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 inv展开更多
Over the past decade,systems biology and plant-omics have increasingly become the main stream in plant biology research.New developments in mass spectrometry and bioinformatics tools,and methodological schema to inte-...Over the past decade,systems biology and plant-omics have increasingly become the main stream in plant biology research.New developments in mass spectrometry and bioinformatics tools,and methodological schema to inte-grate multi-omics data have leveraged recent advances in proteomics and metabolomics.These progresses are driv-ing a rapid evolution in the field of plant research,greatly facilitating our understanding of the mechanistic aspects of plant metabolisms and the interactions of plants with their external environment.Here,we review the recent progresses in MS-based proteomics and metabolomics tools and workflows with a special focus on their applications to plant biology research using several case studies related to mechanistic understanding of stress response,gene/protein function characterization,metabolic and signaling pathways exploration,and natural product discovery.We also present a projection concerning future perspectives in MS-based proteomics and metabolomics development including their applications to and challenges for system biology.This review is intended to provide readers with an overview of how advanced MS technology,and integrated application of proteomics and metabolomics can be used to advance plant system biology research.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
Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identi...Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.展开更多
Increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacte...Increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. Recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaus- tive site-specific bacterial phosphoproteomes. The high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen-host interactions; the high abundance of multi-phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions.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
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 inv
基金This research was supported by the Key Realm R&D Program of Guangdong Province(No.2020B0202090005)the Science and Technology Program of Guangdong Province(2021A0505030050)+2 种基金the Project of Collaborative Innovation Center of Guangdong Academy of Agricultural Sciences(XTXM202203)the Special Fund for Scientific Innovation Strategy-construction of High-Level Academy of Agriculture Science(No.R2020PY-JX019,R2021YJ-QG004)two USDA grants(No.8062-21000-046-00D and No.8062-21000-047-00D)。
文摘Over the past decade,systems biology and plant-omics have increasingly become the main stream in plant biology research.New developments in mass spectrometry and bioinformatics tools,and methodological schema to inte-grate multi-omics data have leveraged recent advances in proteomics and metabolomics.These progresses are driv-ing a rapid evolution in the field of plant research,greatly facilitating our understanding of the mechanistic aspects of plant metabolisms and the interactions of plants with their external environment.Here,we review the recent progresses in MS-based proteomics and metabolomics tools and workflows with a special focus on their applications to plant biology research using several case studies related to mechanistic understanding of stress response,gene/protein function characterization,metabolic and signaling pathways exploration,and natural product discovery.We also present a projection concerning future perspectives in MS-based proteomics and metabolomics development including their applications to and challenges for system biology.This review is intended to provide readers with an overview of how advanced MS technology,and integrated application of proteomics and metabolomics can be used to advance plant system biology research.
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(31620103917 31970459 32070441 31702054 and 31930014)the Shenzhen Science and Technology Program(20180411143628272)the Natural Science Foundation of Guangdong Province(2019A1515011899)。
文摘Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.
基金supported by the National Natural Science Foundation of China(Grant No.20801061)Guangdong Natural Science Foundation(Grant No.8451027501001233)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars,Ministry of Educationthe Fundamental Research Funds for the Central Universities(Grant No.10lgpy19)
文摘Increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. Recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaus- tive site-specific bacterial phosphoproteomes. The high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen-host interactions; the high abundance of multi-phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions.
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.
