Absolute Quantitation Proteomic Analysis Research Articles

Circulating Exosomes from Septic Mice Activate NF-κB/MIR17HG Pathway in Macrophages.

Circulating exosomes derived from polymicrobial sepsis contain various non-coding RNAs and proteins. Isobaric tags for a relative or absolute quantitation proteomic analysis of the exosomal content revealed 70 dysregulated proteins in the circulating exosomes from septic mice. Next-generation sequencing was used to profile the long non-coding RNA expression in primary cultured macrophages treated with exosomes obtained from the blood of septic C57BL/6 mice, and it was discovered that the nuclear factor-kappa B (NF-κB)/miR-17-92a-1 cluster host gene (MIR17HG) pathways were activated in the macrophages. The inhibition of MIR17HG expression by RNA interference resulted in significantly decreased cell viability. RNA pull-down assays of MIR17HG revealed that ten protein targets bind to MIR17HG. Interaction networks of proteins pulled down by MIR17HG were constructed using GeneMANIA, and their functions were mainly involved in ribonucleoprotein granules, type I interferons, the regulation of organelle assembly, the biosynthesis of acetyl coenzyme A, as a signal transducer and activator of transcription (STAT) protein phosphorylation, and mRNA splicing. Furthermore, RNA interference inhibited MIR17HG expression, resulting in significantly decreased cell survival. In conclusion, this work discovered considerable MIR17HG overexpression in macrophages treated with circulating exosomes from sepsis-affected animals. This study's findings assist us in comprehending the role of exosomes in modulating inflammatory responses and mediating pathogenic pathways in macrophages during sepsis.

Proteomic analysis of near-isogenic lines reveals key biomarkers on wheat chromosome 4B conferring drought tolerance.

Drought is a major constraint for wheat production that is receiving increased attention due to global climate change. This study conducted isobaric tags for relative and absolute quantitation proteomic analysis on near-isogenic lines to shed light on the underlying mechanism of qDSI.4B.1 quantitative trait loci (QTL) on the short arm of chromosome 4B conferring drought tolerance in wheat. Comparing tolerant with susceptible isolines, 41 differentially expressed proteins were identified to be responsible for drought tolerance with a p-value of < 0.05 and fold change >1.3 or <0.7. These proteins were mainly enriched in hydrogen peroxide metabolic activity, reactive oxygen species metabolic activity, photosynthetic activity, intracellular protein transport, cellular macromolecule localization, and response to oxidative stress. Prediction of protein interactions and pathways analysis revealed the interaction between transcription, translation, protein export, photosynthesis, and carbohydrate metabolism as the most important pathways responsible for drought tolerance. The five proteins, including 30S ribosomal protein S15, SRP54 domain-containing protein, auxin-repressed protein, serine hydroxymethyltransferase, and an uncharacterized protein with encoding genes on 4BS, were suggested as candidate proteins responsible for drought tolerance in qDSI.4B.1 QTL. The gene coding SRP54 protein was also one of the differentially expressed genes in our previous transcriptomic study.

The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease

BackgroundNeurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear. PurposeTo investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model. MethodsEsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins. ResultsEsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3β activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3β, and blocked autophagy. ConclusionsTo our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

Comparative Proteomics Analysis of Serum Proteins in Gestational Diabetes during Early and Middle Stages of Pregnancy.

This study is designed to screen serum proteins that may serve as biomarkers for gestational diabetes mellitus (GDM), and clarify the mechanisms of disease. By using isobaric tags for relative and absolute quantitation proteomics analysis, serum proteins levels were quantified in pregnant women who subsequently developed GDM (12-16 weeks), GDM patients (24-28 weeks), and their corresponding controls. The strategy of mixing samples is used in proteomic analysis. Thirty-one and 27 differentially expressed proteins are identified in the serum of pregnant women with developed GDM at 12-16 weeks and GDM patients during 24-28 weeks, respectively. Thirty eight and 28 proteins are identified in 24-28 weeks versus 12-16 weeks controls (24/12 CTR group), and 24-28 weeks GDM patients versus 12-16 weeks women with subsequently developed GDM (24/12 GDM group), respectively. Most of these proteins in the case and control subjects are associated with diabetes and maternal and perinatal short- and long-term complications. The results highlight the roles of complement system and the blood clotting cascade in the pathogenesis of GDM. Differentially expressed proteins may serve as potential biomarkers for GDM prediction and diagnosis in the future.

Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy

Argininosuccinate synthetase 1 (ASS1) is a rate-limited enzyme in arginine biosynthesis. The oncogenic potential of ASS1 in terms of prognosis and cancer metastasis in arginine prototrophic gastric cancer (GC) remains unclear at present. We identify differentially expressed proteins in microdissected GC tumor cells relative to adjacent nontumor epithelia by isobaric mass tag for relative and absolute quantitation proteomics analysis. GC cells with stable expression or depletion of ASS1 were further analyzed to identify downstream molecules. We investigated their effects on chemoresistance and cell invasion in the presence or absence of arginine. ASS1 was highly expressed in GC and positively correlated with GC aggressiveness and poor outcome. Depletion of ASS1 led to inhibition of tumor growth and decreased cell invasion via induction of autophagy-lysosome machinery, resulting in degradation of active β-catenin, Snail, and Twist. Ectopic expression of ASS1 in GC cells reversed these effects and protected cancer cells from chemotherapy drug-induced apoptosis via activation of the AKT-mammalian target of rapamycin signaling pathway. ASS1 contributes to GC progression by enhancing aggressive potential resulting from active β-catenin, Snail, and Twist accumulation. Our results propose that ASS1 might contribute to GC metastasis and support its utility as a prognostic predictor of GC.-Tsai, C.-Y., Chi, H.-C., Chi, L.-M., Yang, H.-Y., Tsai, M.-M., Lee, K.-F., Huang, H.-W., Chou, L.-F., Cheng, A.-J., Yang, C.-W., Wang, C.-S., Lin, K.-H. Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Corilagin sensitizes epithelial ovarian cancer to chemotherapy by inhibiting Snail‑glycolysis pathways.

We identified that corilagin is a major component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthusniruri L with antitumor activity. Our previous study found that corilagin inhibited the growth of ovarian cancer cells via the TGF-β/AKT/ERK signaling pathways. Recently, we demonstrated that corilagin enhanced the sensitivity of ovarian cancer cells to chemotherapy. Ovarian cancer cell lines, SKOv3ip, Hey and HO-8910PM-Snail, were treated with different concentrations of corilagin in combination with paclitaxel and carboplatin. Corilagin distinctly enhanced the inhibitory effects of paclitaxel and carboplatin. To understand the mechanisms involved in the chemo-sensitization by corilagin, we performed reverse phase protein array analysis to determine the signaling networks induced by corilagin. We observed that both paclitaxel and carboplatin upregulated the expression levels of several apoptotic and death-related proteins, such as caspase3,caspase7 and PDCD4, which were further enhanced when combined with corilagin. Meanwhile, corilagin induced distinct pathways to paclitaxel and carboplatin treatment. We also performed isobaric tags for relative and absolute quantitation proteomics analysis in corilagen-treated ovarian cancer cells. This analysis indicated that corilagin is mainly involved in the glycolysis pathway. Seahorse XF96 extracellular acidification rate analysis confirmed that corilagin inhibited glycolysis by downregulation of CD44 and STAT3. In summary, our observations indicate that corilagin sensitized epithelial ovarian cancer cells to paclitaxel and carboplatin treatment by primarily inhibiting Snail-glycolysis pathways. Corilagin is a herbal medicine with low toxic effects to normal cells, particularly hepatoprotective, and may be an ideal complimentary medicine when combined with highly toxic chemotherapeutic agents.

Isobaric Tags for Relative and Absolute Quantitation Proteomic Analysis of Germinating Barley under Gibberellin and Abscisic Acid Treatments.

The degradation of starch in barley grains is a primary step of beer production. The addition of an appropriate amount of gibberellin (GA) promotes the production of fermentable sugars, beneficial to the brewing industry. However, the response of proteomics in germinating barley to GA and abscisic acid (ABA) treatments is not thoroughly understood. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) proteomics analysis was performed to illustrate the change of proteins in Tibetan wild barley XZ72 and XZ95 under GA and ABA treatments during germination. XZ72 had more proteins upregulated than XZ95 under GA treatment, while under ABA treatments, XZ95 had more proteins upregulated than XZ72. Concerning the proteins involved in energy metabolism under GA treatment, XZ72 had more proteins upregulated than XZ95. Among the 174 proteins related to starch metabolism, 31 proteins related to starch hydrolysis, such as α-amylase, α-glucosidase, and β-fructofuranosidase, showed higher relative abundance in control and GA treatments in XZ72 than in XZ95. Analysis of correlation between proteins and metabolites indicated that higher hydrolase activity is beneficial for the accumulation of fermentable sugars during germination. On the other hand, 26 starch-synthesis-related proteins were upregulated in XZ95 under ABA treatment. It may be suggested that GA-induced proteins act as accelerators of starch degradation, while ABA-induced proteins inhibit starch degradation. The current results showed that XZ72 is highly capable of allocating the starch-hydrolyzing enzymes, which play important roles in starch breakdown.

Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro–produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.

Proteomic Analyses of PKA and AKAP Signaling in Cocaine Addiction

The development and application of proteomics techniques allows for ab initio identification of changes in protein expression and modifications which drive cellular processes. In the case of behavioral neuroscience, these techniques may be applied toward identification of candidate proteins and cellular pathways within specific nuclei that are affected by experience or training, and testing of subsequent hypotheses in behavioral models. Accordingly, proteomic techniques have been applied to identify protein changes following chronic cocaine abuse. These studies have examined changes in the nucleus accumbens (NAc) from human overdose victims, as well as non-human primates and rodents (for review, see Hemby (2010)). From these, a number of important themes have emerged; for example, changes in cellular metabolism, cytoskeletal dynamics, and signal transduction were highly represented. Of particular note, elements of the cAMP, adenylate cyclase, and PKA signaling pathway have been identified by traditional biochemical methods and genomic analyses, as well as proteomic methods (Hemby, 2010). For example, ChIP-chip microarray analysis of genes influenced by cocaine exposure has indicated increased histone acetylation at the promoters of the PKA catalytic and RII regulatory subunits, as well as A kinase anchoring protein 9 (AKAP9) (also known as Yotiao) and AKAP8 (AKAP95) (Renthal et al, 2009). Moreover, increased binding of transcription factor ΔFosB was identified at the promoters for PKA RIIα, as well as AKAP8 following cocaine vs saline exposure. AKAPs constitute a family of more than 50 proteins across vertebrates and invertebrates, which mediate scaffolding and localization of PKA and other signaling molecules within specific cellular subcompartments (Sanderson and Dell'Acqua, 2011). An isobaric tag for relative and absolute quantitation proteomic analysis of a rat postsynaptic density-enriched subfraction following cocaine self-administration and extinction identified upregulation of AKAP5 (also known as AKAP79/150) (Reissner et al, 2011). AKAP150 serves to complex PKA and other signaling molecules with ionotropic glutamate receptors (Sanderson and Dell'Acqua, 2011). Disruption of AKAP scaffolding by microinjection of a cell permeable inhibitor peptide in the NAc (vs control peptide) led to decreased reinstatement of cocaine seeking, indicating a functional role for AKAPs in the neuropathology of drug abuse (Reissner et al, 2011). Downstream of PKA, Boudreau et al (2009) observed increased AMPA surface expression, as well as PKA (and pERK2) activation over time during withdrawal in cocaine sensitized rats. They went on to use a mass spectrometry approach to identify PKA substrates whose phosphorylation increased over time of withdrawal, employing proteomics to analyze effector proteins downstream of PKA. The repeated identification of the PKA signaling pathway in the cellular adaptations induced by cocaine underscores the importance of this pathway in the addiction process. However, the ubiquitous nature of AKAP and PKA signaling in cellular pathways and subcompartments (eg, synapse vs nucleus, organelles, etc) complicates application of this pathway toward candidate pharmacotherapeutic targets for psychostimulant abuse. In the specific case of AKAP150, however, proteomic and functional studies indicate that synaptic upregulation promotes reinstatement behavior; thus, development of a structurally specific inhibitor targeting this member of the AKAP family may aid inhibition of craving and drug seeking, as a pharmacological adjuvant to cognitive therapy and counseling.