Tandem Mass Tag-based Quantitative Proteomics Research Articles

Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alphaglucosaminidase to Parkinson's disease.

Parkinson's disease is the second most common progressive neurodegenerative disorder, and few reliable biomarkers are available to track disease progression. The proteins, DNA, mRNA, and lipids carried by exosomes reflect intracellular changes, and thus can serve as biomarkers for a variety of conditions. In this study, we investigated alterations in the protein content of plasma exosomes derived from patients with Parkinson's disease and the potential therapeutic roles of these proteins in Parkinson's disease. Using a tandem mass tag-based quantitative proteomics approach, we characterized the proteomes of plasma exosomes derived from individual patients, identified exosomal protein signatures specific to patients with Parkinson's disease, and identified N-acetyl-alpha-glucosaminidase as a differentially expressed protein. N-acetyl-alpha-glucosaminidase expression levels in exosomes from the plasma of patients and healthy controls were validated by enzyme-linked immunosorbent assay and western blot. The results demonstrated that the exosomal N-acetyl-alpha-glucosaminidase concentration was not only lower in Parkinson's disease, but also decreased with increasing Hoehn-Yahr stage, suggesting that N-acetyl- alpha-glucosaminidase could be used to rapidly evaluate Parkinson's disease severity. Furthermore, western blot and immunohistochemistry analysis showed that N-acetyl-alpha-glucosaminidase levels were markedly reduced both in cells treated with methyl-4-phenylpyridinium (MPP+) and cells overexpressing a-synuclein (α-syn) compared with control cells. Additionally, N-acetyl-alpha-glucosaminidase overexpression significantly increased cell viability and inhibited α-syn expression in MPP+-treated cells. Taken together, our findings demonstrate for the first time that exosomal N-acetyl-alpha-glucosaminidase may serve as a biomarker for Parkinson's disease diagnosis, and that N-acetyl-alpha- glucosaminidase may reduce α-syn expression and MPP+-induced neurotoxicity, thus providing a new therapeutic target for Parkinson's disease.

Schisandra chinensis Bee Pollen Extract Inhibits Proliferation and Migration of Hepatocellular Carcinoma HepG2 Cells via Ferroptosis-, Wnt-, and Focal Adhesion-Signaling Pathways.

Bee pollen possesses favorable anticancer activities. As a medicinal plant source, Schisandra chinensis bee pollen (SCBP) possesses potential pharmacological properties, such as reducing cisplatin-induced liver injury, but its anti-liver cancer effect is still rarely reported. This paper aims to investigate the effect and mechanism of SCBP extract (SCBPE) on hepatocellular carcinoma HepG2 cells. The effect of SCBPE on cell proliferation and migration of HepG2 cells was evaluated based on MTT assay, morphology observation, or scratching assay. Furthermore, tandem mass tag-based quantitative proteomics was used to study the effect mechanisms. The mRNA expression levels of identified proteins were verified by RT-qPCR. Tandem mass tag-based quantitative proteomics showed that 61 differentially expressed proteins were obtained in the SCBPE group compared with the negative-control group: 18 significantly downregulated and 43 significantly upregulated proteins. Bioinformatic analysis showed the significantly enriched KEGG pathways were predominantly ferroptosis-, Wnt-, and hepatocellular carcinoma-signaling ones. Protein-protein interaction network analysis and RT-qPCR validation revealed SCBPE also downregulated the focal adhesion-signaling pathway, which is abrogated by PF-562271, a well-known inhibitor of FAK. This study confirmed SCBPE suppressed the cell proliferation and migration of hepatocellular carcinoma HepG2 cells, mainly through modulation of ferroptosis-, Wnt-, hepatocellular carcinoma-, and focal adhesion-signaling pathways, providing scientific data supporting adjuvant treatment of hepatocellular carcinoma using SCBP.

Comparative transcriptomics and proteomics analysis of the symbiotic germination of Paphiopedilum barbigerum with Epulorhiza sp. FQXY019.

Paphiopedilum barbigerum is currently the rarest and most endangered species of orchids in China and has significant ornamental value. The mature seeds of P. barbigerum are difficult to germinate owing to the absence of an endosperm and are highly dependent on mycorrhizal fungi for germination and subsequent development. However, little is known about the regulation mechanisms of symbiosis and symbiotic germination of P. barbigerum seeds. Herein, transcriptomics and proteomics were used to explore the changes in the P. barbigerum seeds after inoculation with (FQXY019 treatment group) or without (control group) Epulorhiza sp. FQXY019 at 90 days after germination. Transcriptome sequencing revealed that a total of 10,961 differentially expressed genes (DEGs; 2,599 upregulated and 8,402 downregulated) were identified in the control and FQXY019 treatment groups. These DEGs were mainly involved in carbohydrate, fatty acid, and amino acid metabolism. Furthermore, the expression levels of candidate DEGs related to nodulin, Ca2+ signaling, and plant lectins were significantly affected in P. barbigerum in the FQXY019 treatment groups. Subsequently, tandem mass tag-based quantitative proteomics was performed to recognize the differentially expressed proteins (DEPs), and a total of 537 DEPs (220 upregulated and 317 downregulated) were identified that were enriched in processes including photosynthesis, photosynthesis-antenna proteins, and fatty acid biosynthesis and metabolism. This study provides novel insight on the mechanisms underlying the in vitro seed germination and protocorm development of P. barbigerum by using a compatible fungal symbiont and will benefit the reintroduction and mycorrhizal symbiotic germination of endangered orchids.

Vitamin D receptor induces oxidative stress to promote esophageal squamous cell carcinoma proliferation via the p53 signaling pathway

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). ObjectiveWe investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. MethodsESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and Western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. ResultsHigh VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. ConclusionsVDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.

Tandem mass tag-based quantitative proteomics analyses of the spermatogenesis-ameliorating effect of Youjing granule on rats.

Male infertility is a common reproductive system disease manifested as aberrant spermatogenesis and identified as "kidney deficiency and dampness" in Chinese traditional medicine. Youjing granule (YG) is a Chinese material medica based on tonifying kidneys and removing dampness. It has proven to be able to regulate semen quality in clinical application, but the underlying mechanism has not been clarified. Using serum containing YG to treat primarily cultured spermatogonial stem cells (SSCs), the apoptotic rate and mitosis phase ratio of SSCs were measured. The liquid chromatography-tandem mass spectrometry with tandem mass tags method was applied for analyzing the serum of rats treated with YG/distilled water, and proteomic analyses were performed to clarify the mechanisms of YG. Totally, 111 proteins in YG-treated serum samples were differentially expressed compared with control groups, and 43 of them were identified as potential target proteins, which were further annotated based on their enrichment in Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. Proteomic analyses showed that the mechanisms of YG may involve regulation of glycolysis, gluconeogenesis and nucleotide-binding and oligomerization domain-like receptor signaling pathway. In addition, RhoA and Lamp2 were found to be possible responders of YG through reviewing the literature. The results demonstrate that our serum proteomics platform is clinically useful in understanding the mechanisms of YG.

Tandem mass tag-based quantitative proteomics elucidates the inactivation mechanisms of high-power pulsed microwave treatment on Pseudomonas aeruginosa PAO1

This study explored the effect of HPPM treatment (200 Hz for 3, 5, 7, 9, and 11 min) on P. aeruginosa PAO1 inactivation using tandem mass tag-based quantitative proteomics. HPPM demonstrated marked nucleic acid and protein constituents (p < 0.05) and inhibited ATPase activity and intercellular ATP concentration time-dependently. Hence, 9 min was selected for proteomic profiling to avoid any potential thermal effect. A total of 452 proteins (309 = down-regulated; 143 = up-regulated) were identified as differentially accumulated proteins (DAPs). The DAPs were primarily related to energy production and conversion; DNA replication, transcription, and translation, cell membrane composition; amino acid (AA) transport and metabolism, and biofilm formation and quorum sensing (QS). HPPM significantly disrupted cell membrane composition, altered AA metabolism, impeded cell energy generation, and drastically inhibited biofilm formation in P. aeruginosa. This study provided insights into the underlying mechanisms of HPPM against P. aeruginosa at the proteomic level. Industrial relevancePseudomonas aeruginosa is a key spoiler implicated in food contamination. High-power pulsed microwave (HPPM) is a potential non-thermal inactivation technology. However, the underlying mechanism to validate its efficacy remains unclear. This study provides the industry with the inactivation efficacy of HPPM against P. aeruginosa, highlighting insights into the molecular and biochemical changes after treatment and its remarkable potential to control cross-contamination in food processing, food services, and clinical environments.

Tandem mass tag-based quantitative proteomics analysis of plasma and plasma exosomes in Parkinson&amp;rsquo;s disease

Tandem mass tag-based quantitative proteomics analysis of plasma and plasma exosomes in Parkinson&amp;rsquo;s disease

Electroacupuncture ameliorates cardiac dysfunction in myocardial ischemia model rats: a potential role of the hypothalamic-pituitary-adrenal axis.

To verify the hypothesis that electroacupuncture inhibits the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis regulating the expression of glial fibrillary acidic protein (GFAP) in the hippocampus of acute myocardial ischemia (AMI) rats. Sixty-six healthy male Sprague-Dawley rats were randomly divided into five groups: Sham, AMI (Model), electroacupuncture at Shenmen (HT7)-Tongli (HT5) segment (EA), non-acupoint electroacupuncture (Control), and Model + corticosterone (Model + CORT). AMI was induced occlusion of the left anterior descending coronary artery, followed by 3 d of electroacupuncture at Shenmen (HT7)-Tongli (HT5) segment. In the Control group, electroacupuncture was applied at points lying 5 and 10 mm from the base of the tail. The AMI + CORT group was injected with CORT (20 mg/kg) in saline. Hemorheology, electrocardiography (ECG), hematoxylin and eosin staining, and expression of glycogen phosphorylase BB (GPBB) and heart-type fatty acid-binding protein (H-FABP) were used to assess cardiac function. The effects of adrenocorticotropic hormone (ACTH) and CORT were evaluated by enzyme-linked immunosorbent assay. Protein expression in the Sham and Model groups were screened by tandem mass tag-based quantitative proteomics analysis. Protein expression was evaluated by Western blotting (vimentin and GFAP) and immunofluorescence staining (GFAP). Compared with the Sham group, the hemorheology indicators, heart rate, ECG-ST segment elevation, and GPBB and H-FABP levels were higher in Model rats. The EA group showed reductions in these indicators compared with the Model group. Similarly, in Model rats, the expression of ACTH and CORT were significantly increased compared with the Sham group. The EA group also showed reduced expression of ACTH and CORT. Importantly, proteomics analysis showed that vimentin was differentially expressed in Model rats. Compared with the Sham group, vimentin and GFAP expression in the hippocampus was increased in the Model group but decreased in the AMI + EA group. Additionally, intraperitoneal injection of CORT aggravated the expression of GPBB, H-FABP and GFAP. Our results suggested that electroacupuncture may protect against cardiac injury induced by AMI through regulation of HPA axis hyperactivity, and that hippocampal GFAP may play an important role in the regulation.

Molecular and biochemical changes in Locusta migratoria (Orthoptera: Acrididae) infected with Paranosema locustae.

Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen's life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.

188. VITAMIN D RECEPTOR INDUCES OXIDATIVE STRESS TO PROMOTE ESOPHAGEAL SQUAMOUS CELL CARCINOMA PROLIFERATION VIA THE P53 SIGNALING PATHWAY

Abstract Background Esophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). We investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. Methods ESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. Results High VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. Conclusions VDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.

Panax notoginseng saponin alleviates pulmonary fibrosis in rats by modulating the renin-angiotensin system

Ethnopharmacological relevancePulmonary fibrosis (PF) is a chronic, progressive, and often fatal interstitial lung disease. Traditional Chinese medicine formulations and their active ingredients have shown potential in the treatment of PF. Panax notoginseng saponin (PNS) is extracted from the widely used traditional Chinese medicinal herb Panax notoginseng (Burkill) F. H. Chen, exhibiting therapeutic effects in pulmonary diseases treatment. Aim of the studyThis study aimed to investigate the effects and elucidate possible potential mechanisms of PNS on bleomycin (BLM)-induced PF in rats. Materials and methodsPF was induced in rats by intratracheal administration of bleomycin (BLM, 5 mg/kg). After disease model induction, the rats were treated with PNS (50, 100, or 200 mg/kg per day) or pirfenidone (PFD, 50 mg/kg per day) for 28 days. Lung function, histopathological changes, collagen deposition, and E− and N-cadherin levels in lung tissue were evaluated. The mechanism of action of PNS was investigated using tandem mass tag-based quantitative proteomics analysis. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were performed to verify the proteomic results. ResultsPNS treatment improved lung function, ameliorated the BLM-induced increase in the lung coefficient, attenuated the degree of alveolar inflammation and fibrosis, and reduced the elevated collagen level in PF rats. PNS treatment also down-regulated the expression of N-cadherin while up-regulating the expression of E-cadherin. Proteomic and bioinformatic analyses revealed that the renin-angiotensin system (RAS) was closely related to the therapeutic effect of PNS. Immunohistochemistry, Western blot, and ELISA results indicated that PNS exerted its anti-fibrotic effect via regulation of the balance between the angiotensin-converting enzyme (ACE)-angiotensin (Ang)II-AngII receptor type 1 (AT1R) and ACE2-Ang(1–7)-MasR axes. ConclusionsPNS ameliorates BLM-induced PF in rats by modulating the RAS homeostasis, and is a new potential therapeutic agent for PF.

Tandem mass tag-based quantitative proteomics reveals osmotic adaptation mechanisms in Alkalicoccus halolimnae BZ-SZ-XJ29T , a halophilic bacterium with a broad salinity range for optimal growth.

The moderate halophilic bacterium Alkalicoccus halolimnae BZ-SZ-XJ29T exhibits optimum growth over a wide range of NaCl concentrations (8.3-12.3%, w/v; 1.42-2.1 mol L-1 ). However, its adaptive mechanisms to cope with high salt-induced osmotic stress remain unclear. Using TMT-based quantitative proteomics, the cellular proteome was assessed under low (4% NaCl, 0.68 mol L-1 NaCl, control (CK) group), moderate (8% NaCl, 1.37 mol L-1 NaCl), high (12% NaCl, 2.05 mol L-1 NaCl), and extremely high (16% NaCl, 2.74 mol L-1 NaCl) salinity conditions. Digital droplet PCR confirmed the transcription of candidate genes related to salinity. A. halolimnae utilized distinct adaptation strategies to cope with different salinity conditions. Mechanisms such as accumulating different amounts and types of compatible solutes (i.e., ectoine, glycine betaine, glutamate, and glutamine) and the uptake of glycine betaine and glutamate were employed to cope with osmotic stress. Ectoine synthesis and accumulation were critical to the salt adaptation of A. halolimnae. The expression of EctA, EctB, and EctC, as well as the intracellular accumulation of ectoine, significantly and consistently increased with increasing salinity. Glycine betaine and glutamate concentrations remained constant under the four NaCl concentrations. The total content of glutamine and glutamate maintained a dynamic balance and, when exposed to different salinities, may play a role in low salinity-induced osmoadaptation. Moreover, cellular metabolism was severely affected at high salt concentrations, but the synthesis of amino acids, carbohydrate metabolism, and membrane transport related to haloadptation was preserved to maintain cytoplasmic concentration at high salinity. These findings provide insights into the osmoadaptation mechanisms of moderate halophiles and can serve as a theoretical underpinning for industrial production and application of compatible solutes.

Tandem mass tag-based quantitative proteomics analyses of a chicken-original virulent and its attenuated Histomonas meleagridis strain in China.

Histomonas meleagridis can cause histomonosis in poultry. Due to the prohibition of effective drugs, the prevention and treatment of the disease requires new strategies. Questions about its pathogenic mechanisms and virulence factors remain puzzling. To address these issues, a tandem mass tag (TMT) comparative proteomic analysis of a virulent strain and its attenuated strain of Chinese chicken-origin was performed. A total of 3,494 proteins were identified in the experiment, of which 745 proteins were differentially expressed (fold change ≥1.2 or ≤0.83 and p < 0.05), with 192 up-regulated proteins and 553 down-regulated proteins in the virulent strain relative to the attenuated strain. Surface protein BspA like, digestive cysteine proteinase, actin, and GH family 25 lysozyme were noted among the proteins up regulated in virulent strains, and these several proteins may be directly related to the pathogenic capacity of the histomonad. Ferredoxin, 60S ribosomal protein L6, 40S ribosomal protein S3, and NADP-dependent malic enzyme which associated with biosynthesis and metabolism were also noted, which have the potential to be new drug targets. The up-regulation of alpha-amylase, ras-like protein 1, ras-like protein 2, and involucrin in attenuated strains helps to understand how it is adapted to the long-term in vitro culture environment. The above results provide some candidate protein-coding genes for further functional verification, which will help to understand the molecular mechanism of pathogenicity and attenuation of H. meleagridis more comprehensively.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid β-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

Comparative Tandem Mass Tag-Based Quantitative Proteomics Analysis of Liver Against Chronic Hypoxia: Molecular Insights Into Metabolism in Rats.

Xu, Jin, Shenhan Gao, Mingyuan Xin, Wenjie Chen, Kaikun Wang, Wenjing Liu, Xinzong Yan, Sinan Peng, and Yanming Ren. Comparative tandem mass tag-based quantitative proteomics analysis of liver against chronic hypoxia: molecular insights into metabolism in rats. High Alt Med Biol. 24:49-58, 2023. Objective: Using a metabolomic approach, we uncovered key regulators in metabolism from tandem mass tag (TMT)-based proteomic analysis in animals chronically exposed to hypoxia. Methods: Sixteen Sprague-Dawley rats (n = 8 per group) were exposed to chronic normoxia or hypoxia (380 mmHg corresponding to a simulated altitude of 5,500 m) for 35 consecutive days. Hypoxia-induced alterations in metabolic pathways were analyzed from TMT-based proteomic analysis, complemented by western blot validation of key regulators. Results: We profiled biochemical parameters and serum lipids, found that serum alanine aminotransferase and blood glucose were not significantly changed due to chronic hypoxia. However, serum triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein (LDL) were significantly affected by chronic hypoxia. And the levels of LDL nearly doubled (p < 0.05) after hypoxia exposure for 35 days. Through Kyoto Encyclopedia of Genes and Genomes classification, we found several metabolic pathways were enriched, including lipid metabolism, cofactor and vitamin metabolism, amino acids metabolism, carbohydrate metabolism, and energy metabolism. To explore the potential functions of proteins in metabolic pathways that become a coordinated shift under chronic hypoxic conditions, Gene Ontology and pathway analysis were carried out on differentially expressed proteins. As the co-expression network shown in Figure, we identified the most significant differentially expressed proteins after chronic hypoxic changes in the livers of rats. Furthermore, we validated the gene expression profiles at the protein level using western blot. Results of western blot were in accordance with our quantitative polymerase chain reaction findings. The levels of fatty acid synthase and aquaporin 1 were significantly downregulated after 35 days and the levels of ATP citrate lyase, 2'-5'-oligoadenylate synthetase 1A, aldehyde dehydrogenase 2, and Ras-related protein Rap-1A were significantly upregulated after 35 days. Conclusions: Although this study cannot completely account for all the molecular mechanisms in rats, we provide a good analysis of protein expression and profiling of rats under chronic hypoxia conditions.

Tandem mass tag-based quantitative proteomics analysis reveals the new regulatory mechanism of progranulin in influenza virus infection.

Progranulin (PGRN) plays an important role in influenza virus infection. To gain insight into the potential molecular mechanisms by which PGRN regulates influenza viral replication, proteomic analyzes of whole mouse lung tissue from wild-type (WT) versus (vs) PGRN knockout (KO) mice were performed to identify proteins regulated by the absence vs. presence of PGRN. Our results revealed that PGRN regulated the differential expression of ALOX15, CD14, CD5L, and FCER1g, etc., and also affected the lysosomal activity in influenza virus infection. Collectively these findings provide a panoramic view of proteomic changes resulting from loss of PGRN and thereby shedding light on the functions of PGRN in influenza virus infection.

TMT-based quantitative proteomics analysis of the effects of Jiawei Danshen decoction myocardial ischemia-reperfusion injury

BackgroundEvery year, approximately 17 million people worldwide die due to coronary heart disease, with China ranking second in terms of the death toll. Myocardial ischemia-reperfusion injury (MIRI) significantly influences cardiac function and prognosis in cardiac surgery patients. Jiawei Danshen Decoction (JWDSD) is a traditional Chinese herbal prescription that has been used clinically for many years in China to treat MIRI. The underlying molecular mechanisms, however, remain unknown. To investigate the proteomic changes in myocardial tissue of rats given JWDSD for MIRI therapy-based proteomics.MethodsMIRI rat model was created by ligating/releasing the left anterior descending coronary artery. For seven days, the drugs were administered twice daily. The model was created following the last drug administration. JWDSD's efficacy in improving MIRI was evaluated using biochemical markers and cardiac histology. Tandem mass tag-based quantitative proteomics (TMT) technology was also used to detect proteins in the extracted heart tissue. To analyze differentially expressed proteins (DEPs), bioinformatics analysis, including gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways, were employed. Furthermore, western blotting confirmed the potential targets regulated by JWDSD.ResultsThe histopathologic characteristics and biochemical data showed JWDSD's protective effects on MIRI rats. A total of 4549 proteins were identified with FDR (false discovery rate) ≤1%. Twenty overlapping were identified (162 DEPs and 45 DEPs in Model/Control or JWDSD/Model group, respectively). Of these DEPs, 16 were regulated by JWDSD. GO analysis provided a summary of the deregulated protein expression in the categories of biological process (BP), cell component (CC), and molecular function (MF). KEGG enrichment analysis revealed that the signaling pathways of neutrophil extracellular trap formation, RNA polymerase, serotonergic synapse, and linoleic acid metabolism are all closely related to JWDSD effects in MIRI rats. Furthermore, T-cell lymphoma invasion and metastasis 1 (TIAM1) was validated using western blotting, and the results were consistent with proteomics data.ConclusionsOur study suggests that JWDSD may exert therapeutic effects through multi-pathways regulation in MIRI treatment. This work may provide proteomics clues for continuing research on JWDSD in treating MIRI.

Functional proteomics identify mannitol metabolism in serum resistance and therapeutic implications in Vibrio alginolyticus

Serum resistance is recognized as one of the most important pathogenic traits of bacterial pathogens, and no control measure is available. Based on our previous discovery that pathogenic Escherichia coli represses glycine, serine, and threonine metabolism to confer serum resistance and that the reactivation of this pathway by exogenous glycine could restore serum sensitivity, we further investigate the mechanism underlying the action of glycine in Vibrio alginolyticus. Thus, V. alginolyticus is treated with glycine, and the proteomic change is profiled with tandem mass tag-based quantitative proteomics. Compared to the control group, glycine treatment influences the expression of a total of 291 proteins. Among them, a trap-type mannitol/chloroaromatic compound transport system with periplasmic component, encoded by N646_0992, is the most significantly increased protein. In combination with the pathway enrichment analysis showing the altered fructose and mannitol metabolism, mannitol has emerged as a possible metabolite in enhancing the serum killing activity. To demonstrate this, exogenous mannitol reduces bacterial viability. This synergistic effect is further confirmed in a V. alginolyticus-Danio rerio infection model. Furthermore, the mechanism underlying mannitol-enabled serum killing is dependent on glycolysis and the pyruvate cycle that increases the deposition of complement components C3b and C5b-9 on the bacterial surface, whereas inhibiting glycolysis or the pyruvate cycle significantly weakened the synergistic effects and complement deposition. These data together suggest that mannitol is a potent metabolite in reversing the serum resistance of V. alginolyticus and has promising use in aquaculture.

Phosphorylation-mediated PI3K-Art signalling pathway as a therapeutic mechanism in the hydrogen-induced alleviation of brain injury in septic mice.

Our previous studies illustrated that 2% H2 inhalation can protect against sepsis-associated encephalopathy (SAE) which is characterized by high mortality and has no effective treatment. To investigate the underlying role of protein phosphorylation in SAE and H2 treatment, a mouse model of sepsis was constructed by caecal ligation and puncture (CLP), then treated with H2 (CLP + H2 ). Brain tissues of the mice were collected to be analysed with tandem mass tag-based quantitative proteomics coupled with IMAC enrichment of phosphopeptides and LC-MS/MS analysis. In proteomics and phosphoproteomics analysis, 268 differentially phosphorylated proteins (DPPs) showed a change in the phosphorylated form in the CLP + H2 group (p < 0.05). Gene ontology analysis revealed that these DPPs were enriched in multiple cellular components, biological processes, and molecular functions. KEGG pathway analysis revealed that they were enriched in glutamatergic synapses, tight junctions, the PI3K-Akt signalling pathway, the HIF-1 signalling pathway, the cGMP-PKG signalling pathway, the Rap1 signalling pathway, and the vascular smooth muscle contraction. The phosphorylated forms of six DPPs, including ribosomal protein S6 (Rps6), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (Ywhag/14-3-3), phosphatase and tensin homologue deleted on chromosome ten (Pten), membrane-associated guanylate kinase 1 (Magi1), mTOR, and protein kinase N2 (Pkn2), were upregulated and participated in the PI3K-Akt signalling pathway. The WB results showed that the phosphorylation levels of Rps6, Ywhag, Pten, Magi1, mTOR, and Pkn2 were increased. The DPPs and phosphorylation-mediated molecular network alterations in H2 -treated CLP mice may elucidate the biological roles of protein phosphorylation in the therapeutic mechanism of H2 treatment against SAE.

6-Methylcoumarin rescues bacterial quorum sensing induced ribosome-inactivating stress in Caenorhabditis elegans

IntroductionBacterial pathogenicity has for long posed severe effects on patient care. Pseudomonas aeruginosa is a common cause of hospital-acquired infections and nosocomial illnesses. It is known to infect the host by colonizing through quorum sensing and the production of exotoxins. MethodsThe current effort is an analysis of proteomic alterations caused by P. aeruginosa PAO1 to study the effects of quorum sensing inhibitor 6-Methylcoumarin on PAO1 infectivity in the Caenorhabditis elegans model. ResultsThrough tandem mass tag-based quantitative proteomics approaches, 229 proteins were found to be differentially regulated in infection and upon inhibition. Among these, 34 proteins were found to be dysregulated in both infection and quorum-sensing inhibition conditions. Along with the dysregulation of proteins involved in host-pathogen interaction, PAO1 was found to induce ribosome-inactivating stress accompanied by the downregulating mitochondrial proteins. This in turn caused dysregulation of apoptosis. The expression of multiple proteins involved in ribosome biogenesis and structure, oxidative phosphorylation, and mitochondrial enzymes were altered due to infection. This mechanism, adapted by PAO1 to survive in the host, was inhibited by 6-Methylcoumarin by rescuing the downregulation of ribosomal and mitochondrial proteins. ConclusionsTaken together, the data reflect the molecular alterations due to quorum sensing and the usefulness of inhibitors in controlling pathogenesis.