FBG), 2-hour plasma glucose (2hPG), and glycated hemoglobin (HbA1c) in individuals diagnosed with prediabetes. PubMed, Embase, Web of Science, and the Cochrane Library were searched as of January 7, 2023. The Cochrane Risk of Bias, version 2 (ROB 2) tool was used to assess the risk of bias. Results: A total of 10 RCTs with 815 prediabetic patients were included. The average age of the participants was 56.1 years, with a standard deviation of 5.1 years. Among the participants, 39.2% were male. The interventions consisted of aerobic dance, treadmill running, walking, and a combination of aerobic exercises. The training sessions occurred three or four times per week. In prediabetic patients, aerobic exercise demonstrated a significant reduction in BMI compared to the control group, with a weighted mean difference (WMD) of -1.44 kg/m2 (95% confidence interval [CI] -1.89, -0.98). There was a decrease in FBG levels, with WMD of -0.51 mmol/L (95% CI -0.70, -0.32). Additionally, aerobic training led to significant improvements in 2hPG levels, with a WMD of -0.76 mmol/L (95% CI -1.14, -0.38). Furthermore, prediabetic patients showed a decrease in HbA1c levels after engaging in aerobic training compared to the control group, with a WMD of -0.34% (95% CI -0.45, -0.23). Conclusion: In summary, engaging in aerobic exercise can have a significant positive impact on glycemic levels in individuals with prediabetes. It can also lead to reductions in BMI, FBG, 2hPG, HbA1c, and other relevant indicators. The extent of these improvements may vary slightly depending on the duration of the aerobic exercise intervention. Systematic review registration: PROSPERO https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023395515.
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Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exercício Físico , Terapia por Exercício , Hemoglobinas Glicadas , Estado Pré-Diabético/terapiaRESUMO
Aim: The oleanolic acid derivatives containing electrophilic warheads were synthesized, and their antitumor activities were investigated. Materials & methods: The cytotoxicity of compounds against tumor cells were determined by the MTT method. The antitumor effects of compounds 27a, Y03 and Y04 were evaluated in vitro through a wound-healing assay, apoptosis and cell circle analysis, and cellular reactive oxide species determination. The levels of related proteins in MCF-7 cells treated with Y03 was determined through Western blot analysis. Results & conclusion: Compounds 27a, Y03 and Y04 displayed high cytotoxicity against breast cancer cells and inhibited cell migration, induced apoptosis, arrest cell circle at G0/G1 and promoted cellular reactive oxide species generation. The antitumor mechanism involved inhibition of Akt/mTOR and induction of ferroptosis.
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Antineoplásicos , Ácido Oleanólico , Humanos , Ácido Oleanólico/farmacologia , Antineoplásicos/farmacologia , Células MCF-7 , Apoptose , Proliferação de Células , Linhagem Celular TumoralRESUMO
Soil heavy metal contamination is crucial due to menacing food safety and mortal health. At present, with the fast advancement of urbanization and industrialization, heavy metals are increasingly released into the soil by anthropogenic activities, and the soil ecosystem contamination around the Danjiangkou Reservoir is directly associated with water quality security of the reservoir. In this paper, using 639 soil samples from the Danjiangkou Reservoir, Henan Province, China, we studied a variety of space distribution characteristics of heavy metals in soil. Geographic information system analysis (GIS), geo-accumulation index (Igeo), contamination factor (CF), principal component analysis (PCA) model, and positive matrix factorization (PMF) model were used together to recognize and quantify the distribution, contamination, and origin of heavy metals. We uncovered an exceptional variety of heavy metal concentrations among the tested soils: the mean arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), manganese (Mn), nickel (Ni), zinc (Zn), lead (Pb) and mercury (Hg) concentrations (14.54, 0.21, 18.69, 81.69, 898.42, 39.37, 79.50, 28.11, 0.04 mg/kg, respectively, in the topsoil (0-20 cm depth)), all exceed their background values. The mean Igeo value and CF values of these trace elements are both in descending order: Cd > Co > Mn > Ni > Pb > Zn > Cr > As > Hg. Cd was the highest contributor to the assessment of heavy metal pollution, with an average Igeo value over three, indicating that the study area is modestly contaminated by Cd. The PCA analysis and PMF model revealed three potential sources, including natural sources (PC1) for Cr, Co, Mn and Ni; agricultural sources (PC2) for Cd, Zn and Hg; and industrial emissions and transportation sources (PC3) for Pb. This study displays a map of heavy metal contamination in the eastern area topsoil of the Danjiangkou Reservoir, showing the most severe pollutant is Cd, which poses a threat to the water quality security of Danjiangkou Reservoir and provides a significant source identification for future contamination control.
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Arsênio , Mercúrio , Metais Pesados , Poluentes do Solo , Solo , Ecossistema , Cádmio/análise , Chumbo/análise , Monitoramento Ambiental , Metais Pesados/análise , Arsênio/análise , Qualidade da Água , Zinco/análise , Mercúrio/análise , Cromo/análise , Cobalto/análise , Manganês/análise , Níquel/análise , China , Poluentes do Solo/análise , Medição de RiscoRESUMO
Huangqi decoction, also known as Huangqi Liuyi decoction, was first recorded in the prescriptions of the Bureau of Taiping People's Welfare Pharmacy. It comprises astragalus and licorice, which is a commonly used prescription in traditional Chinese medicine for the clinical treatment of chronic liver disease, especially liver cirrhosis. Total astragalus saponins (AST) is the main component of astragalus, and glycyrrhizic acid (GA) is the main component of licorice. In this study, normal macrophage exosomes were extracted, and the exosomes incubated with lipopolysaccharides (LPS) and those incubated with LPS + AST + GA were co-cultured with JS1 cells (hepatic stellate cell line). The survival rate and the activation of key signaling pathways of JS1 cells in each group were detected and compared. We found that the co-culture of LPS-induced macrophage exosomes with JS1 cells could significantly increase the expression levels of Collagen-1 (Col-1) and Alpha smooth muscle actin (α-SMA)in JS1 cells. However, a significant reversal effect was observed after pretreatment with AST combined with GA. Further evaluation found that the expression levels of phospho (p)-Smad2 and p-Smad3 in the JS1 cells were significantly increased after macrophages were induced with LPS, whereas pretreatment with AST + GA could significantly decrease the expression levels of p-Smad2 and p-Smad3. Preliminary results of this study indicated that LPS-induced macrophage exosomes can promote the activation of hepatic stellate cells, and the pretreatment of AST combined with GA can exert a significant intervention effect. In this study, the new mechanism of anti-hepatic fibrosis effect of traditional Chinese medicine components of Huangqi Decoction was analyzed from the perspective of exosomes.
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Exossomos , Saponinas , Humanos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/metabolismo , Lipopolissacarídeos/toxicidade , Saponinas/farmacologia , Saponinas/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , MacrófagosRESUMO
Immune evasion is a common reason causing the failure of anticancer immune therapy. Small interfering RNA (siRNA), which can activate the innate and adaptive immune system responses by silencing immune-relevant genes, have been demonstrated to be a powerful tool for preventing or reversing immune evasion. However, siRNAs show poor stability in biological fluids and cannot efficiently cross cell membranes. Nanotechnology has shown great potential for intracellular siRNA delivery in recent years. Nano-immunotherapy can efficiently penetrate the tumor microenvironment (TME) and deliver multiple immunomodulatory agents simultaneously, which appears to be a promising method for combination therapy. Therefore, it provides a new perspective for siRNA delivery in immunomodulation and cancer immunotherapy. The current advances and challenges in nanotechnology-based siRNA delivery strategies for overcoming immune evasion will be discussed in this review. In addition, we also offer insights into therapeutic options, which may expand its applications in clinical cancer treatment.
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BACKGROUND: Certain "star intestinal bacteria" have been found to act as a contributor to the development of colorectal cancer (CRC). Besides, given that the gut microbiome can be detected in a diverse range of samples (stool, tissue, blood, etc), it is categorized into fecal microbiome, blood microbiome, and tissue microbiome. METHODS: To provide an overview of the recent research progress, this review summarizes the characteristics of the gut microbiome in different samples at each stage of CRC and their screening efficiency. RESULTS: The screening models constructed from different sample microbiomes (healthy/colorectal adenoma, healthy/CRC, and colorectal adenoma/CRC) have both strengths and constraints in terms of biomarker reproducibility and area under the receiver-operating characteristic curve (AUC) of the screening models. Many bacteria, such as Bifidobacteria, Fusobacterium nucleatum (F. n), Geotrichum candidum, Porphyromonas asaccharolytica, Escherichia coli, Rhodococcus, Anaerostipes caccae, Enhydrobacter, Lachnoclostridiumsp. m3, Bacteroides clarus, Clostridium hathewayi, Ruminococcaceae, Bacteroides thetaiotaomicron, Culinariside, and enterotoxigenic Bacteroides fragilis (ETBF), show favorable diagnostic efficacy in early screening of colorectal cancer. CONCLUSIONS: This review highlights stool, blood, tissue, and bowel fluid are the main sample sources for biomarkers, each with its own advantages and limitations. Moreover, other samples such as extracellular vesicles and biofilms also should been deserved further attention.
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Adenoma , Neoplasias Colorretais , Microbioma Gastrointestinal , Adenoma/diagnóstico , Biomarcadores , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Fezes/microbiologia , Humanos , Reprodutibilidade dos TestesRESUMO
The serotonin transporter (SERT) terminates serotonin signaling by using sodium and chloride gradients to drive reuptake of serotonin into presynaptic neurons and is the target of widely used medications to treat neuropsychiatric disorders. Despite decades of study, the molecular mechanism of serotonin transport, the coupling to ion gradients, and the role of the allosteric site have remained elusive. Here, we present cryoelectron microscopy structures of SERT in serotonin-bound and serotonin-free states, in the presence of sodium or potassium, resolving all fundamental states of the transport cycle. From the SERT-serotonin complex, we localize the substrate-bound allosteric site, formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule, connected to the central site via a short tunnel. Together with elucidation of multiple apo state conformations, we provide previously unseen structural understanding of allosteric modulation, demonstrating how SERT binds serotonin from synaptic volumes and promotes unbinding into the presynaptic neurons.
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Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [3H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.
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Antidepressivos/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Cloridrato de Vilazodona/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Humanos , Cinética , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/ultraestruturaRESUMO
OBJECTIVE: To investigate the effect of 3.0T MRI and ultrasonography in the diagnosis of uterine scar pregnancy (CSP) after caesarean section, and to compare their diagnostic value for CSP. MATERIALS AND METHODS: A retrospective analysis was conducted on 60 patients with CSP treated in our hospital over a period of July 2018 to March 2020. All patients underwent 3.0T MRI, ultrasonography, and surgical termination of pregnancy and pathological analysis. The value of 3.0T MRI and ultrasonography in the diagnosis of CSP was analyzed. RESULTS: (1) The 60 patients were pathologically analyzed. Among these patients, 2 of whom were trophoblastic diseases, 5 were pregnancy abortion, 8 were cervical pregnancy, and 45 were CSP. (2) The results of ultrasound detection were 37 cases of CSP, 7 cases of misdiagnosis, and 8 cases of missed diagnosis; 3.0T MRI results were 44 cases of CSP, 1 case of misdiagnosis, and 1 case of missed diagnosis. (3) The sensitivity (97.78%), specificity (93.33%), coincidence rate (96.67%), positive diagnosis rate (97.78%), negative diagnosis rate (93.33%), AUC (0.973), and 95% CI (0.914-0.996) of 3.0T MRI in diagnosing CSP were significantly higher than those of ultrasound diagnosis (82.22%, 53.33%, 84.09%, 84.09%, 50%, 0.681, 0.051-0.776) (P<0.05). CONCLUSION: The coincidence rate of 3.0T MRI in the diagnosis of CSP after caesarean section is significantly better than that of ultrasound diagnosis, and it can be used to provide reference for clinical diagnosis of CSP after cesarean section.
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Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.
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Paroxetina/química , Inibidores Seletivos de Recaptação de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Estrutura Molecular , Paroxetina/metabolismo , Estrutura Terciária de Proteína , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/metabolismoRESUMO
The functional relevance and mechanistic basis of the effects of the pyroptosis executioner Gasdermin D (GSDMD) on colitis remain unclear. In this study, we observed that GSDMD protein was activated during intestinal inflammation in a model of chemically induced colitis. GSDMD deficiency exacerbated experimental colitis independent of changes in the microbiota and without affecting the production of antimicrobial peptides. GSDMD deficiency in macrophages, but not epithelial cells, was sufficient to drive this exacerbated experimental colitis. We further demonstrate that GSDMD functions in macrophages as a negative regulator to control cyclic GMP-AMP synthase (cGAS)-dependent inflammation, thereby protecting against colitis. Moreover, the administration of cGAS inhibitor can rescue the colitogenic phenotype in GSDMD-deficient mice. Collectively, these findings provide the first demonstration of GSDMD's role in controlling colitis and a detailed delineation of the underlying mechanism.
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An α-galactosidase designated as TAG was purified from the dried fruit bodies of Tremella aurantialba with 182.5-fold purification. The purification procedure involved ion exchange chromatography on Q-sepharose, DEAE-Cellulose, and Mono Q and gel filtration by FPLC on Superdex 75. The purified α-galactosidase was a monomeric protein with a molecular mass of 88 kDa. The optimal pH of TAG was 5.0 and more than 60% of the original enzyme activity remained at pH 2.0 and 3.0. Its optimal temperature was 54 °C with good thermo-stability, 30.8% of the original activity was retained after exposure to a temperature of 70 °C for 1 h. The metal ions Hg2+, Cu2+, Fe3+ and Mg2+ strongly inhibited the enzyme activity. The enzyme activity was found to be inhibited by N-bromosuccinimide indicating that tryptophan was essential to the catalytic activity of α-galactosidase. The enzyme completely hydrolysed stachyose and partially hydrolysed raffinose to galactose at 50 °C within 6 h as detected by thin layer chromatography and the dinitrosalicylic acid method and the content of reducing sugar reached 4.36 mg/mL.
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Basidiomycota/enzimologia , Proteínas Fúngicas , Oligossacarídeos/química , Rafinose/química , alfa-Galactosidase , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Temperatura Alta , Hidrólise , Metais/química , alfa-Galactosidase/química , alfa-Galactosidase/isolamento & purificaçãoRESUMO
The NLRP3 inflammasome is critical for EAE pathogenesis; however, the role of gasdermin D (GSDMD), a newly identified pyroptosis executioner downstream of NLRP3 inflammasome, in EAE has not been well defined. Here, we observed that the levels of GSDMD protein were greatly enhanced in the CNS of EAE mice, especially near the areas surrounding blood vessels. GSDMD was required for the pathogenesis of EAE, and GSDMD deficiency in peripheral myeloid cells impaired the infiltration of immune cells into the CNS, leading to the suppression of neuroinflammation and demyelination. Furthermore, the loss of GSDMD reduced the activation and differentiation of T cell in the secondary lymphoid organs and prevented T cell infiltration into CNS of EAE. The administration of inflammasome-related cytokines partially rescued the impairment of pathogenesis of EAE in GSDMD KO mice. Collectively, these findings provide the first demonstration of GSDMD in peripheral myeloid cells driving neuroinflammation during EAE pathogenesis.
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Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Mieloides/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/patologia , Citocinas/metabolismo , Doenças Desmielinizantes/genética , Encefalomielite Autoimune Experimental/genética , Feminino , Inflamassomos/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/genéticaRESUMO
Although mitochondria are known to be involved in host defense against viral infection, the physiological role of mitophagy, a crucial mechanism for maintaining mitochondrial homeostasis, in antiviral immunity remains poorly defined. Here, we show that Parkin, a central player in mitophagy, has a vital function in regulating host antiviral responses. Parkin-knockout mice exhibit improved viral clearance and survival after viral infection. However, Parkin deficiency does not affect antiviral signaling and interferon production. Instead, Parkin deficiency augments innate antiviral inflammation by enhancing mitochondrial ROS (mtROS)-mediated NLRP3 inflammasome activation and promoting viral clearance. Loss of NLRP3 can reverse the enhanced antiviral responses in Parkin knockout mice. Furthermore, we find that Parkin expression is downregulated in peripheral blood mononuclear cells of patients infected with virus. Collectively, our results suggest that Parkin plays an important role in antiviral immunity by controlling mtROS-NLRP3 axis-mediated inflammation. These findings provide physiological insight of the importance of mitophagy in regulating host antiviral response.