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
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
Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphopro...Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.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
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
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
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
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
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
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
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
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
In a recent article published in Cell,Kjaergaard et al.show that the skeletal muscle proteome and phosphoproteome are associated with whole-body insulin sensitivity,and identify selective insulin resistance(IR)signatu...In a recent article published in Cell,Kjaergaard et al.show that the skeletal muscle proteome and phosphoproteome are associated with whole-body insulin sensitivity,and identify selective insulin resistance(IR)signatures in individuals with and without type 2 diabetes(T2D).1 Their elegant work provides compelling evidence for more personalised therapeutic strategies to treat T2D.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
1.INTRODUCTION Erythropoiesis,a major branch of hematopoiesis,is responsible for producing specialized red blood cells(RBCs)that play a vital role in systemic oxygen transport.1 A defining feature of mammalian termina...1.INTRODUCTION Erythropoiesis,a major branch of hematopoiesis,is responsible for producing specialized red blood cells(RBCs)that play a vital role in systemic oxygen transport.1 A defining feature of mammalian terminal erythropoiesis is the expulsion of the highly condensed nucleus from the cytoplasm during the final stage of differentiation.2 This process,termed enucleation,not only creates additional cytoplasmic space for hemoglobin synthesis but also provides RBCs with the flexibility required to traverse narrow capillaries.3 Following enucleation,reticulocytes undergo further maturation through sequential membrane remodeling,proteomic reprogramming,and organelle clearance.展开更多
De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light.During this process light,an important environmental signal,triggers the inhibition of mesocoty...De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light.During this process light,an important environmental signal,triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts,and etiolated leaves transition from the"sink"stage to the"source"stage.De-etiolation has been extensively studied in maize(Zea mays L.).However,little is known about how this transition is regulated.In this study,we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize.We identified 16,420 proteins in proteome,among which 14,168 proteins were quantified.In addition,8746 phosphorylation sites within 3110 proteins were identified.From the combined proteomic and phosphoproteomic data,we identified a total of 17,436 proteins.Only 7.0%(998/14,168)of proteins significantly changed in abundance during de-etiolation.In contrast,26.6%of phosphorylated proteins exhibited significant changes in phosphorylation level;these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions.Based on phosphoproteomic analysis,34.0%(1057/3110)of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites,and 37 proteins contained more than 16 phosphorylation sites,indicating that multi-phosphorylation is ubiquitous during the de-etiolation process.Our results suggest that plants might preferentially regulate the level of posttranslational modifications(PTMs)rather than protein abundance for adapting to changing environments.The study of PTMs could thus better reveal the regulation of de-etiolation.展开更多
Brassinosteroid(BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3 s are the main negative regulator kinases. BRs have been sh...Brassinosteroid(BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3 s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1(pag1), and its corresponding wild-type(ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage(10 days after pollination(DPA)) and secondary cell wall synthesis stage(20DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs(GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor GhDNFYB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.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
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support from the Ministry of Science and Technology of the People's Republic of China(2019YFA0707100,2019YFA0802203)Strategic Priority Research Program of Chinese Academy of Sciences(XDA24040202)National Key Research and Development Program of China(2022YFF1001704)。
文摘Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.
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.
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.
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.
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.
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supported by the Medical Research Council(MR/W01937X/1).
文摘In a recent article published in Cell,Kjaergaard et al.show that the skeletal muscle proteome and phosphoproteome are associated with whole-body insulin sensitivity,and identify selective insulin resistance(IR)signatures in individuals with and without type 2 diabetes(T2D).1 Their elegant work provides compelling evidence for more personalised therapeutic strategies to treat T2D.
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/.
基金supported by the National Natural Science Foundation of China(82270127 to J.L.)the Hunan Provincial Natural Science Foundation of China(no.2024JJ3037 to J.L.,and 2025JJ60692 to Y.P.)+2 种基金the Postdoctoral Fellowship Program of CPSF(GZC20233178 to Y.P.)the Hunan Province Clinical Medical Technology Innovation Guidance Project(2021SK50917 to L.Z.)the Innovation Platform and Talent Plan Project of Hunan Provincial Department of Science and Technology(2023SK4056 to L.Z.).
文摘1.INTRODUCTION Erythropoiesis,a major branch of hematopoiesis,is responsible for producing specialized red blood cells(RBCs)that play a vital role in systemic oxygen transport.1 A defining feature of mammalian terminal erythropoiesis is the expulsion of the highly condensed nucleus from the cytoplasm during the final stage of differentiation.2 This process,termed enucleation,not only creates additional cytoplasmic space for hemoglobin synthesis but also provides RBCs with the flexibility required to traverse narrow capillaries.3 Following enucleation,reticulocytes undergo further maturation through sequential membrane remodeling,proteomic reprogramming,and organelle clearance.
基金supported by the National Key R&D Program of China(Grant No.2016YFD0101003)the Heilongjiang Provincial Outstanding Youth Science Foundation,China(Grant No.JC2017008)
文摘De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light.During this process light,an important environmental signal,triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts,and etiolated leaves transition from the"sink"stage to the"source"stage.De-etiolation has been extensively studied in maize(Zea mays L.).However,little is known about how this transition is regulated.In this study,we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize.We identified 16,420 proteins in proteome,among which 14,168 proteins were quantified.In addition,8746 phosphorylation sites within 3110 proteins were identified.From the combined proteomic and phosphoproteomic data,we identified a total of 17,436 proteins.Only 7.0%(998/14,168)of proteins significantly changed in abundance during de-etiolation.In contrast,26.6%of phosphorylated proteins exhibited significant changes in phosphorylation level;these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions.Based on phosphoproteomic analysis,34.0%(1057/3110)of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites,and 37 proteins contained more than 16 phosphorylation sites,indicating that multi-phosphorylation is ubiquitous during the de-etiolation process.Our results suggest that plants might preferentially regulate the level of posttranslational modifications(PTMs)rather than protein abundance for adapting to changing environments.The study of PTMs could thus better reveal the regulation of de-etiolation.
基金supported by the National Natural Science Foundation of China(31971987 and 31601067)。
文摘Brassinosteroid(BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3 s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1(pag1), and its corresponding wild-type(ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage(10 days after pollination(DPA)) and secondary cell wall synthesis stage(20DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs(GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor GhDNFYB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.
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.
