Quantitative proteomics provides insight into the response of the marine dinoflagellate Prorocentrum donghaiense to changes in ambient phosphorus

Dinofl agellates are the major causative agents of harmful algal blooms in the global ocean and they usually form blooms under conditions of very low dissolved inorganic phosphorus(DIP).However,the mechanisms underpinning the dinofl agellate blooms remain unclear.Here,we quantitatively compared protein expression profi les of a marine dinofl agellate,Prorocentrum donghaiense,grown in inorganic P-replete,P-defi cient,and DIP-and dissolved organic phosphorus(DOP)-resupplied conditions by employing a Tandem Mass Tag(TMT)-based quantitative proteomic approach.Proteins involved in intracellular P reallocation,organic P,and non-P lipid utilization were up-regulated under the P-defi cient condition,while inorganic phosphate transporters varied insignifi cantly.In response to the P resupplementation,nitrogen metabolism,ribosome,porphyrin,and chlorophyll metabolism were up-regulated,while lysosome,and starch and sucrose metabolism were down-regulated.Notably,photosynthesis was up-regulated and secondary metabolism was down-regulated only in the DIP-resupplied cells,whereas amino acid metabolism and vitamin B6 metabolism were up-regulated in the DOP-resupplied cells,indicating diff erential response mechanisms of P.donghaiense to DIP or DOP resupplementation.Our results indicated that P.donghaiense initiated multiple strategies in response to an ambient inorganic P-defi ciency,and its efficient DOP assimilation by providing both P and carbon sources might be a key factor driving bloom formations of P.donghaiense in a low DIP environment.

Quantitative Proteomics Analysis Identifies the Potential Mechanism Underlying Yellow-Green Leave Mutant in Wheat

Enhancing photosynthesis efficiency is considered as one of the most crucial targets during wheat breeding.However,the molecular basis underlying high photosynthesis efficiency is not well understood up to now.In this study,we investigated the protein expression profile of wheat Jimai5265yg mutant,which is a yellow-green mutant with chlorophylls b deficiency but high photosynthesis efficiency.Though TMT-labeling quantitative proteomics analysis,a total of 72 differential expressed proteins(DEPs)were obtained between the mutant and wild type(WT).GO analysis found that they significantly enriched in thylakoid membrane,pigment binding,magnesium chelatase activity and response to light intensity.KEGG analysis showed that they involved in photosynthesis-antenna protein as well as porphyrin and chlorophyll metabolism.Finally,118 RNA editing events were found between mutant and WT genotype.The A to C editing in the 3-UTR of TraesCS6D02G401500 lead to its high expression in mutant through removing the inhibition of tae-miR9781,which might have vital role in regulating the yellow-green mutant.This study provided some useful clues about the molecular basis of Jimai5265yg mutant as well as chlorophylls metabolism in wheat.

Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤)ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study

OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.

Quantitative proteomics reveals key pathways in the symbiotic interface and the likely extracellular property of soybean symbiosome

An effective symbiosis between legumes and rhizobia relies largely on diverse proteins at the plantrhizobium interface for material transportation and signal transduction during symbiotic nitrogen fixation.Here,we report a comprehensive proteome atlas of the soybean symbiosome membrane(SM),peribacteroid space(PBS),and root microsomal fraction(RMF)using state-of-the-art label-free quantitative proteomic technology.In total,1759 soybean proteins with diverse functions are detected in the SM,and 1476 soybean proteins and 369 rhizobial proteins are detected in the PBS.The diversity of SM proteins detected suggests multiple origins of the SM.Quantitative comparative analysis highlights amino acid metabolism and nutrient uptake in the SM,indicative of the key pathways in nitrogen assimilation.The detection of soybean secretory proteins in the PBS and receptor-like kinases in the SM provides evidence for the likely extracellular property of the symbiosome and the potential signaling communication between both symbionts at the symbiotic interface.Our proteomic data provide clues for how some of the sophisticated regulation between soybean and rhizobium at the symbiotic interface is achieved,and suggest approaches for symbiosis engineering.

Circulating proteomic biomarkers for diagnosing sporadic amyotrophic lateral sclerosis:a cross-sectional study

Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.

Remote ischemic conditioning-induced hyperacute and acute responses of plasma proteome in healthy young male adults:a quantitative proteomic analysis

Background: Long-term remote ischemic conditioning (RIC) has been proven to be beneficial in multiple diseases, such as cerebral and cardiovascular diseases. However, the hyperacute and acute effects of a single RIC stimulus are still not clear. Quantitative proteomic analyses of plasma proteins following RIC application have been conducted in preclinical and clinical studies but exhibit high heterogeneity in results due to wide variations in experimental setups and sampling procedures. Hence, this study aimed to explore the immediate effects of RIC on plasma proteome in healthy young adults to exclude confounding factors of disease entity, such as medications and gender. Methods: Young healthy male participants were enrolled after a systematic physical examination and 6-month lifestyle observation. Individual RIC sessions included five cycles of alternative ischemia and reperfusion, each lasting for 5 min in bilateral forearms. Blood samples were collected at baseline, 5 min after RIC, and 2 h after RIC, and then samples were processed for proteomic analysis using liquid chromatography-tandem mass spectrometry method. Results: Proteins related to lipid metabolism (e.g., Apolipoprotein F), coagulation factors (hepatocyte growth factor activator preproprotein), members of complement cascades (mannan-binding lectin serine protease 1 isoform 2 precursor), and inflammatory responses (carboxypeptidase N catalytic chain precursor) were differentially altered at their serum levels following the RIC intervention. The most enriched pathways were protein glycosylation and complement/coagulation cascades. Conclusions: One-time RIC stimulus may induce instant cellular responses like anti-inflammation, coagulation, and fibrinolysis balancing, and lipid metabolism regulation which are protective in different perspectives. Protective effects of single RIC in hyperacute and acute phases may be exploited in clinical emergency settings due to apparently beneficial alterations in plasma proteome profile. Furthermore, the beneficial effects of long-term (repeated) RIC interventions in preventing chronic cardiovascular diseases among general populations can also be expected based on our study findings.

Quantitative Proteomics Using Isobaric Labeling:A Practical Guide

In the past decade,relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples.Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions,its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results.Here,we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method.We also discuss which precautions should be taken at each step of the isobaric labeling workflow,to obtain reliable quantification results in large-scale quantitative proteomics experiments.In the last section,we discuss the broad applications of the isobaric labeling technology in biological and clinical studies,with an emphasis on thermal proteome profiling and proteogenomics.

Label-free quantitative proteomics reveals fibrinopeptide B and heparin cofactorⅡas potential serum biomarkers in respiratory syncytial virus-infected mice treated with Qingfei oral liquid formula

Respiratory syncytial virus(RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid(QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group(M), QFOL-treated group(Q) and the control group(C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins(DEPs) were identified(15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B(FpB) and heparin cofactor Ⅱ(HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the Fp B level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.

Quantitative proteomics revealed extensive microenvironmental changes after stem cell transplantation in ischemic stroke

The local microenvironment is essential to stem cell-based therapy for ischemic stroke,and spatiotemporal changes of the microenvironment in the pathological process provide vital clues for understanding the therapeutic mechanisms.However,relevant studies on microenvironmental changes were mainly confined in the acute phase of stroke,and long-term changes remain unclear.This study aimed to investigate the microenvironmental changes in the subacute and chronic phases of ischemic stroke after stem cell transplantation.Herein,induced pluripotent stem cells(iPSCs)and neural stem cells(NSCs)were transplanted into the ischemic brain established by middle cerebral artery occlusion surgery.Positron emission tomography imaging and neurological tests were applied to evaluate the metabolic and neurofunctional alterations of rats transplanted with stem cells.Quantitative proteomics was employed to investigate the protein expression profiles in iPSCs-transplanted brain in the subacute and chronic phases of stroke.Compared with NSCs-transplanted rats,significantly increased glucose metabolism and neurofunctional scores were observed in iPSCs-transplanted rats.Subsequent proteomic data of iPSCs-transplanted rats identified a total of 39 differentially expressed proteins in the subacute and chronic phases,which are involved in various ischemic stroke-related biological processes,including neuronal survival,axonal remodeling,antioxidative stress,and mitochondrial function restoration.Taken together,our study indicated that iPSCs have a positive therapeutic effect in ischemic stroke and emphasized the wide-ranging microenvironmental changes in the subacute and chronic phases.

An integrated quantitative proteomics strategy reveals the dual mechanisms of celastrol against acute inflammation

Inflammation is a defense mechanism associated with a wide range of diseases.Celastrol is a small molecule isolated from traditional Chinese medicine with potent anti-inflammation activity.In this study,we established an integrated quantitative proteomics strategy to investigate the acute response to celastrol treatment in a rat macrophage cell line challenged with lipopolysaccharide(LPS).Both stableisotopic based non-targeted quantitative profiling and PRM-based targeted quantitation methods were employed.Dimethyl-labeling based non-targeted profiling revealed 28 and 52 proteins that significantly up-and down-regulated by celastrol.Bioinformatics analysis pinpoint key signaling pathways affected.Seven proteins were selected for examining their time-dependent regulatory pattern in response to celastrol using targeted PRM quantitation.The abundance of mRNA at multiple time-points of selected proteins was also examined.Celastrol induced an acute response of selected key transcriptional factors in terms of mRNA or protein abundance within one hour.Interestingly,regulatory trend of mRNA and protein abundance suggested a novel dual mechanism of celastrol in the terms of acute antiinflammation.The integrated quantitative proteomic strategy established in this study constitutes an efficient workflow to characterize key components and their time-dependent regulatory pattern for monitoring drug response.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.

Global Quantitative Proteomics Analysis Reveals the Downstream Signaling Networks of Msx1 and Msx2 in Myoblast Differentiation

The msh homeobox 1(Msx1)and msh homeobox 2(Msx2)coordinate in myoblast differentiation and also contribute to muscle defects if altered during development.Deciphering the downstream signaling networks of Msx1 and Msx2 in myoblast differentiation will help us to understand the molecular events that contribute to muscle defects.Here,the proteomics characteristics in Msx1-and Msx2-mediated myoblast differentiation was evaluated using isobaric tags for the relative and absolute quantification labeling technique(iTRAQ).The downstream regulatory proteins of Msx1-and Msx2-mediated differentiation were identified.Bioinformatics analysis revealed that these proteins were primarily associated with xenobiotic metabolism by cytochrome P450,fatty acid degradation,glycolysis/gluconeogenesis,arginine and proline metabolism,and apoptosis.In addition,our data show Acta1 was probably a core of the downstream regulatory networks of Msx1 and Msx2 in myoblast differentiation.

Quantitative Proteomic Analysis of Bromotetrandrine and Tetrandrine in K562 Cell Line Using ^(18)O-labeling Method

Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class I new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method. Methods To illustrate its mechanism, a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study. Compared to normal chronic leukemia cell line K562 and K562 induced by Tet, the proteomic changes of K562 induced by W198 were investigated. In order to validate the quantitation by the 18O-labeling, the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample. Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments. Comparing the 105 identified soluble proteins’ expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells, 16 proteins were found with significantly altered expression levels after W198 treatment. Eight proteins were up-expressed including HMGB2, peroxiredoxin-2, and eIF4A-I, etc. Eight proteins were down-expressed including TCP-1, GRP94, GST-π, and SFGHs, etc. Compared to K562 induced by Tet, eight proteins of K562 were found with significantly altered expression levels after W198 treatment. Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a, etc. Three proteins were down-expressed including phosphoglycerate kinase 1, isoform 5 of interleukin enhancer-binding factor 3, etc. Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool, but it is not suitable for validation using a large number of samples. Other more effective methods, such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples. In total, 105 soluble proteins were discovered, and 16 proteins were found with significantly altered expression levels after W198 treatment. These repressed or activated proteins are the potential drug targets of W198, which may provide novel targets for future development of biomarkers for cancer therapy.

Label-Free Quantitative Proteomics Analysis of the Sorafenib Resistance in HepG2 Cells

Drug resistance of sorafenib seriously affects the treatment effect of late-stage hepatocellular carcinoma(HCC)patients.However,the precise mechanism of resistance to sorafenib remains unclear.Therefore,to obtain a deep understand of sorafenib resistance mechanisms and find potential therapeutic targets are very important for improving the clinical prognosis of HCC patients.In this study,a label-free quantitative proteomics method was performed to investigate the proteins differentially expressed between HepG2 and the sorafenib-acquired resistance HepG2(HepG2-R)cells.In total,84 differential expressed proteins were identified between the two cell lines.Bioinformatics analysis results demonstrated the dysregulated metabolic processes have a significant impact on the drug resistance of HepG2-R cells.Among them,the expression of Microsomal glutathione S-transferase 1(MGST1)in two cell lines was further confirmed by western blot method.Moreover,colony formation assay and trypan blue dye assay results revealed that MGST1 is closely connected with the sorafenib resistance of HepG2-R cells,and the knockdown of MGST1 increased the sensitivity of sorafenib resistance HepG2-R cells to sorafenib treatment.In conclusion,these results lay a foundation for deciphering the mechanism for HCC sorafenib resistance and present a possibility of MGST1 serving as a therapeutic target for the treatment of sorafenib resistance HCC.

Comparative proteomic analysis reveals the effects of different fatty acid forms on high-fat diet mice

Long-chain omega-3 polyunsaturated fatty acids(LC-PUFAs),known for having many health benefits,are usually present in three forms:triglycerides(TG),ethyl esters(EE),and phospholipid(PL).In this study,the effects of these three LC-PUFAs forms(fish oil for TG and EE,krill oil for PL)on the obese mice were compared,and the proteomic changes that focused on lipid metabolism were evaluated via label-free quantitative proteomics analysis.Compared with the model group,all three of the LC-PUFA form supplementations(labeled as the FO-TG group,FO-EE group and KO-PL groups)could significantly reduce body weight gain(P<0.01).Low-density lipoprotein cholesterol levels were significantly decreased,whereas high-density lipoprotein cholesterol levels were significantly increased in the FO-TG group and FO-EE group(P<0.01),and especially in the PL group(P<0.001).Furthermore,proteomics analysis results suggested that some differentially expressed genes involved in the fatty acid degradation and oxidation pathways had a higher expression fold in the KO-PL group than in the FO-TG or FO-EE groups.Our results showed that dietary LC-PUFAs can reduce fat deposition and inhibit lipogenesis in the liver by upregulating the expression of proteins that are involved in the fatty acid degradation and oxidation pathways.Additionally,KO-PL elicits stronger effects than FO-TG or FO-EE.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA

Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes

Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.

Functional annotation and enrichment analysis of differentially expressed serum proteins in patients with type 2 diabetes after dapagliflozin

BACKGROUND Only 50% of patients with type 2 diabetes mellitus(T2DM) can control their blood glucose levels. Dapagliflozin is a selective inhibitor of sodium-glucose cotransporter 2(SGLT-2) that improves the insulin sensitivity of the liver and peripheral tissues. Many studies confirmed that SGLT2 inhibitors reduce blood glucose and have multiple beneficial effects such as weight loss, lipid regulation, and kidney protection. Nevertheless, the mechanisms of the renal and cardiovascular protective effects of dapagliflozin from the perspective of differentially expressed proteins in the serum of T2DM patients have not been intensively explored so far.AIM To identify differentially expressed proteins associated with dapagliflozin treatment in patients with T2DM.METHODS Twenty T2DM patients [hemoglobin A1c(HbA1c) 7.0%-10.0%] were enrolled at The Affiliated Hospital of Inner Mongolia Medical University between January 1, 2017 and December 1, 2018. They received dapagliflozin(10 mg/d) for 3 mo, and the HbA1c < 7.0% target was achieved. The changes in clinical indexes were compared before and after treatments. Label-free quantitative proteomics was used to identify differentially expressed proteins using the serum samples of five patients. The identified differentially expressed proteins were analyzed using various bioinformatics tools.RESULTS Dapagliflozin significantly improved the clinical manifestation of the patients. There were 18 downregulated proteins and one upregulated protein in the serum samples of patients after dapagliflozin administration. Bioinformatics analyses, including subcellular localization, Eu Karyotic Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes annotations, were used to profile the biological characteristics of the 19 differentially expressed proteins. Based on the literature and function enrichment analysis, two downregulated proteins, myeloperoxidase(MPO) and alpha Ⅱ B integrin(ITGA2B), and one upregulated protein, podocalyxin(PCX), were selected for enzyme linked immunosorbent assay validation. These validated differentially expressed proteins had multiple correlations with clinical indexes, including Hb Ac1 and fasting C-peptide.CONCLUSION Dapagliflozin has hypoglycemic effects and regulates the serum expressions of MPO, ITGA2B, and PCX, possibly contributing to the effects of dapagliflozin on oxidative stress, insulin resistance, and lipid metabolism.

miRNA-451 regulates rhesus choroid-retinal endothelial cell function and proteome profile

AIM:To evaluate the effect of miRNA-451 on rhesus macaque choroid-retinal endothelial(RF/6A)cell function and proteome profile.METHODS:The RF/6A cells were transfected with miRNA-451 mimic and inhibitor.The role of miRNA-451 on proliferation ability was evaluated by CCK-8 assay.Furthermore,iTRAQ quantitative proteomic analysis was applied to comprehensively illuminate the change of cellular proteins and biological function between different groups.RESULTS:In miRNA-451 overexpression group,cell proliferation of RF/6A decreased both at 24 h and 48 h;while in miRNA-451 inhibition group,on the contrary,RF/6A cell proliferation was increased at 48 h.Based on iTRAQ quantitative proteomic analysis,23 differentially expressed proteins(DEPs)were detected in the comparison of miRNA-451 mimic and mimic control-transfected RF/6A cells,and 30 DEPs were identified in the comparison of RF/6A cells transfected with miRNA-451 inhibitor and inhibitor control.DEPs such as GORASP2,KRT1,SLC7 A2,RIC8 A,DDX42,CAP1,PCBP2 might be closely related to the inhibitory effect of miRNA-451 on RF/6A cell proliferation,while PCYT1 A,MGAT1,TUBB,MCU,SIL1,BID,MSH6 might account for the positive effect of miRNA-451 inhibitor on RF/6A cell growth.PTPN1,as the only protein exhibiting an opposite trend between miRNA-451 mimic and inhibitortransfected cells,was most likely accountable for the inhibition of miRNA-451 mimic on RF/6A cell growth,and the promotion of miRNA-451 inhibitor on RF/6A cell proliferation.CONCLUSION:miRNA-451 overexpression can suppress the growth of RF/6A cells while knockdown of miRNA-451 can promote RF/6A cell viability.Among all DEPs,increased PTPN1 is most likely to account for the negative regulation of miRNA-451 on RF/6A proliferation.miRNA-451 can be a protective factor for neovascular disease of fundus via regulating choroid retinal endothelial cell function.

Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis

Ethylene participates in the regulation of numerous cellular events and biological processes, including wa- ter loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a lSN stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eill-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene- regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-O and ein3eill genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up- regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.