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phosphoproteomics Reveals the AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

Genomics, Proteomics & Bioinformatics 2022年第4期20卷 597-613页

作者： Yuejing Jiang Xiaoji Cong Shangwen Jiang Ying Dong Lei Zhao Yi Zang Minjia Tan Jia Li National Center for Drug Screening State Key Laboratory of Drug ResearchShanghai Institute of Materia MedicaChinese Academy of SciencesShanghai 201203China Chemical Proteomics Center State Key Laboratory of Drug ResearchShanghai Institute of Materia MedicaChinese Academy of SciencesShanghai 201203China University of Chinese Academy of Sciences Beijing 100049China Open Studio for Druggability Research of Marine Natural Products Pilot National Laboratory for Marine Science and Technology(Qingdao)Qingdao 266237China

AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various *** studies have demonstrated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully ***,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone *** with these findings,we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradiation(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent ***,we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPKdeficient cells to ***,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.

关键词： AMPK phosphoproteomics Histone modification DNA damage Apoptosis

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Na_(2)CO_(3)-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by phosphoproteomics

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Genomics, Proteomics & Bioinformatics 2020年第3期18卷 271-288页

作者： Jinwei Suo Heng Zhang Qi Zhao Nan Zhang Yongxue Zhang Ying Li Baohua Song Juanjuan Yu Jianguo Cao Tai Wang Ji Luo Lihai Guo Jun Ma Xumin Zhang Yimin She Lianwei Peng Weimin Ma Siyi Guo Yuchen Miao Sixue Chen Zhi Qin Shaojun Dai Development Center of Plant Germplasm Resources College of Life and Environmental SciencesShanghai Normal UniversityShanghai 200234China Alkali Soil Natural Environmental Science Center Northeast Forestry UniversityKey Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil FieldMinistry of EducationHarbin 150040China State Key Laboratory of Subtropical Silviculture Zhejiang A&F UniversityHangzhou 311300China Institute of Botany Chinese Academy of SciencesBeijing 100093China AB Sciex Asia Pacific Application Support Center Shanghai 200233China Shanghai Center for Plant Stress Biology Chinese Academy of SciencesShanghai 201602China School of Life Sciences Fudan UniversityShanghai 200433China Institute of Plant Stress Biology State Key Laboratory of Cotton BiologyDepartment of BiologyHenan UniversityKaifeng 475004China Department of Biology Genetics InstitutePlant Molecular and Cellular Biology ProgramInterdisciplinary Center for Biotechnology ResearchUniversity of FloridaGainesvilleFL 32610USA

Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to *** understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were *** addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,*** addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis *** PCC 6803,leading to enhanced Na_(2)CO_(3)*** these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.

关键词： Chloroplasts Na_(2)CO_(3)stress ROS scavenging phosphoproteomics Puccinellia tenuiflora

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Effects of calcium application on apple fruit softening during storage revealed by proteomics and phosphoproteomics

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Horticultural Plant Journal 2022年第4期8卷 408-422页

作者： Yaxiu Xu Jiaojiao Liu Nannan Zang Zepeng Yin Aide Wang Key Laboratory of Fruit Postharvest Biology Key Laboratory of Protected HorticultureNational and Local Joint Engineering Research Center of Northern Horticultural Facilities Design and Application TechnologyCollege of HorticultureShenyang Agricultural UniversityShenyang 110866 China

Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely *** 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 *** results indicated that Ca inhibited the abundance of polygalacturonase(PG)activity and increased the phosphorylation level of *** and phosphorylation were involved in cell wall degradation,thereby delaying fruit *** a secondary messenger,Ca-mediated signaling subsequently triggered downstream mitogen-activated protein kinases(MAPK)cascades and activated the membrane,transport,and ROS ***,MdEIN2,a key enzyme involved in the ubiquitin of protein modification,increased at Ser753 and Ser758 in Ca-treated ***,diverse primary and secondary metabolisms including glycolysis,fatty acid metabolism,and oxidation respiratory chain were modulated to prevent fruit *** results provide basic information from protein and phosphorylation levels for apple fruit ripening during storage,which may be helpful for apple fruit storage control.

关键词： Apple Proteomics phosphoproteomics Calcium Fruit ripening

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Mass spectrometry-based phosphoproteomics in cancer research

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Frontiers in Biology 2012年第6期7卷 566-586页

作者： Xiao-Shan YUE Amanda B. HUMMON Department of Chemistry and Biochemistry University of Notre Dame Notre Dame IN 46556 USA

Phosphorylation is one of the most common post translational modifications （PTM）, participating in a large number of processes to regulate cellular functions. Phosphorylation is also one of the key factors in the origin and development of cancer. The rapid development of mass spectrometric-based phosphoproteomic technologies has made it possible for high-throughput identification and quantification of phosphorylation events. In this review, we provide a general introduction and summary of the achievements made in mass spectrometry based phosphoproteomic research, including the approaches for phosphopeptide identification and quantification, as well as instrumentation and data interpretation methods. We also review some discoveries in cancer research made possible by phosphoproteomic analysis technologies.

关键词： phosphoproteomics mass spectrometry cancer research phosphopeptide enrichment quantitative phosphoproteomics data interpretation

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Mechanisms of Dangua Fang(丹瓜方)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics

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Journal of Traditional Chinese Medicine 2024年第2期44卷 334-344页

作者： HENG Xianpei WANG Zhita LI Liang YANG Liuqing HUANG Suping JIN Lang HE Weidong Department of Endocrinology People's Hospital Affiliated to Fujian University of Traditional Chinese MedicineFuzhou 350004China Academy of Integrative Medicine Fujian University of Traditional Chinese MedicineFuzhou 350122China Faculty of Humanities and Management Fujian University of Traditional Chinese MedicineFuzhou 350122China Department of Geriatrics People's Hospital Affiliated to Fujian University of Traditional Chinese MedicineFuzhou 350004China

OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on ***: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 ***,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 ***,cell experiments were used to verify the results of *** mitogen-activated protein kinase kinase kinase kinase 4(MAP4k4)and phosphorylated adducin 1(ADD1)were detected using western ***:DGR effectively reduced PBG,TG,and the liver index(P1.2),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 includ

关键词： phosphoproteomics glycolipid metabolism therapeutic mechanism compound formula Dangua Fang

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The divide and conquer strategies for deep phosphoproteomics analysis

The divide and conquer strategies for deep phosphoproteomics...

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2018年中国质谱学术大会（CMSC2018）

作者： Mingliang Ye CAS Key Laboratory of Separation Sciences for Analytical Chemistry National Chromatographic R&A CenterDalian Institute of Chemical PhysicsChinese Academy of Sciences (CAS)

With the advance of mass spectrometry based proteomics techniques, thousands of phosphorylation sites can be identified from a single cell line or a single tissue sample by large scale global analysis. However, such large scale analysis is time consuming. And, due to the high complexity of the phosphoproteome sample, this approach is often unable to identify low abundance regulatory phosphorylation sites. Alternatively, the "divide and conquer" strategy by targeted analysis of a subset of phosphoproteome, i.e. subphosphoproteome, can improve the detection sensitivity of these phosphorylation sites. Recently, we have developed several subphosphoproteomics analysis methods. We develop a negative enrichment strategy, in which a high pH SAX was applied to deplete high abundant acidic phosphopeptides from phosphopeptide mixture, to improved the coverage for the detection of basophilic kinase substrates. To improve the the identification of phosphopeptides with unknown cleavage specificity, a de novo assisted database search strategy was developed, which has higher speed and higher sensitivity. More importantly, to improve the identification of low abundant tyrosine phosphosites, an SH2 domain-derived pTyrsuperbinder was used as the affinity *** superbinder-based AP-MS/MS approach led to the identification of 19,570 pTyr peptides and 10,030 unique pTyr sites from nine human cell lines, both of which are unprecedented.

关键词： phosphoproteomics tyrosine phosphorylation endogenous phosphopeptides

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Quantitative phosphoproteomics Analysis of the Insulin/IGF-1 Signaling Mutants in C. elegans

Quantitative Phosphoproteomics Analysis of the Insulin/IGF-1...

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2018年中国质谱学术大会（CMSC2018）

作者： Wen-Jun Li Li Tao Yong-Hong Yan Chen-Wei Wang Ze-Xian Liu Yu-Xin Li Mei-Jun Zhang Xue-Mei Li Yu Xue Meng-Qiu Dong National Institute of Biological Sciences School of Life Science Peking University Collage of Life Science & Technology HUST

It is well established that the C. elegans insulin/IGF-1 receptor tyrosine kinase DAF-2 acts though a highly conserved kinase cascade consisting of AGE-1(PI 3-kinase), PDK-1, AKT-1, and AKT-2 to inhibit the FoxO transcription factor DAF-16, thereby maintaining a relatively short, wild-type lifespan(1). Surprisingly, little is known about the protein phosphorylation events in this pathway in C. elegans. To fill this void, we carried out quantitative phosphoproteomics analyses(2) of synchronized wild-type, daf-2(e1370), daf-16(mu86), and daf-2(e1370);daf-16(mu86) worms on adult day 1, each in three or four independent experiments. This led to the largest C. elegans phosphoproteomics datasets to date, with high-confidence identification of 15,443 phosphosites from 22,536 unique phosphopeptides. As expected, mutation in the daf-2 gene decreased phosphorylation of the conserved AKT sites on DAF-16(3). Interestingly, we found that phosphorylation of a newly identified site on AKT-1, which turned out to activate DAF-16, was higher in the daf-2 mutant, so it suggested the existence of a feedback mechanism that tries to compensate the reduction of daf-2 activity. We also found that the daf-2 mutation may slow down protein synthesis by modulating the phosphorylation levels of translation initiation factors. We verified that phosphorylation of eIF2α at S49 indeed increased in the long-lived daf-2 mutant and this change contributed to daf-2 longevity. The phosphoproteomics data also led to the discoveries that inhibition of CK2 and inhibition of CDK-1 phosphorylation at a conserved residue T179 can lengthen C. elegans lifespan. In sum, this study uncovered new signaling branches downstream of the insulin receptor DAF-2 and shed light on the mechanistic details underlying the extraordinary longevity of the daf-2 mutant. In this talk, I will share our experience in handling various technical difficulties that may be common to quantitative phosphoproteomics analyses.

关键词： phosphoproteomics proteomics quantitation insulin signaling phosphorylation

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基于数据独立采集的磷酸化蛋白质组学全面揭示I型酪蛋白激酶在植物发育中的作用

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Science Bulletin 2023年第18期68卷 2077-2093,M0004页

作者：渠莉刘默洋郑玲丽王旭薛红卫 Shanghai Collaborative Innovation Center of Agri-Seeds Joint Center for Single Cell BiologySchool of Agriculture and BiologyShanghai Jiao Tong UniversityShanghai 200240China

I型酪蛋白激酶(CKI)是一类真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶,通过介导底物的磷酸化调节生长发育和信号转导:拟南芥蛋白AELs,是植物特异的CK1,具有多种功能,但其内源底物的准确识别和验证极大地限制了对CK1生理功能的系统阐明... 详细信息

I型酪蛋白激酶(CKI)是一类真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶,通过介导底物的磷酸化调节生长发育和信号转导:拟南芥蛋白AELs,是植物特异的CK1,具有多种功能,但其内源底物的准确识别和验证极大地限制了对CK1生理功能的系统阐明.本研究通过数据独立采集模式(DIA)开展的磷酸化蛋白质组学研究系统揭示了CKI的内源底物:从AELs过表达及多种突变体的幼苗中提取蛋白并进行质谱检测,与野生型比较分析获取了3985个在AELs过表达材料中丰度高于野生型(Col-0)以及在缺失突变体中丰度低于Col-0的磷酸化肽段(AEL依赖的磷酸化肽段).对应获得1032个磷酸化蛋白。进一步从磷酸化肽段中富集了8个底物识别基序并对其中4个新基序进行了实验验证,用于预测CK1的潜在候选底物,基于富集到的一个参与生殖发育的锌指转录因子,C3H17,我们通过生化和遗传分析进行了底物验证和功能鉴定,证明AEL通过介导C3H17磷酸化以增强蛋白稳定性和转录活性进而调控拟南芥的胚胎发育,基于CK1在真核生物中的高度保守性,进一步利用CK1的底物识别基序搜索了水稻、小鼠和人类的蛋白数据库,获得了远超已知数目的众多候选底物,极大地扩展了对CK1在动植物中发挥的共同和独特功能的理解,为跨物种全面揭示CK1的保守功能提供了重要线索.

关键词： Casein kinase 1(CK1) Arabidopsis EL1-like(AEL) Data-independent acquisition(DIA) phosphoproteomics C3H17 Embryo development

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Identification of a sensor histidine kinase (BfcK) controlling biofilm formation in Clostridium acetobutylicum

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Chinese Journal of Chemical Engineering 2022年第6期35卷 84-93页

作者： Dong Liu Shikai Ge Zhenyu Wang Mengting Li Wei Zhuang Pengpeng Yang Yong Chen Hanjie Ying State Key Laboratory of Materials-Oriented Chemical Engineering College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjing 211816China School of Chemical Engineering and Energy Zhengzhou UniversityZhengzhou 450001China

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the ***,genetic control of *** biofilm has not been dissected so ***,to identify potential genes controlling *** 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 *** of these disruptants formed robust biofilm still,or suffered both growth and biofilm *** a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent *** analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein *** bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm *** on these findings,possible bfcK-mediated mechanisms for biofilm formation were *** is a big step toward understanding the biofilm formation in *** and will help further engineering of its biofilm-based industrial processes.

关键词： Clostridium acetobutylicum Biofilm Histidine kinases CA_C2730 phosphoproteomics Flagellar motility

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Reversible protein phosphorylation, a central signaling hub to regulate carbohydrate metabolic networks

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The Crop Journal 2023年第5期11卷 1279-1286页

作者： Fengyi Gao Liang Zhang James RLloyd Wenbin Zhou Yanmei Chen State Key Laboratory of Plant Environmental Resilience College of Biological SciencesChina Agricultural UniversityBeijing 100193China College of Biology and Food Shangqiu Normal UniversityShangqiu 476000HenanChina College of Life Science Henan Normal UniversityXinxiang 453007HenanChina Department of Genetics Institute for Plant BiotechnologyStellenbosch University7600 StellenboschSouth Africa Institute of Crop Sciences Chinese Academy of Agricultural SciencesBeijing 100081China

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.

关键词： Crop plants Metabolomics SnRK kinase TOR kinase Transcription factor phosphoproteomics

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