Isobaric Tags Research Articles

Comparative Proteomic Analyses of Susceptible and Resistant Maize Inbred Lines at the Stage of Enations Forming following Infection by Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) is the main pathogen causing maize rough dwarf disease (MRDD) in China. Typical enation symptoms along the abaxial leaf veins prevail in RBSDV-infected maize inbred line B73 (susceptible to RBSDV), but not in X178 (resistant to RBSDV). Observation of the microstructures of epidermal cells and cross section of enations from RBSDV-infected maize leaves found that the increase of epidermal cell and phloem cell numbers is associated with enation formation. To identify proteins associated with enation formation and candidate proteins against RBSDV infection, comparative proteomics between B73 and X178 plants were conducted using isobaric tags for relative and absolute quantitation (iTRAQ) with leaf samples at the enation forming stage. The proteomics data showed that 260 and 316 differentially expressed proteins (DEPs) were identified in B73 and X178, respectively. We found that the majority of DEPs are located in the chloroplast and cytoplasm. Moreover, RBSDV infection resulted in dramatic changes of DEPs enriched by the metabolic process, response to stress and the biosynthetic process. Strikingly, a cell number regulator 10 was significantly down-regulated in RBSDV-infected B73 plants. Altogether, these data will provide value information for future studies to analyze molecular events during both enation formation and resistance mechanism to RBSDV infection.

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.

Effects of electroacupuncture on rats with cognitive impairment: An iTRAQ-based proteomics analysis

ObjectiveThe study explores the effects of electroacupuncture (EA) at the governing vessel (GV) on proteomic changes in the hippocampus of rats with cognitive impairment. MethodsHealthy male rats were randomly divided into 3 groups: sham, model and EA. Cognitive impairment was induced by left middle cerebral artery occlusion in the model and EA groups. Rats in the EA group were treated with EA at Shenting (GV24) and Baihui (GV20) for 7 d. Neurological deficit was scored using the Longa scale, the learning and memory ability was detected using the Morris water maze (MWM) test, and the proteomic profiling in the hippocampus was analyzed using protein-labeling technology based on the isobaric tag for relative and absolute quantitation (iTRAQ). The Western blot (WB) analysis was used to detect the proteins and validate the results of iTRAQ. ResultsCompared with the model group, the neurological deficit score was significantly reduced, and the escape latency in the MWM test was significantly shortened, while the number of platform crossings increased in the EA group. A total of 2872 proteins were identified by iTRAQ. Differentially expressed proteins (DEPs) were identified between different groups: 92 proteins were upregulated and 103 were downregulated in the model group compared with the sham group, while 142 proteins were upregulated and 126 were downregulated in the EA group compared with the model group. Most of the DEPs were involved in oxidative phosphorylation, glycolipid metabolism and synaptic transmission. Furthermore, we also verified 4 DEPs using WB technology. Although the WB results were not exactly the same as the iTRAQ results, the expression trends of the DEPs were consistent. The upregulation of heat-shock protein β1 (Hspb1) was the highest in the EA group compared to the model group. ConclusionEA can effect proteomic changes in the hippocampus of rats with cognitive impairment. Hspb1 may be involved in the molecular mechanism by which acupuncture improves cognitive impairment.

Three types of Leishmania mexicana amastigotes: Proteome comparison by quantitative proteomic analysis.

Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

Abstract 13188: Co-Translational Profiling in the Cardiac Endothelium During the Development of Sepsis

Introduction: The production of functional proteins is a multistep process whereby key maturation and regulatory processes occur co-translationally by coupling mRNA translation and nascent polypeptide chain maturation on the ribosome. However, limited data exist regarding co-translational regulation in the cells of the intact cardiovascular system. To overcome this limitation, we adapted the translating ribosome affinity purification (TRAP) approach using the HA-tagged ribosomal protein L22 reporter mouse (RiboTag) crossed with an inducible endothelial-specific Cdh5creERT2 mouse ( RiboTag EC ). Hypothesis: We hypothesized this model would permit the simultaneous immunoprecipitation (IP) of mRNA and nascent proteins for RNAseq and proteomic analysis from the coronary endothelium. Methods: Mice were injected with E. coli lipopolysaccharide (LPS) (6mg/Kg, i.p. 12 hours) to induce sepsis and perturb endothelial cell function. Hearts were homogenized, a small amount (~10%) was used for RNA-Seq and proteomics input controls (IN), and the remainder was used to IP ribosomal bound polyA+ mRNA and nascent proteins. Eluted proteins were digested, labeled with isobaric tags for quantification, and analyzed by MS3 with a Thermo Scientific Fusion Lumos Orbitrap. Results: Principal components analysis of the RNAseq data showed clear separation of the IP (endothelial cells) and IN (whole heart) transcriptomes between control and LPS groups. Transcripts characteristic of endothelial cells (EC) were enriched, whereas markers characteristic of cardiomyocytes, fibroblasts, and smooth muscle cells were depleted. Overall, >7000 proteins were identified, and initial analysis showed similar depletion and enrichment of proteins in the control and LPS hearts. Alignment of transcript and protein datasets was used for bioinformatic analysis. A hierarchical view of one of the top networks in which concordant expression of both EC transcripts and proteins identified interferon-gamma (IFNG) as an upstream signal in the pathogenesis of sepsis. Conclusions: Our “functional translatome” approach is a new discovery platform for a deeper predictive analysis of functionally relevant signaling networks and disease pathways in the intact cardiovascular system.

ITRAQ-based proteomic analysis of Deinococcus radiodurans in response to 12C6+ heavy ion irradiation

BackgroundDeinococcus radiodurans (D. radiodurans) is best known for its extreme resistance to diverse environmental stress factors, including ionizing radiation (IR), ultraviolet (UV) irradiation, oxidative stress, and high temperatures. Robust DNA repair system and antioxidant system have been demonstrated to contribute to extreme resistance in D. radiodurans. However, practically all studies on the mechanism underlying D. radiodurans’s extraordinary resistance relied on the treated strain during the post-treatment recovery lag phase to identify the key elements involved. The direct gene or protein changes of D. radiodurans after stress have not yet been characterized.ResultsIn this study, we performed a proteomics profiling on D. radiodurans right after the heavy ion irradiation treatment, to discover the altered proteins that were quickly responsive to IR in D. radiodurans. Our study found that D. radiodurans shown exceptional resistance to 12C6+ heavy ion irradiation, in contrast to Escherichia coli (E.coli) strains. By using iTRAQ (Isobaric Tags for Relative and Absolute Quantitation)-based quantitative mass spectrometry analysis, the kinetics of proteome changes induced by various dosages of 12C6+ heavy ion irradiation were mapped. The results revealed that 452 proteins were differentially expressed under heavy ion irradiation, with the majority of proteins being upregulated, indicating the upregulation of functional categories of translation, TCA cycle (Tricarboxylic Acid cycle), and antioxidation regulation under heavy ion irradiation.ConclusionsThis study shows how D. radiodurans reacts to exposure to 12C6+ heavy ion irradiation in terms of its overall protein expression profile. Most importantly, comparing the proteome profiling of D. radiodurans directly after heavy ion irradiation with research on the post-irradiation recovery phase would potentially provide a better understanding of mechanisms underlying the extreme radioresistance in D. radiodurans.

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p<0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

Quantitative proteomics analysis of tomato root cell wall proteins in response to salt stress.

Cell wall proteins perform diverse cellular functions in response to abiotic and biotic stresses. To elucidate the possible mechanisms of salt-stress tolerance in tomato. The 30d seedlings of two tomato genotypes with contrasting salt tolerances were transplanted to salt stress (200 mM NaCl) for three days, and then, the cell wall proteins of seedling roots were analyzed by isobaric tags for relative and absolute quantification (iTRAQ). There were 82 and 81 cell wall proteins that changed significantly in the salt-tolerant tomato IL8-3 and the salt-sensitive tomato M82, respectively. The proteins associated with signal transduction and alterations to cell wall polysaccharides were increased in both IL8-3 and M82 cells wall in response to salt stress. In addition, many different or even opposite metabolic changes occurred between IL8-3 and M82 in response to salt stress. The salt-tolerant tomato IL8-3 experienced not only significantly decreased in Na+ accumulation but also an obviously enhanced in regulating redox balance and cell wall lignification in response to salt stress. Taken together, these results provide novel insight for further understanding the molecular mechanism of salt tolerance in tomato.

Mass spectrometry-based proteomics analysis of human globus pallidus from progressive supranuclear palsy patients discovers multiple disease pathways.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder clinically characterized by progressive postural instability, supranuclear gaze palsy, parkinsonism, and cognitive decline caused by degeneration in specific areas of the brain including globus pallidus (GP), substantia nigra, and subthalamic nucleus. However, the pathogenetic mechanism of PSP remains unclear to date.Unbiased global proteome analysis of patients' brain samples is an important step toward understanding PSP pathogenesis, as proteins serve as workhorses and building blocks of the cell. In this study, we conducted unbiased mass spectrometry-based global proteome analysis of GP samples from 15 PSP patients, 15 Parkinson disease (PD) patients, and 15 healthy control (HC) individuals. To analyze 45 samples, we conducted 5 batches of 11-plex isobaric tandem mass tag (TMT)-based multiplexing experiments. The identified proteins were subjected to statistical analysis, such as a permutation-based statistical analysis in the significance analysis of microarray (SAM) method and bootstrap receiver operating characteristic curve (ROC)-based statistical analysis. Subsequently, we conducted bioinformatics analyses using gene set enrichment analysis, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) protein-protein interaction (PPI) analysis, and weighted gene co-expression network analysis (WGCNA). We have identified 10,231 proteins with ∼1,000 differentially expressed proteins. The gene set enrichment analysis results showed that the PD pathway was the most highly enriched, followed by pathways for oxidative phosphorylation, Alzheimer disease, Huntington disease, and non-alcoholic fatty liver disease (NAFLD) when PSP was compared to HC or PD. Most of the proteins enriched in the gene set enrichment analysis were mitochondrial proteins such as cytochrome c oxidase, NADH dehydrogenase, acyl carrier protein, succinate dehydrogenase, ADP/ATP translocase, cytochrome b-c1 complex, and/or ATP synthase. Strikingly, all of the enriched mitochondrial proteins in the PD pathway were downregulated in PSP compared to both HC and PD. The subsequent STRING PPI analysis and the WGCNA further supported that the mitochondrial proteins were the most highly enriched in PSP. Our study showed that the mitochondrial respiratory electron transport chain complex was the key proteins that were dysregulated in GP of PSP, suggesting that the mitochondrial respiratory electron transport chain complex could potentially be involved in the pathogenesis of PSP. This is the first global proteome analysis of human GP from PSP patients, and this study paves the way to understanding the mechanistic pathogenesis of PSP.

Comparative proteomics analysis of Schistosoma japonicum developed in different Oncomelania snails as intermediate hosts.

Schistosomiasis is a tropical parasitic disease that seriously endangers humans and animals. In this study, two Oncomelania snails, Oncomelania hupensis (O. hupensis) and Oncomelania weishan (O. weishan), were infected with Schistosoma japonicum (S. japonicum) cercariae during the early period, and ICR mice were subsequently infected with two kinds of miracidia that developed in male and female adult worms. In this study, isobaric tags for relative and absolute quantification (iTRAQ) were used to identify four channels: 113, 115, 117, and 119.A total of 2364 adult schistosome proteins were identified, and 1901 proteins were quantitative. Our results revealed 68 differentially expressed proteins (DEPs) in female adult worms, including 24 upregulated proteins and 44 downregulated proteins, and 55 DEPs in male adult worms, including 25 upregulated proteins and 30 downregulated proteins. LC-MS/MS and bioinformatics analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function and catabolism pathways. In summary, this proteomics analysis of adult schistosomes that hatched in two intermediate hosts helps to improve our understanding of the growth and developmental mechanisms of S. japonicum.

A simple and direct approach for cysteine-based redox proteomics by differential isobaric labeling validated in human and rat aortic smooth muscle cells

A simple and direct approach for cysteine-based redox proteomics by differential isobaric labeling validated in human and rat aortic smooth muscle cells

Assessing the effects of Leishmania (Leishmania) infantum and L. (L.) amazonensis infections in macrophages using a quantitative proteome approach

Leishmania (Leishmania) infantum is the causative agent of visceral leishmaniasis, while L. (L.) amazonensis is associated with localized cutaneous and diffuse leishmaniasis, which can affect different organ tissues leading to visceral manifestations in some hosts. The wide range of clinical manifestations of leishmaniasis depends on host factors such as the immune response and on the species of Leishmania involved in the infection. Macrophages are the main infected cells in the vertebrate host, and proteins play a pivotal role in Leishmania-macrophage interactions. Here, we performed difference gel electrophoresis (DIGE) and shotgun quantitative mass spectrometry-based proteomics by means of tandem mass tags (TMT) isobaric peptide labeling followed by LC-MS/MS to investigate differentially abundant proteins in BALB/c macrophages infected with these Leishmania species. Using DIGE for comparison, we found that 2.34% spots (29/1240) were differentially intense in infected murine macrophages. Leishmania (L.) infantum and L. (L.) amazonensis induced similar changes in the host cells; 11 spots were selected as differentially intense in each species and seven in the uninfected control group. Using TMT, 5939 Mus musculus proteins were identified, of which 410 and 433 were differentially abundant in L. (L.) infantum and L. (L.) amazonensis infections, respectively, while 170 proteins were commonly regulated by both the species. Gene ontology enrichment analysis indicated that Leishmania infection interfered with apoptotic mechanisms in macrophages and induced epigenetic changes that may affect gene transcription. Moreover, downregulation of proteins such as PYCARD and MyD88 seemed to influence the inflammatory process in L. (L.) amazonensis infection, whereas upregulation of TAP1 and ERAP1 was involved in the adaptive immune response in L. (L.) infantum infection. Differentially abundant proteins identified in this study may contribute to a better understanding of the factors that determine the course of infection. Our results suggest several possible targets for vaccines, drugs, and diagnosis of leishmaniasis.

Identification of Cofilin-1 as a novel biomarker of atopic dermatitis using iTRAQ quantitative proteomics.

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin condition; however, little is known about the pathogenesis and serum biomarker of this disease. Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic assay was adopted to identify and quantify the differentially expressed proteins (DEPs) in the serum of AD patients. Bioinformatic analysis, including GO, Reactome, GSEA, PPI, and ssGSEA analysis, were used to identified the enriched pathways, hub proteins and immune cells. The expression level and distribution of hub proteins were confirmed by ELISA and IHC. Sixty-six DEPs were identified with iTRAQ proteomic assay by analyzing serum from AD patients and normal subjects. GO and Reactome analysis shown the alternated pathway were mainly involved in immunity, oxidative stress, and actin cytoskeleton. The GSEA and PPI network analysis among the DEPs were carried out and identified Cofilin-1 and profilin-1 as the core components of this network. Additionally, the disruption of Th1/Th2/Th17 cell balance and the significantly reducing of Treg, MDSC, and γδT cells was also found in AD patients using the ssGSEA analysis. Further ELISA and IHC assay validated the significantly elevated expression of Cofilin-1 in AD patients. Our results suggested that Cofilin-1 may serve as a novel biomarker for AD diagnosis.

ITRAQ Proteomics Identified the Potential Biomarkers of Coronary Artery Lesion in Kawasaki Disease and In Vitro Studies Demonstrated That S100A4 Treatment Made HCAECs More Susceptible to Neutrophil Infiltration.

Coronary artery lesions (CAL) are a major complication of Kawasaki disease (KD). The early prediction of CAL enables the medical personnel to apply adequate medical intervention. We collected the serum samples from the KD patients with CAL (n = 32) and those without CAL (n = 31), followed by a global screening with isobaric tagging for relative and absolute quantification (iTRAQ) technology and specific validation with an enzyme-linked immunosorbent assay (ELISA). iTRAQ identified 846 proteins in total in the serum samples, and four candidate proteins related to CAL were selected for ELISA validation as follows: Protein S100-A4 (S100A4), Catalase (CAT), Folate receptor gamma (FOLR3), and Galectin 10 (CLC). ELISA validation showed that the S100A4 level was significantly higher in KD patients with CAL than in those without CAL (225.2 ± 209.5 vs. 143.3 ± 83 pg/mL, p &lt; 0.05). In addition, KD patients with CAL had a significantly lower CAT level than those without CAL (1.6 ± 1.5 vs. 2.7 ± 2.3 ng/mL, p &lt; 0.05). Next, we found that S100A4 treatment on human coronary artery endothelial cells (HCAECs) reduced the abundance of cell junction proteins, which promoted the migration of HCAECs. Further assays also demonstrated that S100A4 treatment enhanced the permeability of the endothelial layer. These results concluded that S100A4 treatment resulted in an incompact endothelial layer and made HCAECs more susceptible to in vitro neutrophil infiltration. In addition, both upregulated S100A4 and downregulated CAT increased the risk of CAL in KD. Further in vitro study implied that S100A4 could be a potential therapeutic target for CAL in KD.

Single-gene knockout-coupled omics analysis identifies C9orf85 and CXorf38 as two uncharacterized human proteins associated with ZIP8 malfunction.

Human transmembrane protein metal cation symporter ZIP8 (SLC39A8) is a member of the solute carrier gene family responsible for intracellular transportation of essential micronutrients, including manganese, selenium, and zinc. Previously, we established a ZIP8-knockout (KO) human cell model using the CRISPR/Cas9 system and explored how the expression of ZIP8 could possibly contribute to a wide range of human diseases. To further assess the biophysiological role of ZIP8, in the current study, we employed isobaric tags for relative and absolute quantitation (iTRAQ) and detected the changes of the proteome in ZIP8-KO cells (proteomic data are available via ProteomeXchange with identifier PXD036680). A total of 286 differentially expressed proteins (206 downregulated and 80 upregulated proteins) were detected in the ZIP8-KO cell model, and subsequent bioinformatics analyses (GO, KEGG, KOG, and PPI) were performed on these proteins. Interestingly, four "uncharacterized" proteins (proteins with unknown biological function) were identified in the differentially expressed proteins: C1orf198, C9orf85, C17orf75, and CXorf38-all of which were under-expressed in the ZIP8-KO cells. Notably, C9orf85 and CXorf38 were amongst the top-10 most downregulated proteins, and their expressions could be selectively induced by essential micronutrients. Furthermore, clinical-based bioinformatic analysis indicated that positive correlations between the gene expressions of ZIP8 and C9orf85 or CXorf38 were observed in multiple cancer types. Overall, this study reveals the proteomic landscape of cells with impaired ZIP8 and uncovers the potential relationships between essential micronutrients and uncharacterized proteins C9orf85 and CXorf38. The differentially expressed proteins identified in ZIP8-KO cells could be the potential targets for diagnosing and/or treating human ZIP8-associated diseases, including but not limited to malnutrition, viral infection, and cancers.

Nuclear Proteomics of Induced Leukemia Cell Differentiation.

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3–12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Proteomics Evidence of the Role of TDMQ20 in the Cholinergic System and Synaptic Transmission in a Mouse Model of Alzheimer's Disease.

The interaction between copper ions and amyloid peptide Aβ has been reported to be involved in Alzheimer's disease (AD) pathology. Based on copper coordination biochemistry, we designed specific copper chelators [tetradentate monoquinolines (TDMQs)] in order to regulate copper homeostasis in the AD brain and inhibit the deleterious oxidative stress catalyzed by copper-Aβ complexes. We previously reported that TDMQ20, a highly selective copper chelator selected as a drug candidate, was able to extract copper from the Cu-Aβ1-16 complex and restore cognitive and behavioral deficits in AD mouse models. For a better understanding of the mechanism of action of TDMQ20, we decided to investigate the change of profile of proteins expressed in 5xFAD mice after an oral treatment of TDMQ20 (dose = 10 mg/kg, once every two days for 3 months, in total 45 times). Clioquinol (CQ), a non-specific chelator, has been used as a comparator. Here, we report the proteomic alterations in the cortex of 5xFAD mice using iTRAQ (isobaric tags for relative and absolute quantification) proteomics technology. The results indicated that 178 differentially expressed proteins (DEPs) have been identified in the AD mouse group with respect to wild type (WT) animals (AD/WT). After treatment by TDMQ20, 35 DEPs were found common in AD/WT and TDMQ20/AD groups in an opposite change manner (up- or down-regulated, respectively). In addition, among the 35 DEPs mentioned above, 10 common target proteins have been identified in AD/WT, TDMQ20/AD, and CQ/AD groups, among which 3 target proteins were successfully validated by western blot analysis. In particular, the expression levels of ChAT and CHRM4 are significantly increased upon TDMQ20 treatment with respect to 5xFAD mice, while CQ did not significantly change the expression of these proteins. Our study suggests the involvement of the copper chelator TDMQ20 on the cholinergic system, a feature that may explain the improved cognitive and behavioral performance in AD mice upon oral treatment of TDMQ20.

A comparative proteomic analysis provides insight into the molecular mechanism of bud break in longan

BackgroundThe timing of bud break is very important for the flowering and fruiting of longan. To obtain new insights into the underlying regulatory mechanism of bud break in longan, a comparative analysis was conducted in three flower induction stages of two longan varieties with opposite flowering phenotypes by using isobaric tags for relative and absolute quantification (iTRAQ).ResultsIn total, 3180 unique proteins were identified in 18 samples, and 1101 differentially abundant proteins (DAPs) were identified. “SX” (“Shixia”), a common longan cultivated variety that needs an appropriate period of low temperatures to accumulate energy and nutrients for flower induction, had a strong primary inflorescence, had a strong axillary inflorescence, and contained high contents of sugars, and most DAPs during the bud break process were enriched in assimilates and energy metabolism. Combined with our previous transcriptome data, it was observed that sucrose synthase 6 (SS6) and granule-bound starch synthase 1 (GBSSI) might be the key DAPs for “SX” bud break. Compared to those of “SX”, the primary inflorescence, axillary inflorescence, floral primordium, bract, and prophyll of “SJ” (“Sijimi”) were weaker. In addition, light, rather than a high sugar content or chilling duration, might act as the key signal for triggering bud break. In addition, catalase isozyme 1, an important enzyme in the redox cycle, and RuBisCO, a key enzyme in the Calvin cycle of photosynthetic carbon assimilation, might be the key DAPs for SJ bud break.ConclusionOur results present a dynamic picture of the bud break of longan, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this fruit tree species.

Physiological and protein profiling analysis provides insight into the underlying molecular mechanism of potato tuber development regulated by jasmonic acid in vitro

BackgroundJasmonates (JAs) are one of important phytohormones regulating potato tuber development. It is a complex process and the underlying molecular mechanism regulating tuber development by JAs is still limited. This study attempted to illuminate it through the potential proteomic dynamics information about tuber development in vitro regulated by exogenous JA.ResultsA combined analysis of physiological and iTRAQ (isobaric tags for relative and absolute quantification)-based proteomic approach was performed in tuber development in vitro under exogenous JA treatments (0, 0.5, 5 and 50 μΜ). Physiological results indicated that low JA concentration (especially 5 μM) promoted tuber development, whereas higher JA concentration (50 μM) showed inhibition effect. A total of 257 differentially expressed proteins (DEPs) were identified by iTRAQ, which provided a comprehensive overview on the functional protein profile changes of tuber development regulated by JA. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that low JA concentration (especially 5 μM) exhibited the promotion effects on tuber development in various cellular processes. Some cell wall polysaccharide synthesis and cytoskeleton formation-related proteins were up-regulated by JA to promote tuber cell expansion. Some primary carbon metabolism-related enzymes were up-regulated by JA to provide sufficient metabolism intermediates and energy for tuber development. And, a large number of protein biosynthesis, degradation and assembly-related were up-regulated by JA to promote tuber protein biosynthesis and maintain strict protein quality control during tuber development.ConclusionsThis study is the first to integrate physiological and proteomic data to provide useful information about the JA-signaling response mechanism of potato tuber development in vitro. The results revealed that the levels of a number of proteins involved in various cellular processes were regulated by JA during tuber development. The proposed hypothetical model would explain the interaction of these DEPs that associated with tuber development in vitro regulated by JA.