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3',5'-cyclic uridine monophosphate (cUMP) and 3',5'-cyclic cytidine monophosphate (cCMP) have been established as bacterial second messengers in the phage defense system, named pyrimidine cyclase system for anti-phage resistance (Pycsar). This system consists of a pyrimidine cyclase and a cyclic pyrimidine receptor protein. However, the molecular mechanism underlying cyclic pyrimidine synthesis and recognition remains unclear. Herein, we determine the crystal structures of a uridylate cyclase and a cytidylate cyclase, revealing the conserved residues for cUMP and cCMP production, respectively. In addition, a distinct zinc-finger motif of the uridylate cyclase is identified to confer substantial resistance against phage infections. Furthermore, structural characterization of cUMP receptor protein PycTIR provides clear picture of specific cUMP recognition and identifies a conserved N-terminal extension that mediates PycTIR oligomerization and activation. Overall, our results contribute to the understanding of cyclic pyrimidine-mediated bacterial defense.
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Pirimidinas , Pirimidinas/química , Pirimidinas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Bacteriófagos/metabolismo , Uridina Monofosfato/metabolismo , Uridina Monofosfato/química , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Moleculares , Secuencia de Aminoácidos , Dedos de ZincRESUMEN
BACKGROUND: Protein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. RESULTS: Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. SIGNIFICANCE: This platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.
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Neoplasias Ováricas , Paclitaxel , Espectrometría de Masas en Tándem , Paclitaxel/farmacología , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Femenino , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/química , Supervivencia Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Nanotecnología , Ensayos de Selección de Medicamentos Antitumorales , Proteínas Quinasas/metabolismo , Proliferación Celular/efectos de los fármacosRESUMEN
Objectives: The protective effects and related mechanisms of Jing-Si herbal tea (JSHT) were investigated in cellular damage mediated by pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α, on normal human lung fibroblast by multiomic platform analysis. Materials and Methods: The in silico high-throughput target was analyzed using pharmacophore models by BIOVIA Discovery Studio 2022 with ingenuity pathway analysis software. To assess cell viability, the study utilized the MTT assay technique. In addition, the IncuCyte S3 ZOOM System was implemented for the continuous monitoring of cell confluence of JSHT-treated cytokine-injured HEL 299 cells. Cytokine concentrations were determined using a Quantibody Human Inflammation Array. Gene expression and signaling pathways were determined using next-generation sequencing. Results: In silico high-throughput target analysis of JSHT revealed ingenuity in canonical pathways and their networks. Glucocorticoid receptor signaling is a potential signaling of JSHT. The results revealed protective effects against the inflammatory cytokines on JSHT-treated HEL 299 cells. Transcriptome and network analyses revealed that induction of helper T lymphocytes, TNFSF12, NFKB1-mediated relaxin signaling, and G-protein coupled receptor signaling play important roles in immune regulatory on JSHT-treated cytokine-injured HEL 299 cells. Conclusion: The findings from our research indicate that JSHT holds promise as a therapeutic agent, potentially offering advantageous outcomes in treating virus infections through various mechanisms. Furthermore, the primary bioactive components in JSHT justify extended research in antiviral drug development, especially in the context of addressing coronavirus.
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The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20 mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20 kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0 µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52 µg/mL, and the concentration limit of quantitation was 4.60 µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.
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Lipoproteínas HDL , Fosfolípidos , Humanos , Células Cultivadas , Espectrometría de Masas , Electroforesis CapilarRESUMEN
BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are crucial components of brain function involved in memory and neurotransmission. Sodium benzoate is a promising NMDAR enhancer and has been proven to be a novel, safe, and efficient therapy for patients with Alzheimer disease (AD). However, in addition to the role of sodium benzoate as an NMDA enhancer, other mechanisms of sodium benzoate in treating AD are still unclear. To elucidate the potential mechanisms of sodium benzoate in Alzheimer disease, this study employed label-free quantitative proteomics to analyze serum samples from AD cohorts with and without sodium benzoate treatment. METHODS: The serum proteins from each patient were separated into 24 fractions using an immobilized pH gradient, digested with trypsin, and then subjected to nanoLCâMS/MS to analyze the proteome of all patients. The nanoLCâMS/MS data were obtained with a label-free quantitative proteomic approach. Proteins with fold changes were analyzed with STRING and Cytoscape to find key protein networks/processes and hub proteins. RESULTS: Our analysis identified 861 and 927 protein groups in the benzoate treatment cohort and the placebo cohort, respectively. The results demonstrated that sodium benzoate had the most significant effect on the complement and coagulation cascade pathways, amyloidosis disease, immune responses, and lipid metabolic processes. Moreover, Transthyretin, Fibrinogen alpha chain, Haptoglobin, Apolipoprotein B-100, Fibrinogen beta chain, Apolipoprotein E, and Alpha-1-acid glycoprotein 1 were identified as hub proteins in the proteinâprotein interaction networks. CONCLUSIONS: These findings suggest that sodium benzoate may exert its influence on important pathways associated with AD, thus contributing to the improvement in the pathogenesis of the disease.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Benzoato de Sodio/farmacología , Benzoato de Sodio/uso terapéutico , Proteómica , Espectrometría de Masas en Tándem , Fibrinógeno/uso terapéuticoRESUMEN
Thirty-four phytochemicals were isolated from dry tubers of Bletilla striata Rchb.f. The compounds were classified as bibenzyls 1-14, dihydrophenanthrenes 15, 17, 20, 21, phenanthrenes 16, 18, 19, simple benzoids 22-24, a fatty acid 25, glucosyloxybenzyl 2-isobutylmalates 26-32, and glucosyloxybenzyl cinnamates 33, 34. Compounds 1-4, 7, 8, 11, 12, and 16 inhibited melanogenesis (17.96-55.27%) induced by α-MSH in B16F10 cells at 10-40 µM. However, compounds 9, 10, 17, 18, and 21 exhibited significant cytotoxicity against melanomas, with IC50 values of 12-34 µM. Additionally, compounds 15, 17, 19, 20, 23, 31, and 33 reduced the ROS generation induced by H2O2 in HaCaT cells at 6.25-100 µM. In particular, compounds 15, 19, and 20 strongly inhibited ROS generation, with IC50 values of 2.15-9.48 µM. Consequently, compounds 1-4, 7-12, and 15-21 may be the strongest leads to follow in B. striata extract for further research on the skin disorders, hyperpigmentation, melanoma, and ageing.
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Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2'3'-phosphodiester linkage through intermediate pppU[3'-5']pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases.
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Nucleotidiltransferasas , Pirimidinas , Nucleotidiltransferasas/genética , Nucleótidos , Cromogranina A , Nucleótidos de PurinaRESUMEN
PURPOSE: Stroke is the sudden death of brain cells in a localized area due to an inadequate blood flow or blood vessel rupture, and it seriously affects the quality of life. The metabolite biomarkers are needed for predicting the functional outcome of acute ischemic stroke (AIS). EXPERIMENTAL DESIGN: To identify biomarkers for AIS, untargeted LC/MS metabolomics was performed on plasma samples from subjects with favorable prognosis (mRS ≤ 2) and unfavorable prognosis (mRS > 2). The identified markers were further absolutely quantified by a targeted MRM approach. RESULTS: There were 10 upregulated and 26 downregulated markers. Among these candidates, one was successfully identified as glycocholic acid and then absolutely quantified in plasma samples. Glycocholic acid could discriminate between subjects with favorable and unfavorable prognosis with an area under the curve (AUC) of 0.68 and odds ratio of 5.88. CONCLUSIONS AND CLINICAL RELEVANCE: Glycocholic acid was identified as a potential plasma metabolite marker of non-progressive outcomes after ischemic stroke and could serve as predictive prognostic markers for clinical acute stroke outcomes.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Pronóstico , Calidad de Vida , Cromatografía Liquida , Espectrometría de Masas en Tándem , Accidente Cerebrovascular/diagnóstico , Biomarcadores , Ácido Glicocólico , MetabolómicaRESUMEN
BACKGROUND AND AIMS: Alzheimer's disease (AD), the most common type of dementia, is hard to recognize early, resulting in delayed treatment and poor outcome. At present, there is neither reliable, non-invasive methods to diagnose it accurately and nor effective drugs to recover it. Discovery and quantification of novel metabolite markers in plasma of AD patients and investigation of the correlation between the markers and AD assessment scores. MATERIALS AND METHODS: Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics with LC-quadrupole- time-of-flight (Q-TOF) was performed in plasma samples of age-matched AD patients and healthy controls. The potential markers were further quantified with targeted multiple reaction monitoring (MRM) approach. RESULTS: Among the candidates, progesterone, and 3-indoleacetic acid (3-IAA) were successfully identified and then validated in 50 plasma samples from 25 AD patients and 25 matched normal controls with MRM approach. As a result, 3-IAA was significantly altered in AD patients and correlated with some AD assessment scores. CONCLUSION: By using untargeted LC-MS metabolomic and LC-MRM approaches to analyze plasma metabolites of AD patients and normal subjects, 3-IAA was discovered and quantified to be significantly altered in AD patients and correlated with several AD assessment scores.
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Enfermedad de Alzheimer , Humanos , Cromatografía Liquida/métodos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Espectrometría de Masas en Tándem/métodos , Metabolómica/métodos , Ácidos Indolacéticos , BiomarcadoresRESUMEN
In bottom-up proteomic profiling, the complexity of proteome composition and wide dynamic range has created challenges on the limited number of protein identification and proteome coverage, especially in sample input-limited nanoflow (nano) LC-MS/MS analysis. Herein, we developed a fully automatic online 2D nano-LC-MS/MS system using both high-pH and low-pH reverse phase (RP) LCs on a single LC instrument toward comprehensive proteomics analysis. Compared to conventional microflow 2D-LC, the high-pH RP trapping column demonstrated a low sample requirement of cellular protein digest at the µg level with good fractionation resolution of >90% peptides in a single fraction. Compared to the offline 2D RP-RP nano-LC-QTOF using C18-HPLC column and C18-Stage Tip, and 1D nano-LC-QTOF system, superior coverage was observed on the higher number of identified protein groups/unique peptides by 1.35-/1.68-, 1.46-/1.75-, and 3.21-/4.35-fold, respectively, using an online 2D RP-RP nano-LC-QTOF mass spectrometer. On the evolution of quantitation performance, the online 2D high-/low-pH RP data-independent acquisition (DIA) showed a higher reproducibility in protein groups intensity (R2 > 0.977) and more quantified proteins than that obtained using the offline 2D high-/low-pH RP DIA approach. Using an advanced Orbitrap Exploris 480 mass spectrometer, â¼1.9-fold higher proteome coverage was also observed in our 2D online RP-RP system (6039 protein groups) compared to the 1D nano-LC system (3133 protein groups). In summary, the online 2D nano-LC-MS/MS platform can be a sensitive and robust approach compatible with conventional nano-LC instruments for deep proteome coverage of trace amounts of samples.
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Proteoma , Espectrometría de Masas en Tándem , Proteoma/análisis , Proteómica , Reproducibilidad de los Resultados , Péptidos/análisis , Concentración de Iones de HidrógenoRESUMEN
The effects of transglutaminase (TGase, 1.0 unit/mL) with heat (95 °C, 5 min), 2-mercaptoethanol (2-ME, 0.83 %), and l-cysteine (l-Cys, 50 mM) pretreatment on the cross-linking of ovalbumin (OVA) and ovotransferrin (OVT) were investigated. SDS-PAGE revealed that although the polymerization of OVA and OVT did not occur after 3 h of incubation at 40 °C with TGase, OVA polymerized into high molecular weight polymers following TGase with 2-ME and heat pretreatment after 3 h of incubation. The surface hydrophobicity and reactive sulfhydryl (SH) groups of OVA samples significantly increased from 4065.7 ± 136.7 and 89.3 ± 1.2 SH groups (µmol/g) to 31483.6 ± 342.7 and 119.5 ± 3.7 SH groups (µmol/g), respectively. Similar results were obtained for OVT with TGase and l-Cys pretreatment and a 3-h incubation at 40 °C. The use of TGase, a reducing agent, and/or heat pretreatment can be used for the polymerization of OVA and OVT.
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Sustancias Reductoras , Transglutaminasas , Ovalbúmina , Transglutaminasas/metabolismo , Conalbúmina , Calor , MercaptoetanolRESUMEN
A simple and fast low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human very low-density lipoproteins (VLDLs). Native PAPC standard was analyzed to optimize the low-flow CE-MS method. The optimal CE conditions included separation buffer (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 0.5% (v/v) formic acid, 20 mM ammonium acetate), sheath liquid (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 20 mM ammonium acetate), separation voltage (20 kV), separation capillary internal diameter (i.d.) (75 µm), separation capillary temperature (23ËC) and sample injection time (6 s). The selected MS conditions included heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). Sheath gas was not used in this study. The total ion chromatograms (TICs), extracted ion chromatograms (EICs) and MS spectra of native PAPC standard and its in vitro oxidation products showed good repeatability and sensitivity. To determine the ox-PAPC products in human VLDLs, the EICs and MS spectra of VLDLs were compared with the in vitro oxidation products of native PAPC standard. For native PAPC standard, the measured linear range was 2.5 - 100.0 µg/mL, and the coefficients of determination (R2) was 0.9994. The concentration limit of detection (LOD) was 0.44 µg/mL, and the concentration limit of quantitation (LOQ) was 1.34 µg/mL. A total of 21 ox-PAPC products were analyzed for the VLDLs of healthy and uremic subjects. The levels of 7 short-chain and 5 long-chain ox-PAPC products on uremic VLDLs were significantly higher than healthy VLDLs. This simple low-flow CE-MS method might be a good alternative for LC-MS for the analysis of ox-PAPC products. Furthermore, it might also help scientists to expedite the search for uremic biomarkers.
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Lipoproteínas VLDL , Metanol , Humanos , Espectrometría de Masas , Lipoproteínas LDL , Electroforesis CapilarRESUMEN
The employment of liquid chromatography-mass spectrometry (LC-MS) untargeted and targeted metabolomics has led to the discovery of novel biomarkers and improved the understanding of various disease mechanisms. Numerous strategies have been reported to expand the metabolite coverage in LC-MS-untargeted and targeted metabolomics. To improve the sensitivity of low-abundance or poor-ionized metabolites for reducing the amount of clinical sample, chemical derivatization methods are used to target different functional groups. Proper sample preparation is beneficial for reducing the matrix effect, maintaining the stability of the LC-MS system, and increasing the metabolite coverage. Machine learning has recently been integrated into the workflow of LC-MS metabolomics to accelerate metabolite identification and data-processing automation, and increase the accuracy of disease classification and clinical outcome prediction. Due to the rapidly growing utility of LC-MS metabolomics in discovering disease markers, this review will address the recent advances in the field and offer perspectives on various strategies for expanding metabolite coverage, chemical derivatization, sample preparation, clinical disease markers, and machining learning for disease modeling.
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Hereditary Hemolytic Anemias (HHAs) are a rare but heterogeneous group of erythrocytic diseases, characterized by intrinsic cellular defects due to inherited genetic mutations. We investigated the efficacy of Chinese herbal medicine (CHM) in reducing the overall, diabetes-related, and cardiovascular diseases (CVDs)-related mortalities among patients with HHAs using a nationwide population database. In total, we identified 33,278 patients with HHAs and included 9,222 non-CHM and 9,222 CHM matched pairs after matching. The Cox proportional hazards model was used to compare the risk of mortality between non-CHM and CHM users. The Kaplan-Meier method and log-rank test were used to compare the cumulative incidence mortality between non-CHM and CHM users. The CHM prescription patterns were presented by the association rules and network analyses, respectively. The CHM prescription patterns were presented by the association rules and network analyses, respectively. CHM users showed significant reduced risks for of overall (adjusted hazard ratio [aHR]: 0.67, 95% confidence interval [CI]: 0.61-0.73, p < 0.001), diabetes-related (aHR: 0.57, 95% CI: 0.40-0.82, p < 0.001), and CVDs-related (aHR: 0.59, 95% CI: 0.49-0.72, p < 0.001) mortalities compared with non-CHM users. Two CHM clusters are frequently used to treat Taiwanese patients with HHAs. Cluster 1 is composed of six CHMs: Bei-Mu (BM; Fritillaria cirrhosa D.Don), Gan-Cao (GC; Glycyrrhiza uralensis Fisch.), Hai-Piao-Xiao (HPX; Endoconcha Sepiae), Jie-Geng (JG; Platycodon grandiflorus (Jacq.) A.DC.), Yu-Xing-Cao (YXC; Houttuynia cordata Thunb.), and Xin-Yi-Qing-Fei-Tang (XYQFT). Cluster 2 is composed of two CHMs, Dang-Gui (DG; Angelica sinensis (Oliv.) Diels) and Huang-Qi (HQi; Astragalus membranaceus (Fisch.) Bunge). Further randomized clinical trials are essential to evaluate the safety and effectiveness of above CHM products and to eliminate potential biases in the current retrospective study.
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Rapidly identifying methicillin-resistant Staphylococcus aureus (MRSA) with high integration in the current workflow is critical in clinical practices. We proposed a matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based machine learning model for rapid MRSA prediction. The model was evaluated on a prospective test and four external clinical sites. For the data set comprising 20,359 clinical isolates, the area under the receiver operating curve of the classification model was 0.78 to 0.88. These results were further interpreted using shapely additive explanations and presented using the pseudogel method. The important MRSA feature, m/z 6,590 to 6,599, was identified as a UPF0337 protein SACOL1680 with a lower binding affinity or no docking results compared with UPF0337 protein SA1452, which is mainly detected in methicillin-susceptible S. aureus. Our MALDI-TOF MS-based machine learning model for rapid MRSA identification can be easily integrated into the current clinical workflows and can further support physicians in prescribing proper antibiotic treatments. IMPORTANCE Over 20,000 clinical MSSA and MRSA isolates were collected to build a machine learning (ML) model to identify MSSA/MRSA and their markers. This model was tested across four external clinical sites to ensure the model's usability. We report the first discovery and validation of MRSA markers on the largest scale of clinical MSSA and MRSA isolates collected to date, covering five different clinical sites. Our developed approach for the rapid identification of MSSA and MRSA can be highly integrated into the current workflows.
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Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Aprendizaje Automático , Estudios Prospectivos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Infecciones Estafilocócicas/diagnóstico , Staphylococcus aureus/químicaRESUMEN
One new indazole alkaloid, indigodole E (1), was isolated from a traditional Chinese medicine Qing Dai prepared from the aerial parts of Strobilanthes cusia. The structure of 1 was elucidated by NMR, MS, UV, and IR spectra as well as optical rotation. Additionally, compound 1 could obviously inhibit not only IL-17A protein production at concentrations from 1.25 to 2.5 µg/mL, but also IL-17 gene expression at concentrations from 5.0 to 10.0 µg/mL without cytotoxicity toward Th17 and Jukat cells, respectively. Overall, indazole analogue 1 could be the anti-IL 17 A contributor of Qing Dai in this investigation.
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Acanthaceae , Acanthaceae/química , Medicina Tradicional China , Espectroscopía de Resonancia Magnética , IndazolesRESUMEN
Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in ß-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.
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Bacterias/metabolismo , Inmunidad Innata , Proteínas de la Membrana/química , Cristalografía por Rayos X , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Fosfatos de Dinucleósidos , Humanos , Interferones , Ligandos , Proteínas de la Membrana/genética , Nucleotidiltransferasas/metabolismo , PrevotellaRESUMEN
The complexity of the proteome often limits the number of identified proteins in the nanoflow LC-MS (nanoLC-MS) analysis of samples. Therefore, peptide fractionation is essential for reducing the sample complexity and improving the proteome coverage. In this study, to achieve high-pH reversed-phase (RP)-well plate fractionation for high-throughput proteomics analysis, C18 particles were coated on a 96-well plate, and the sample-loading processes were optimized for high-pH fractionation. The sample capacity of the high-pH RP-well plate was estimated to be ~6 µg of protein. There were 1.85- and 1.71-fold increases in the number of protein groups and peptides identified, respectively, with high-pH RP-well plate fractionation, compared to those without fractionation. In addition, with alkaline C18 well plate fractionation, exosome markers could be detected using ~1 µg of a protein digest of exosomes by microflow LC-MS (microLC-MS). These results illustrate that high-pH RP-well plate fractionation has superior sensitivity and effectiveness in preparing trace amounts of proteins for deep proteome analysis.
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Exosomas , Proteoma , Cromatografía de Fase Inversa/métodos , Exosomas/química , Concentración de Iones de Hidrógeno , Péptidos/análisis , Proteoma/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND/AIM: Helicobacter pylori, a gram-negative bacterium, causes chronic stomach diseases in humans. Heat shock proteins (HSPs) are involved in cell integrity, cell growth, and gastric mucosa colonization by H. pylori. This study aimed to investigate HSP expression levels in H. pylori-infected gastric adenocarcinoma AGS cells. MATERIALS AND METHODS: We determined protein expression levels using iTRAQ proteomics analysis. We analyzed the possible network interactions for H. pylori targets in AGS cells using the Ingenuity Pathway Analysis (IPA) software. RESULTS: H. pylori-infected AGS cells potentially targeted EIF2 and BAG2 signaling pathways to regulate cell physiology. In addition, after 3, 6, and 12 h of infection, western blotting revealed significantly decreased HSP70 and HSP105 expression. CONCLUSION: H. pylori decreases HSPs in AGS gastric adenocarcinoma cells, and this is associated with the regulation of EIF2 and BAG2 signaling pathways.