Effects of multiple stressors on pancreatic human islets proteome reveal new insights into the pathways involved.

Pancreatic ß-cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause ß-cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying ß-cell's inability to maintain glucose homeostasis under severe stresses is unknown. This study used proinflammatory cytokines, thapsigargin, and rotenone in the presence of high concentration glucose to mimicking the conditions experienced by dysfunctional ß-cells in human pancreatic islets, and profiled the alterations to the islet proteome with TMT-based proteomics. The results were further verified with label-free quantitative proteomics. The differentially expressed proteins under stress conditions reveal that immune related pathways are mostly perturbed by cytokines, while the respiratory electron transport chains and protein processing in ER pathways by rotenone. Thapsigargin together with high glucose induces dramatic increases of proteins in lipid synthesis and peroxisomal protein import pathways, with energy metabolism and vesicle secretion related pathways downregulated. High concentration glucose, on the other hand, alleviated complex I inhibition induced by rotenone. Our results contribute to a more comprehensive understanding of the molecular events involved in ß-cell dysfunction.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment.

BACKGROUND: In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics. RESULTS: The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 â„ƒ, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins. CONCLUSIONS: PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects.

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 µM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.

Integrating Physiological Features and Proteomic Analyses Provides New Insights in Blue/Red Light-Treated Moso Bamboo (Phyllostachys edulis).

Bamboo is one of the most important nontimber forestry products in the world. Light is not only the most critical source of energy for plant photosynthesis but also involved in regulating the biological processes of plants. However, there are few reports on how blue/red light affects Moso bamboo. This study investigated the growth status and physiological responses of Moso bamboo (Phyllostachys edulis) to blue/red light treatments. The growth status of the bamboo plants was evaluated, revealing that both blue- and red-light treatments promoted plant height and overall growth. Gas exchange parameters, chlorophyll fluorescence, and enzyme activity were measured to assess the photosystem response of Moso bamboo to light treatments. Additionally, the blue light treatment led to a higher chlorophyll content and enzyme activities compared to the red light treatment. A tandem mass tag quantitative proteomics approach identified significant changes in protein abundance under different light conditions with specific response proteins associated with distinct pathways, such as photosynthesis and starch metabolism. Overall, this study provides valuable insights into the physiological and proteomic responses of Moso bamboo to blue/red light treatments, highlighting their potential impact on growth and development.

[Mechanism of electroacupuncture for vascular dementia based on proteomics].

OBJECTIVE: To explore the potential mechanism of electroacupuncture (EA) for vascular dementia (VD) using tandem mass tag (TMT) quantitative proteomics technology. METHODS: Among 80 male SPF SD rats, 78 rats which met the selection criteria through the Morris water maze test were selected and randomly divided into a sham surgery group (18 rats) and a surgery group (60 rats). VD model was established by four-vessel occlusion (4-VO) method in the surgery group, and 36 rats with successful modeling were randomly assigned to a model group (18 rats) and an EA group (18 rats). Each group was further divided into three subgroups based on intervention duration, with each subgroup containing 6 rats. Seven days after model establishment, the EA group received EA intervention at left and right "Sishencong" (EX-HN 1) and bilateral "Fengchi" (GB 20), with continuous wave at a frequency of 2 Hz and current intensity of 1 mA, daily for 30 min, with subgroups receiving EA for 7, 14, or 21 d respectively. Cognitive function before and after interventions was assessed using Morris water maze. Proteomic analysis was conducted on the optimal EA subgroup and corresponding sham surgery and model subgroups, identifying differentially expressed proteins and analyzing them through bioinformatics. Differentially expressed target proteins was performed using parallel reaction monitoring (PRM) and Western blot techniques. RESULTS: Compared to the sham surgery group, the model group exhibited prolonged escape latency and reduced number of platform crossings (P<0.01); compared with model group, the EA group showed reductions in escape latency and increased platform crossings after 7, 14, and 21 days of intervention (P<0.01, P<0.05). Compared to the 7 and 14-day intervention, the rats in the EA group of 21-day intervention showed the most significant improvements in reductions of escape latency and increased platform crossings (P<0.01, P<0.05), and was selected for further proteomic, PRM analyses, and Western blot validation. Compared to the sham surgery group, the model group displayed 71 differentially expressed proteins, with 50 up-regulated and 21 down-regulated proteins; compared to the model group, the EA group had 54 differentially expressed proteins, with 30 up-regulated and 24 down-regulated proteins. Functional enrichment and clustering analyses indicated that these proteins were primarily associated with cellular processes, metabolic processes, phagocytosis recognition, immune response, and regulation of extracellular matrix, etc. Enrichment was observed in the mammalian target of rapamycin (mTOR) signaling pathway and neurotrophic factors signaling pathways, involving glycogen synthase kinase 3ß (GSK3ß) and mitogen-activated protein kinase kinase 2 (Map2k2), with PRM and Western blot findings consistent with the proteomic results. Which meant that compared with the model group, the protein expression of GSK3ß and Map2k2 of hippocampus was increased in the EA group (P<0.01, P<0.05). CONCLUSION: EA at "Sishencong" (EX-HN 1) and "Fengchi" (GB 20) could improve cognitive function in VD rats, with the mechanism involving multiple targets and pathways, potentially related to GSK3ß, Map2k2 proteins, and the mTOR and neurotrophic factor signaling pathways.

Serum proteomic analysis reveals possible mechanism underlying physiological hemostasis of swim bladder.

The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.

Integrative Proteomic and Phosphoproteomic Analyses Revealed Complex Mechanisms Underlying Reproductive Diapause in Bombus terrestris Queens.

Reproductive diapause is an overwintering strategy for Bombus terrestris, which is an important pollinator for agricultural production. However, the precise mechanisms underlying reproductive diapause in bumblebees remain largely unclear. Here, a combination analysis of proteomics and phosphoproteomics was used to reveal the mechanisms that occur during and after diapause in three different phases: diapause (D), postdiapause (PD), and founder postdiapause (FPD). In total, 4655 proteins and 10,600 phosphorylation sites of 3339 proteins were identified. Diapause termination and reactivation from D to the PD stage were characterized by the upregulation of proteins associated with ribosome assembly and biogenesis, transcription, and translation regulation in combination with the upregulation of phosphoproteins related to neural signal transmission, hormone biosynthesis and secretion, and energy-related metabolism. Moreover, the reproductive program was fully activated from PD to the FPD stage, as indicated by the upregulation of proteins related to fat digestion and absorption, the biosynthesis of unsaturated fatty acids, fatty acid elongation, protein processing in the endoplasmic reticulum, and the upregulation of energy-related metabolism at the phosphoproteome level. We also predicted a kinase-substrate interaction network and constructed protein-protein networks of proteomic and phosphoproteomic data. These results will help to elucidate the mechanisms underlying the regulation of diapause in B. terrestris for year-round mass breeding.

Liver proteomics analysis reveals abnormal metabolism of bile acid and arachidonic acid in Chinese hamsters with type 2 diabetes mellitus.

Non-obese, spontaneous, and genetically predisposed type 2 diabetic Chinese hamsters exhibit metabolic abnormalities similar to those observed in human T2DM. Here, tandem mass tag (TMT)-based quantitative proteomics technology was used to screen and identify differentially abundant proteins in the liver that are associated with diabetes in Chinese hamsters. GO and KEGG pathway enrichment analysis were conducted to validate the findings, as well as qRT-PCR and western blotting. In total, 103 proteins were identified in the livers of diabetic hamsters, of which 48 were up-regulated and 55 were down-regulated. KEGG pathway enrichment analysis further demonstrated that linoleic acid metabolism, arachidonic acid metabolism, bile secretion, and other pathways were affected. Moreover, AQP9 and EPHX1 were significantly down-regulated in the bile secretion pathway, whereas PTGES2, Cyp2c27, and Cyp2c70 were associated with the arachidonic acid metabolic pathway. Serum levels of bile acid (BA) and arachidonic acid (AA) in diabetic Chinese hamsters were significantly higher than those in control hamsters. Cumulatively, our findings indicate that the five candidate proteins may be associated with abnormal BA and AA metabolism, suggesting their involvement in pathological changes in the livers of Chinese hamsters with T2DM. SIGNIFICANCE: The liver proteomics of Chinese hamsters describes differentially abundant proteins associated with T2DM, while promoting this animal model as an appropriate and ideal platform for investigating underlying molecular mechanisms of T2DM. This study reveals abnormal bile acid and arachidonic acid metabolism in T2DM hamsters, which may provide insights for studying the relationship between candidate proteins and KEGG pathways to elucidate the underlying molecular mechanism associated with T2DM.

Proteomic analysis to identify differentially expressed proteins between subjects with metabolic healthy obesity and non-alcoholic fatty liver disease.

Obese subjects with non-alcoholic fatty liver disease (NAFLD) and considered metabolically healthy have not been well differentiated. In this study, obese subjects were divided into metabolic healthy obesity (MHO) and NAFLD groups. Liver tissues were sampled from these two types of subjects undergoing bariatric surgery, and proteins in the liver tissues that expressed differently between the two groups of subjects were identified by Tandem Mass Tags (TMT) assay. Compared with the MHO group, 132 proteins were found to be upregulated and 84 proteins were found to be downregulated (mainly localized in mitochondria) in NAFLD group. The KEGG pathway analysis showed that significantly upregulated metabolic pathways include PPAR signaling, ECM-receptor interaction and oxidative phosphorylation was significantly downregulated. The GO analysis revealed that upregulated proteins were involved in extracellular structure organization, extracellular matrix organization and downregulated proteins took part in the oxidation-reduction process and so on. FBLN5 and DHRS2 were further validated by Western blot, immunohistochemistry and ELISA. All results demonstrate that FBLN5 expression was significantly upregulated but DHRS2 was significantly downregulated. SIGNIFICANCE: The variation between MHO and NAFLD was studied by mass spectroscopy to evaluate the mechanism with which MHO subjects resist the harmful effects induced by obesity.

Association of Proteomics Changes with Al-Sensitive Root Zones in Switchgrass.

In this paper, we report on aluminum (Al)-induced root proteomic changes in switchgrass. After growth in a hydroponic culture system supplemented with 400 µM of Al, plants began to show signs of physiological stress such as a reduction in photosynthetic rate. At this time, the basal 2-cm long root tips were harvested and divided into two segments, each of 1-cm in length, for protein extraction. Al-induced changes in proteomes were identified using tandem mass tags mass spectrometry (TMT-MS)-based quantitative proteomics analysis. A total of 216 proteins (approximately 3.6% of total proteins) showed significant differences between non-Al treated control and treated groups with significant fold change (twice the standard deviation; FDR adjusted p-value < 0.05). The apical root tip tissues expressed more dramatic proteome changes (164 significantly changed proteins; 3.9% of total proteins quantified) compared to the elongation/maturation zones (52 significantly changed proteins, 1.1% of total proteins quantified). Significantly changed proteins from the apical 1-cm root apex tissues were clustered into 25 biological pathways; proteins involved in the cell cycle (rotamase FKBP 1 isoforms, and CDC48 protein) were all at a reduced abundance level compared to the non-treated control group. In the root elongation/maturation zone tissues, the identified proteins were placed into 18 pathways, among which proteins involved in secondary metabolism (lignin biosynthesis) were identified. Several STRING protein interaction networks were developed for these Al-induced significantly changed proteins. This study has identified a large number of Al-responsive proteins, including transcription factors, which will be used for exploring new Al tolerance genes and mechanisms. Data are available via ProteomeXchange with identifiers PXD008882 and PXD009125.