Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely unknown.In order to study the changes of proteins and enriched phosphopepti...Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely unknown.In order to study the changes of proteins and enriched phosphopeptides,we conducted TMT labeling,bio-material-based PTM enrichment based on mass spectrometry in Ca-treated‘Golden Delicious’(GD)apple fruit(Malus×domestica).This dataset presents a comprehensive overview of the critical pathways involved in fruit ripening.A total of 47 proteins and 124 phosphoproteins significantly changed in Ca-treated fruit,which are crucial for regulating the cell wall and cytoskeleton,Ca-mediated signaling and transport,ethylene production,protein fate,especially ubiquitination-based protein degradation,and primary and secondary metabolisms.Our results indicated that Ca inhibited the abundance of polygalacturonase(PG)activity and increased the phosphorylation level of CSLD3.PG and phosphorylation were involved in cell wall degradation,thereby delaying fruit softening.As a secondary messenger,Ca-mediated signaling subsequently triggered downstream mitogen-activated protein kinases(MAPK)cascades and activated the membrane,transport,and ROS signaling.Moreover,MdEIN2,a key enzyme involved in the ubiquitin of protein modification,increased at Ser753 and Ser758 in Ca-treated fruit.Furthermore,diverse primary and secondary metabolisms including glycolysis,fatty acid metabolism,and oxidation respiratory chain were modulated to prevent fruit softening.These results provide basic information from protein and phosphorylation levels for apple fruit ripening during storage,which may be helpful for apple fruit storage control.展开更多
OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.METHODS:Sprague-Dawley rats with normal glucose levels were ...OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.METHODS:Sprague-Dawley rats with normal glucose levels were randomly divided into three groups,including a conventional diet control group(Group A),high-fat-highsugar diet model group(Group B),and DGR group(Group C,high-fat-high-sugar diet containing 20.5 g DGR).After 10 weeks of intervention,the fasting blood glucose(FBG),2 h blood glucose[PBG;using the oral glucose tolerance test(OGTT)],hemoglobin A1c(HbA1c),plasma total cholesterol(TC),and triglycerides(TG)were tested,and the livers of rats were removed to calculate the liver index.Then,hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry(LC-MS/MS)analysis followed by database search and bioinformatics analysis.Finally,cell experiments were used to verify the results of phosphoproteomics.Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4(MAP4k4)and phosphorylated adducin 1(ADD1)were detected using western blotting.RESULTS:DGR effectively reduced PBG,TG,and the liver index(P<0.05),and significantly decreased HbA1c,TC,and hepatic portal TG(P<0.01),showed significant hematoxylin and eosin(HE)staining,red oil O staining,and Masson staining of liver tissue.The total spectrum was 805334,matched spectrum was 260471,accounting for accounting 32.3%,peptides were 19995,modified peptides were 14671,identified proteins were 4601,quantifiable proteins were 4417,identified sites were 15749,and quantified sites were 14659.Based on the threshold of expression fold change(>1.2),DGR upregulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins,and downregulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins,which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism.Therefore,DGR improved biological tissue processes,including information storage and processing,cellular processes and signaling,and metabolism.The metabolic functions regulated by DGR mainly include energy production and conversion,carbohydrate transport and metabolism,lipid transport and metabolism,inorganic ion transport and metabolism,secondary metabolite biosynthesis,transport,and catabolism.In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.CONCLUSION:DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders,thereby regulating glycolipid metabolism through a multi-target and multi-method process.展开更多
Phosphorylation is one of the most common post translational modifications （PTM）, participating in a large number of processes to regulate cellular functions. Phosphorylation is also one of the key factors in the or...Phosphorylation is one of the most common post translational modifications （PTM）, participating in a large number of processes to regulate cellular functions. Phosphorylation is also one of the key factors in the origin and development of cancer. The rapid development of mass spectrometric-based phosphoproteomic technologies has made it possible for high-throughput identification and quantification of phosphorylation events. In this review, we provide a general introduction and summary of the achievements made in mass spectrometry based phosphoproteomic research, including the approaches for phosphopeptide identification and quantification, as well as instrumentation and data interpretation methods. We also review some discoveries in cancer research made possible by phosphoproteomic analysis technologies.展开更多
AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demonstrated the regulatory roles of AMPK in...AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully understood.Herein,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation.Together with these findings,we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradiation(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent manner.Besides,we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPKdeficient cells to IR.Therefore,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.展开更多
Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,phys...Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.展开更多
Protein phosphorylation plays an important role in immune signaling transduction in plant resistance to pathogens.Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),severely devastates wheat production...Protein phosphorylation plays an important role in immune signaling transduction in plant resistance to pathogens.Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),severely devastates wheat production.Nonetheless,the molecular mechanism of wheat resistance to stripe rust remains limited.In this study,quantitative phosphoproteomics was employed to investigate the protein phosphorylation changes in wheat challenged by Pst.A total of 1537 and 2470 differentially accumulated phosphoproteins(DAPs)were identified from four early infection stage(6,12,18 and 24 h post-inoculation)in incompatible and compatible wheat-Pst interactions respectively.KEGG analysis revealed that Oxidative Phosphorylation,Phosphatidylinositol Signaling,and MAPK signaling processes are distinctively enriched in incompatible interaction,while Biosynthesis of secondary metabolites and RNA degradation process were significantly enriched in compatible interactions.In particular,abundant changes in phosphorylation levels of chloroplast proteins were identified,suggesting the regulatory role of photosynthesis in wheat-Pst interaction,which is further emphasized by protein-protein interaction(PPI)network analysis.Motif-x analysis identified[xxxxSPxxxx]motif,likely phosphorylation sites for defensive response-related kinases,and a new[xxxxSSxxxx]motif significantly enriched in incompatible interaction.The results shed light on the early phosphorylation events contributing to wheat resistance against Pst.Moreover,our study demonstrated that the phosphorylation levels of Nucleoside diphosphate kinase TaNAPK1 are upregulated at 12 hpi with CYR23 and at 24 hpi with CYR31.Transient silencing of TaNAPK1 was able to attenuate wheat resistance to CYR23 and CYR31.Our study provides new insights into the mechanisms underlying Pst-wheat interactions and may provide database to find potential targets for the development of new resistant varieties.展开更多
Background：The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of ...Background：The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets,we carried out a phosphoproteomics analysis of human endometrium.Methods：A large-scale differential phosphoproteome analysis,using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry,was performed in endometrium tissues from 8 women with or without endometriosis.Results：The phosphorylation profiling of endometrium from endometriosis patients had been obtained,and found that identified 516 proteins were modified at phosphorylation level during endometriosis.Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity.Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways,which might be deregulated during the development of endometriosis.Conclusions：That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.展开更多
The most common phenotype induced by the endosymbiont Wolbachia in in-sects is cytoplasmic incompatibility,where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females.This ...The most common phenotype induced by the endosymbiont Wolbachia in in-sects is cytoplasmic incompatibility,where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females.This suggests that some modi-fications are induced in host sperms during spermatogenesis by Wolbachia.To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster,we applied isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic strategy combined with titanium dioxide(TiO2)enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male re-productive systems in D.melanogaster.We identified 182 phosphopeptides,defining 140 phosphoproteins,that have at least a 1.2 fold change in abundance with a P-value of<0.05.Most of the differentially abundant phosphoproteins(DAPPs)were associated with micro-tubule cytoskeleton organization and spermatid differentiation.The DAPPs included pro-teins already known to be associated with spermatogenesis,as well as many not previously studied during this process.Six genes coding for DAPPs were knocked down,respectively,in Wolbachia-free fly testes.Among them,Slmap knockdown caused the most severe dam-age in spermatogenesis,with no mature sperm observed in seminal vesicles.Immunoflu-orescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted.These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction.By identifying phospho-modulated proteins we also provide a signifi-cant candidate set for future studies on their roles in spermatogenesis.展开更多
Gastric cancer(GC)is a prevalent and devastating disease with a poor prognosis.The lack of biomarkers for early detection and effective targeted therapeutics for GC patients represents two major challenges.Through iso...Gastric cancer(GC)is a prevalent and devastating disease with a poor prognosis.The lack of biomarkers for early detection and effective targeted therapeutics for GC patients represents two major challenges.Through isobaric tags for relative and absolute quantitation(iTRAQ)coupled with liquid chromatography-tandem mass spectrometry(LC-MS/MS)phosphoproteomic analysis of 14 GC and gastric epithelial cell lines,we discovered the discoidin domain receptor tyrosine kinase 1(DDR1)as a top potential drug target out of 40 tyrosine kinases detected along with over 1000 phosphoproteins profiled.The DDR1 protein and mRNA levels were upregulated in GC cells concurrent with DDR1 gene amplification.Immunohistochemistry staining of more than 200 clinical samples revealed that DDR1 was overexpressed in approximately 41%and 48%of the intestinal and diffuse types of GC cases,respectively,compared with only 3.5%in normal tissues.Higher DDR1 expression was associated with poor prognosis.In cellular models,DDR1 overexpression led to accelerated proliferation,invasion,and malignant transformation,putatively via inhibition of the Hippo pathway and consequent activation of YAP-TEAD target gene expression.Notably,DDR1-overexpressing GC cells exhibited high vulnerability to selective DDR1 inhibitors.The present study provides preclinical support for the application of DDR1-selective inhibitors in DDR1-overexpressing GC.展开更多
Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been...Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.展开更多
Plants produce a range of carbohydrates to meet their growth and developmental needs. Protein reversible phosphorylation plays key roles in coordinating multiple metabolic pathways and integrating diverse internal and...Plants produce a range of carbohydrates to meet their growth and developmental needs. Protein reversible phosphorylation plays key roles in coordinating multiple metabolic pathways and integrating diverse internal and external cues. Understanding such regulatory metabolism will provide novel resources for breeding and crop management by modulating metabolic pathways for control of growth and stress response. In this review, we summarize the complex, multifaceted functions of protein phosphorylation and their connections to plant metabolism. We focus particularly on carbohydrate metabolic pathways that are controlled by key kinases and discuss how they are linked to downstream changes in physiology, important agronomic traits and crop quality.展开更多
Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological acti...Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological activities;and regulates cell proliferation and enlargement,phytohormone biosynthesis and signaling,plant disease resistance,and grain filling and quality during rice seed development.Research work on protein phosphorylation started in the 1950 s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme.Over the last decade,rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues,organs and organelles.The progress made in this field is evident in number of research works.However,research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions.In this review,the general description of protein phosphorylation,including history,structure,frequency of occurrence and function,are discussed.This work also elucidates the different methods for identification,qualification and finally,the progress in rice phosphoproteome research and perspectives.展开更多
Salinity severely affects plant growth and development.Thus,it is crucial to identify the genes functioning in salt stress response and unravel the mechanism by which plants against salt stress.This study used the pho...Salinity severely affects plant growth and development.Thus,it is crucial to identify the genes functioning in salt stress response and unravel the mechanism by which plants against salt stress.This study used the phosphoproteomic assay and found that 123 of the 4000 quantitative analyzed phosphopeptides were induced by salt stress.The functional annotation of the non-redundant protein database(NR)showed 23 differentially expressed transcription factors,including a phosphopeptide covering the Serine 31 in the RAV(related to ABI3/VP1)transcription factor(named SiRAV1).SiRAV1 was located in the nucleus.Phenotypic and physiological analysis showed that overexpressing SiRAV1 in foxtail millet enhanced salt tolerance and alleviated the salt-induced increases of H_(2)O_(2) accumulation,malondialdehyde(MDA)content,and percent of electrolyte leakage.Further analysis showed that SiRAV1 positively regulated SiCAT expression to modulate the catalase(CAT)activity by directly binding to the SiCAT promoter in vivo and in vitro.Moreover,we found that phosphorylation of SiRAV1 at the Ser31 site positively regulated salt tolerance in foxtail millet via enhancing its binding ability to SiCAT promoter but did not affect its subcellular localization.Overall,our results define a mechanism for SiRAV1 function in salt response where salt-triggered phosphorylation of SiRAV1 at Ser31 enhances its binding ability to SiCAT promoter,and the increased SiCAT expression contributes to salt tolerance in foxtail millet.展开更多
The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment.This cognitive impairment is thought to result specifically from damage to t...The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment.This cognitive impairment is thought to result specifically from damage to the hippocampus.In this study,we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test.Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury.Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus,as well as in the density of mature dendritic spines.To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage,we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury.The differentially expressed proteins were mainly enriched in inflammation,immunity,and coagulation,suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury.In contrast,differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure,which is more consistent with neurodegeneration.We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury,and western blotting showed that,while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury,its phosphorylation level was significantly increased,which is consistent with the omics results.Administration of GRP78608,an N-methyl-D-aspartate receptor 1 antagonist,to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment.In conclusion,our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.展开更多
We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative ...We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative splicing or alternative start sites to encode more than one RNA/protein.However,specific functions for these different PDEs have not yet been identified in most cell types despite the fact that selective inhibitors to most of the PDE families are available. Conventional approaches to study PDE function typically rely on measurements of global cAMP,or general increases in cAMP-dependent protein kinase A(PKA),or exchange protein activated by cAMP(EPAC) activity.Although newer approaches utilizing subcellularly-targeted FRET reporter sensors have helped to define more compartmentalized regulation of cAMP,PKA,and EPAC,they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem,we have begun to use an unbiased,mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the ″ functional pools″ of cAMP that are regulated by specific cAMP-PDEs(the PDE-regulated phosphoproteomes). In MA-10 Leydig cells we find that in order to detect appreciable increased in either phosphorylation or steroid production,one needs to inhibit both PDE 4 and PDE 8 activity.Using this combination of inhibitors,we find large PDE inhibitor-induced changes in many different proteins that modulate steroid trafficking and biosynthesis. The data are consistent with the idea that cAMP serves to coordinate hormone stimulation of steroid production by altering the phosphorylation of many different proteins at multiple points in the overall pathway rather than just controlling a single rate limiting step. It seems quite likely that many of the proteins phosphorylated in this cell type in response to PDE inhibition,will also be regulated by cAMP in many other cell types.Similarly,in Jurkat cells we find multiple,distinct,PDE regulated phosphoproteomes that differ in response to different PDE inhibitors. Here we also find that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Inhibition of a single PDE produces little effect. Bioinformatics analyses of these phosphoproteomes suggest differing functional roles,mechanisms of action,and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. In this tissue also,the data strongly imply that phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. Overall,the findings illustrate that the approach of using selective,inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.展开更多
Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis ...Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.展开更多
Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cel...Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cell proteome(WCP)of PHCs have been extensively studied,only a small number of studies have addressed the role of posttranslational modifications in this process.To elucidate the underlying mechanisms that induce dedifferentiation,we carried out parallel quantifications of the transcriptome,WCP,ubiquitinome,and phosphoproteome of rat PHCs after 0,6,12,24,and 48 h of in vitro culture.Our data constitute a detailed proteomic analysis of dedifferentiated PHCs including 2196 proteins,2056 ubiquitinated sites,and 4932 phosphorylated peptides.We revealed a low correlation between the transcriptome and WCP during dedifferentiation.A combined analysis of the ubiquitinome with the corresponding WCP indicated that the dedifferentiation of PHCs led to an increase in nondegradative K27 ubiquitination.Functional analysis of the altered phosphoproteins suggested a significant enrichment in ferroptosis.In all,404 proteins with both ubiquitination and phosphorylation were identified to be involved in critical metabolic events.Furthermore,Ptbph Hnqjd,Hnrnpu,and Srrm2 were identified as hub genes.Taken together,our data provide new insights into proteome dynamics during PHC dedifferentiation and potential targets to inhibit the dedifferentiation process.展开更多
Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic...Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.展开更多
The LGS1(Large grain size 1)gene,also known as GS2/GL2/Os GRF4,is involved in regulating grain size and quality in rice,but the mechanism governing grain size has not been elucidated.We performed transcriptomic,proteo...The LGS1(Large grain size 1)gene,also known as GS2/GL2/Os GRF4,is involved in regulating grain size and quality in rice,but the mechanism governing grain size has not been elucidated.We performed transcriptomic,proteomic,and phosphoproteomic analyses of young rice panicles in Samba(a wild-type cultivar with extra-small grain)and NIL-LGS1(a nearly isogenic line of LGS1 with large grain in the Samba genetic background)at three developmental stages(4–6)to identify internal dynamic functional networks determining grain size that are mediated by LGS1.Differentially expressed proteins formed seven highly functionally correlated clusters.The concordant regulation of multiple functional clusters may be key features of the development of grain length in rice.In stage 5,16 and 24 phosphorylated proteins were significantly up-regulated and down-regulated,and dynamic phosphorylation events may play accessory roles in determining rice grain size by participating in protein–protein interaction networks.Transcriptomic analysis in stage 5 showed that differentially expressed alternative splicing events and dynamic gene regulatory networks based on 39 transcription factors and their highly correlated target genes might contribute to rice grain development.Integrative multilevel omics analysis suggested that the regulatory network at the transcriptional and posttranscriptional levels could be directly manifested at the translational level,and this analysis also suggested a regulatory mechanism,regulation of protein translation levels,in the biological process that extends from transcript to protein to the development of grain.Functional analysis suggested that biological processes including MAPK signaling,calcium signaling,cell proliferation,cell wall,energy metabolism,hormone pathway,and ubiquitin-proteasome pathway might be involved in LGS1-mediated regulation of grain length.Thus,LGS1-mediated regulation of grain size is affected by dynamic transcriptional,posttranscriptional,translational and posttranslational changes.展开更多
As one of the most crucial post-translational modifications,protein phosphorylation regulates a broad range of biological processes in eukaryotes.Biocuration,integration,and annotation of reported phosphorylation even...As one of the most crucial post-translational modifications,protein phosphorylation regulates a broad range of biological processes in eukaryotes.Biocuration,integration,and annotation of reported phosphorylation events will deliver a valuable resource for the community.Here,we present an updated database,the eukaryotic phosphorylation site database 2.0(EPSD 2.0),which includes 2,769,163 experimentally identified phosphorylation sites(p-sites)in 362,707 phosphoproteins from 223 eukaryotes.From the literature,873,718 new p-sites identified through high-throughput phosphoproteomic research were first collected,and 1,078,888 original phosphopeptides together with primary references were reserved.Then,this dataset was merged into EPSD 1.0,comprising 1,616,804 p-sites within 209,326 proteins across 68 eukaryotic organisms.We also integrated 362,190 additional known p-sites from 10 public databases.After redundancy clearance,we manually re-checked each p-site and annotated 88,074 functional events for 32,762 p-sites,covering 58 types of downstream effects on phosphoproteins,and regulatory impacts on 107 biological processes.In addition,phosphoproteins and p-sites in 8 model organisms were meticulously annotated utilizing information supplied by 100 external platforms encompassing 15 areas.These areas included kinase/phosphatase,transcription regulators,three-dimensional structures,physicochemical characteristics,genomic variations,functional descriptions,protein domains,molecular interactions,drug-target associations,disease-related data,orthologs,transcript expression levels,proteomics,subcellular localization,and regulatory pathways.We expect that EPSD 2.0 will become a useful database supporting comprehensive studies on phosphorylation in eukaryotes.The EPSD 2.0 database is freely accessible online at https://epsd.biocuckoo.cn/.展开更多
基金supported by the National Natural Science Foundation of China (Grant Nos. 31722047, 31801848)LiaoNing Revitalization Talents Program (Grant No. XLYC1802019)
文摘Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely unknown.In order to study the changes of proteins and enriched phosphopeptides,we conducted TMT labeling,bio-material-based PTM enrichment based on mass spectrometry in Ca-treated‘Golden Delicious’(GD)apple fruit(Malus×domestica).This dataset presents a comprehensive overview of the critical pathways involved in fruit ripening.A total of 47 proteins and 124 phosphoproteins significantly changed in Ca-treated fruit,which are crucial for regulating the cell wall and cytoskeleton,Ca-mediated signaling and transport,ethylene production,protein fate,especially ubiquitination-based protein degradation,and primary and secondary metabolisms.Our results indicated that Ca inhibited the abundance of polygalacturonase(PG)activity and increased the phosphorylation level of CSLD3.PG and phosphorylation were involved in cell wall degradation,thereby delaying fruit softening.As a secondary messenger,Ca-mediated signaling subsequently triggered downstream mitogen-activated protein kinases(MAPK)cascades and activated the membrane,transport,and ROS signaling.Moreover,MdEIN2,a key enzyme involved in the ubiquitin of protein modification,increased at Ser753 and Ser758 in Ca-treated fruit.Furthermore,diverse primary and secondary metabolisms including glycolysis,fatty acid metabolism,and oxidation respiratory chain were modulated to prevent fruit softening.These results provide basic information from protein and phosphorylation levels for apple fruit ripening during storage,which may be helpful for apple fruit storage control.
基金the National Natural Science Foundation of China:Based on the"miR34a/Nampt-NAD+-TAC"Pathway to Study the Mechanism of Simultaneously Treating the Phlegm and Blood Stasis in the Regulation of Glycolipid(No.81873213)Study on the Mechanism of Simultaneously Treating the Phlegm and Blood Stasis on Glycolipid Metabolism Based on Intestinal Fat Absorption Regulated by miR-34a/Stat3-Nfil3 Pathway(82074308)+1 种基金a New Mechanism of Regulating the Amino Acid Metabolism of Type 2 Diabetes Mellitus with Dissipating Phlegm-Stasis:Based on the TCA Cycle-Mediated Transformation of"α-KG→Glutamate"(82274389)by Industry-University Cooperation Project for University in Fujian Province:Preparation of Monomeric Traditional Chinese Medicine Complexes Based on Nampt's Activation of Tricarboxylic Acid Cycle and Respiratory Chain to Interfere with Glycolipid Metabolism(2022Y41010015)。
文摘OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.METHODS:Sprague-Dawley rats with normal glucose levels were randomly divided into three groups,including a conventional diet control group(Group A),high-fat-highsugar diet model group(Group B),and DGR group(Group C,high-fat-high-sugar diet containing 20.5 g DGR).After 10 weeks of intervention,the fasting blood glucose(FBG),2 h blood glucose[PBG;using the oral glucose tolerance test(OGTT)],hemoglobin A1c(HbA1c),plasma total cholesterol(TC),and triglycerides(TG)were tested,and the livers of rats were removed to calculate the liver index.Then,hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry(LC-MS/MS)analysis followed by database search and bioinformatics analysis.Finally,cell experiments were used to verify the results of phosphoproteomics.Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4(MAP4k4)and phosphorylated adducin 1(ADD1)were detected using western blotting.RESULTS:DGR effectively reduced PBG,TG,and the liver index(P<0.05),and significantly decreased HbA1c,TC,and hepatic portal TG(P<0.01),showed significant hematoxylin and eosin(HE)staining,red oil O staining,and Masson staining of liver tissue.The total spectrum was 805334,matched spectrum was 260471,accounting for accounting 32.3%,peptides were 19995,modified peptides were 14671,identified proteins were 4601,quantifiable proteins were 4417,identified sites were 15749,and quantified sites were 14659.Based on the threshold of expression fold change(>1.2),DGR upregulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins,and downregulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins,which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism.Therefore,DGR improved biological tissue processes,including information storage and processing,cellular processes and signaling,and metabolism.The metabolic functions regulated by DGR mainly include energy production and conversion,carbohydrate transport and metabolism,lipid transport and metabolism,inorganic ion transport and metabolism,secondary metabolite biosynthesis,transport,and catabolism.In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.CONCLUSION:DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders,thereby regulating glycolipid metabolism through a multi-target and multi-method process.
文摘Phosphorylation is one of the most common post translational modifications （PTM）, participating in a large number of processes to regulate cellular functions. Phosphorylation is also one of the key factors in the origin and development of cancer. The rapid development of mass spectrometric-based phosphoproteomic technologies has made it possible for high-throughput identification and quantification of phosphorylation events. In this review, we provide a general introduction and summary of the achievements made in mass spectrometry based phosphoproteomic research, including the approaches for phosphopeptide identification and quantification, as well as instrumentation and data interpretation methods. We also review some discoveries in cancer research made possible by phosphoproteomic analysis technologies.
基金supported by the National Natural Science Foundation of China(Grant Nos.81872888,81821005,81673489,and 31871414)the Special Project on Precision Medicine under the National Key R&D Program(Grant No.2017YFC0906600)+2 种基金the Shanghai Science and Technology Development Funds,China(Grant No.19JC1416300)the Key New Drug Creation and Manufacturing Program of China(Grant Nos.2018ZX09711002-004 and 2018ZX09711002-007)the KC Wong Education Foundation。
文摘AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully understood.Herein,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation.Together with these findings,we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradiation(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent manner.Besides,we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPKdeficient cells to IR.Therefore,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.
基金The Foundation of Shanghai Science and Technology Committee(Grant No.17391900600)The Program for Professor of Special Appointment(Eastern Scholar)from The Shanghai Bureau of Higher Education(2011 and 2017)+1 种基金The Natural and Science Foundation of Heilongjiang Provence(Grant No.ZD2019C003)to Shaojun DaiThe Fund of Shanghai Engineering Research Center of Plant Germplasm Resources(Grant No.17DZ2252700)。
文摘Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.
基金supported by the National Natural Science Foundation of China(32161143023,32225041)the earmarked fund for CARS(CARS-03)Science and Technology Assistance Project of Xinjiang Uygur Autonomous Region(2022E02070),and Tang Scholar.
文摘Protein phosphorylation plays an important role in immune signaling transduction in plant resistance to pathogens.Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),severely devastates wheat production.Nonetheless,the molecular mechanism of wheat resistance to stripe rust remains limited.In this study,quantitative phosphoproteomics was employed to investigate the protein phosphorylation changes in wheat challenged by Pst.A total of 1537 and 2470 differentially accumulated phosphoproteins(DAPs)were identified from four early infection stage(6,12,18 and 24 h post-inoculation)in incompatible and compatible wheat-Pst interactions respectively.KEGG analysis revealed that Oxidative Phosphorylation,Phosphatidylinositol Signaling,and MAPK signaling processes are distinctively enriched in incompatible interaction,while Biosynthesis of secondary metabolites and RNA degradation process were significantly enriched in compatible interactions.In particular,abundant changes in phosphorylation levels of chloroplast proteins were identified,suggesting the regulatory role of photosynthesis in wheat-Pst interaction,which is further emphasized by protein-protein interaction(PPI)network analysis.Motif-x analysis identified[xxxxSPxxxx]motif,likely phosphorylation sites for defensive response-related kinases,and a new[xxxxSSxxxx]motif significantly enriched in incompatible interaction.The results shed light on the early phosphorylation events contributing to wheat resistance against Pst.Moreover,our study demonstrated that the phosphorylation levels of Nucleoside diphosphate kinase TaNAPK1 are upregulated at 12 hpi with CYR23 and at 24 hpi with CYR31.Transient silencing of TaNAPK1 was able to attenuate wheat resistance to CYR23 and CYR31.Our study provides new insights into the mechanisms underlying Pst-wheat interactions and may provide database to find potential targets for the development of new resistant varieties.
文摘Background：The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets,we carried out a phosphoproteomics analysis of human endometrium.Methods：A large-scale differential phosphoproteome analysis,using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry,was performed in endometrium tissues from 8 women with or without endometriosis.Results：The phosphorylation profiling of endometrium from endometriosis patients had been obtained,and found that identified 516 proteins were modified at phosphorylation level during endometriosis.Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity.Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways,which might be deregulated during the development of endometriosis.Conclusions：That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.
基金supported by the National Natural Science Foundation of China(no.31672352,31872288).
文摘The most common phenotype induced by the endosymbiont Wolbachia in in-sects is cytoplasmic incompatibility,where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females.This suggests that some modi-fications are induced in host sperms during spermatogenesis by Wolbachia.To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster,we applied isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic strategy combined with titanium dioxide(TiO2)enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male re-productive systems in D.melanogaster.We identified 182 phosphopeptides,defining 140 phosphoproteins,that have at least a 1.2 fold change in abundance with a P-value of<0.05.Most of the differentially abundant phosphoproteins(DAPPs)were associated with micro-tubule cytoskeleton organization and spermatid differentiation.The DAPPs included pro-teins already known to be associated with spermatogenesis,as well as many not previously studied during this process.Six genes coding for DAPPs were knocked down,respectively,in Wolbachia-free fly testes.Among them,Slmap knockdown caused the most severe dam-age in spermatogenesis,with no mature sperm observed in seminal vesicles.Immunoflu-orescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted.These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction.By identifying phospho-modulated proteins we also provide a signifi-cant candidate set for future studies on their roles in spermatogenesis.
基金supported by the National Natural Science Foundation of China(Grant No.32170738)the National Medical Research Council of Singapore(Grant No.NMRC/CBRG/0013/2012).
文摘Gastric cancer(GC)is a prevalent and devastating disease with a poor prognosis.The lack of biomarkers for early detection and effective targeted therapeutics for GC patients represents two major challenges.Through isobaric tags for relative and absolute quantitation(iTRAQ)coupled with liquid chromatography-tandem mass spectrometry(LC-MS/MS)phosphoproteomic analysis of 14 GC and gastric epithelial cell lines,we discovered the discoidin domain receptor tyrosine kinase 1(DDR1)as a top potential drug target out of 40 tyrosine kinases detected along with over 1000 phosphoproteins profiled.The DDR1 protein and mRNA levels were upregulated in GC cells concurrent with DDR1 gene amplification.Immunohistochemistry staining of more than 200 clinical samples revealed that DDR1 was overexpressed in approximately 41%and 48%of the intestinal and diffuse types of GC cases,respectively,compared with only 3.5%in normal tissues.Higher DDR1 expression was associated with poor prognosis.In cellular models,DDR1 overexpression led to accelerated proliferation,invasion,and malignant transformation,putatively via inhibition of the Hippo pathway and consequent activation of YAP-TEAD target gene expression.Notably,DDR1-overexpressing GC cells exhibited high vulnerability to selective DDR1 inhibitors.The present study provides preclinical support for the application of DDR1-selective inhibitors in DDR1-overexpressing GC.
基金This work was supported by the Key Program of the National Natural Science Foundation of China(Grant No.21636003)the Outstanding Youth Foundation of Jiangsu(Grant No.SBK2017010373)+1 种基金the National Key Research and Development Program of China(Grant No.2019YFD1101204)the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.Dong Liu is supported by the Jiangsu Qinglan Talent Program.
文摘Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.
基金supported by the National Natural Science Foundation of China (32170409, 32370430)National Key Research and Development Program of China (2023YFE0109500)。
文摘Plants produce a range of carbohydrates to meet their growth and developmental needs. Protein reversible phosphorylation plays key roles in coordinating multiple metabolic pathways and integrating diverse internal and external cues. Understanding such regulatory metabolism will provide novel resources for breeding and crop management by modulating metabolic pathways for control of growth and stress response. In this review, we summarize the complex, multifaceted functions of protein phosphorylation and their connections to plant metabolism. We focus particularly on carbohydrate metabolic pathways that are controlled by key kinases and discuss how they are linked to downstream changes in physiology, important agronomic traits and crop quality.
文摘Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological activities;and regulates cell proliferation and enlargement,phytohormone biosynthesis and signaling,plant disease resistance,and grain filling and quality during rice seed development.Research work on protein phosphorylation started in the 1950 s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme.Over the last decade,rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues,organs and organelles.The progress made in this field is evident in number of research works.However,research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions.In this review,the general description of protein phosphorylation,including history,structure,frequency of occurrence and function,are discussed.This work also elucidates the different methods for identification,qualification and finally,the progress in rice phosphoproteome research and perspectives.
基金This work was funded by the National Natural Science Foundation of China(31902062)the South China Botanical Garden,Chinese Academy of Sciences(QNXM-02).
文摘Salinity severely affects plant growth and development.Thus,it is crucial to identify the genes functioning in salt stress response and unravel the mechanism by which plants against salt stress.This study used the phosphoproteomic assay and found that 123 of the 4000 quantitative analyzed phosphopeptides were induced by salt stress.The functional annotation of the non-redundant protein database(NR)showed 23 differentially expressed transcription factors,including a phosphopeptide covering the Serine 31 in the RAV(related to ABI3/VP1)transcription factor(named SiRAV1).SiRAV1 was located in the nucleus.Phenotypic and physiological analysis showed that overexpressing SiRAV1 in foxtail millet enhanced salt tolerance and alleviated the salt-induced increases of H_(2)O_(2) accumulation,malondialdehyde(MDA)content,and percent of electrolyte leakage.Further analysis showed that SiRAV1 positively regulated SiCAT expression to modulate the catalase(CAT)activity by directly binding to the SiCAT promoter in vivo and in vitro.Moreover,we found that phosphorylation of SiRAV1 at the Ser31 site positively regulated salt tolerance in foxtail millet via enhancing its binding ability to SiCAT promoter but did not affect its subcellular localization.Overall,our results define a mechanism for SiRAV1 function in salt response where salt-triggered phosphorylation of SiRAV1 at Ser31 enhances its binding ability to SiCAT promoter,and the increased SiCAT expression contributes to salt tolerance in foxtail millet.
基金funded by the National Natural Science Foundation of China,Nos.82171363(to PL),82171321(to XL),82171458(to XJ)the Youth Nova Program of Shaanxi,No.2021KJXX-19(to PL)。
文摘The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment.This cognitive impairment is thought to result specifically from damage to the hippocampus.In this study,we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test.Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury.Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus,as well as in the density of mature dendritic spines.To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage,we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury.The differentially expressed proteins were mainly enriched in inflammation,immunity,and coagulation,suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury.In contrast,differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure,which is more consistent with neurodegeneration.We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury,and western blotting showed that,while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury,its phosphorylation level was significantly increased,which is consistent with the omics results.Administration of GRP78608,an N-methyl-D-aspartate receptor 1 antagonist,to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment.In conclusion,our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.
文摘We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative splicing or alternative start sites to encode more than one RNA/protein.However,specific functions for these different PDEs have not yet been identified in most cell types despite the fact that selective inhibitors to most of the PDE families are available. Conventional approaches to study PDE function typically rely on measurements of global cAMP,or general increases in cAMP-dependent protein kinase A(PKA),or exchange protein activated by cAMP(EPAC) activity.Although newer approaches utilizing subcellularly-targeted FRET reporter sensors have helped to define more compartmentalized regulation of cAMP,PKA,and EPAC,they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem,we have begun to use an unbiased,mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the ″ functional pools″ of cAMP that are regulated by specific cAMP-PDEs(the PDE-regulated phosphoproteomes). In MA-10 Leydig cells we find that in order to detect appreciable increased in either phosphorylation or steroid production,one needs to inhibit both PDE 4 and PDE 8 activity.Using this combination of inhibitors,we find large PDE inhibitor-induced changes in many different proteins that modulate steroid trafficking and biosynthesis. The data are consistent with the idea that cAMP serves to coordinate hormone stimulation of steroid production by altering the phosphorylation of many different proteins at multiple points in the overall pathway rather than just controlling a single rate limiting step. It seems quite likely that many of the proteins phosphorylated in this cell type in response to PDE inhibition,will also be regulated by cAMP in many other cell types.Similarly,in Jurkat cells we find multiple,distinct,PDE regulated phosphoproteomes that differ in response to different PDE inhibitors. Here we also find that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Inhibition of a single PDE produces little effect. Bioinformatics analyses of these phosphoproteomes suggest differing functional roles,mechanisms of action,and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. In this tissue also,the data strongly imply that phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. Overall,the findings illustrate that the approach of using selective,inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.
基金supported by the National Natural Science Foundation of China(32001110)Training Program for Cultivating Highlevel Talents by the China Scholarship Council(2021lxjjw01)Open Project of State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University(2021-KF-004)。
文摘Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.
基金This work was supported by the National Key Research and Development Program of China(2016YFC1101304/3)the National Natural Science Foundation of China(81400589,81790630,and 81790633)Research Unit of Infectious Diseases and Microecology,Chinese Academy of Medical Sciences(2019RU021).
文摘Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cell proteome(WCP)of PHCs have been extensively studied,only a small number of studies have addressed the role of posttranslational modifications in this process.To elucidate the underlying mechanisms that induce dedifferentiation,we carried out parallel quantifications of the transcriptome,WCP,ubiquitinome,and phosphoproteome of rat PHCs after 0,6,12,24,and 48 h of in vitro culture.Our data constitute a detailed proteomic analysis of dedifferentiated PHCs including 2196 proteins,2056 ubiquitinated sites,and 4932 phosphorylated peptides.We revealed a low correlation between the transcriptome and WCP during dedifferentiation.A combined analysis of the ubiquitinome with the corresponding WCP indicated that the dedifferentiation of PHCs led to an increase in nondegradative K27 ubiquitination.Functional analysis of the altered phosphoproteins suggested a significant enrichment in ferroptosis.In all,404 proteins with both ubiquitination and phosphorylation were identified to be involved in critical metabolic events.Furthermore,Ptbph Hnqjd,Hnrnpu,and Srrm2 were identified as hub genes.Taken together,our data provide new insights into proteome dynamics during PHC dedifferentiation and potential targets to inhibit the dedifferentiation process.
基金financially supported by National Key R&D Program of China(No.2018YFA0507501)the National Natural Science Foundation of China(Nos.22074019,21425518 and 22004017)Shanghai Sailing Program(No.20YF1405300)。
文摘Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.
基金the National Key Research and Development Program of China(2017YFD0100103)the Seed Industry Innovation and Industrialization Project of Fujian Province(fjzycxny2017004,zycxny2021004)+1 种基金the Program on Technology of Fujian Province(2020NZ08016,2020N0049)the Open Program of State Key Laboratory of Rice Biology of China(170101)。
文摘The LGS1(Large grain size 1)gene,also known as GS2/GL2/Os GRF4,is involved in regulating grain size and quality in rice,but the mechanism governing grain size has not been elucidated.We performed transcriptomic,proteomic,and phosphoproteomic analyses of young rice panicles in Samba(a wild-type cultivar with extra-small grain)and NIL-LGS1(a nearly isogenic line of LGS1 with large grain in the Samba genetic background)at three developmental stages(4–6)to identify internal dynamic functional networks determining grain size that are mediated by LGS1.Differentially expressed proteins formed seven highly functionally correlated clusters.The concordant regulation of multiple functional clusters may be key features of the development of grain length in rice.In stage 5,16 and 24 phosphorylated proteins were significantly up-regulated and down-regulated,and dynamic phosphorylation events may play accessory roles in determining rice grain size by participating in protein–protein interaction networks.Transcriptomic analysis in stage 5 showed that differentially expressed alternative splicing events and dynamic gene regulatory networks based on 39 transcription factors and their highly correlated target genes might contribute to rice grain development.Integrative multilevel omics analysis suggested that the regulatory network at the transcriptional and posttranscriptional levels could be directly manifested at the translational level,and this analysis also suggested a regulatory mechanism,regulation of protein translation levels,in the biological process that extends from transcript to protein to the development of grain.Functional analysis suggested that biological processes including MAPK signaling,calcium signaling,cell proliferation,cell wall,energy metabolism,hormone pathway,and ubiquitin-proteasome pathway might be involved in LGS1-mediated regulation of grain length.Thus,LGS1-mediated regulation of grain size is affected by dynamic transcriptional,posttranscriptional,translational and posttranslational changes.
基金supported by the National Natural Science Foundation of China(Grant Nos.32341020 and 32341021)the National Key R&D Program of China(Grant Nos.2022YFC2704304 and 2021YFF0702000)+1 种基金the Interdisciplinary Research Program of HUST(Grant Nos.2023JCYJ010 and 2024JCYJ013)the Hubei Province Postdoctoral Outstanding Talent Tracking Support Program,and the Research Core Facilities for Life Science(HUST),China.
文摘As one of the most crucial post-translational modifications,protein phosphorylation regulates a broad range of biological processes in eukaryotes.Biocuration,integration,and annotation of reported phosphorylation events will deliver a valuable resource for the community.Here,we present an updated database,the eukaryotic phosphorylation site database 2.0(EPSD 2.0),which includes 2,769,163 experimentally identified phosphorylation sites(p-sites)in 362,707 phosphoproteins from 223 eukaryotes.From the literature,873,718 new p-sites identified through high-throughput phosphoproteomic research were first collected,and 1,078,888 original phosphopeptides together with primary references were reserved.Then,this dataset was merged into EPSD 1.0,comprising 1,616,804 p-sites within 209,326 proteins across 68 eukaryotic organisms.We also integrated 362,190 additional known p-sites from 10 public databases.After redundancy clearance,we manually re-checked each p-site and annotated 88,074 functional events for 32,762 p-sites,covering 58 types of downstream effects on phosphoproteins,and regulatory impacts on 107 biological processes.In addition,phosphoproteins and p-sites in 8 model organisms were meticulously annotated utilizing information supplied by 100 external platforms encompassing 15 areas.These areas included kinase/phosphatase,transcription regulators,three-dimensional structures,physicochemical characteristics,genomic variations,functional descriptions,protein domains,molecular interactions,drug-target associations,disease-related data,orthologs,transcript expression levels,proteomics,subcellular localization,and regulatory pathways.We expect that EPSD 2.0 will become a useful database supporting comprehensive studies on phosphorylation in eukaryotes.The EPSD 2.0 database is freely accessible online at https://epsd.biocuckoo.cn/.
