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An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

Protein & Cell 2012年第12期3卷 921-928页

作者： Nuoyan zheng xiahe huang Bojiao Yin Dan Wang Qi Xie Department of Nephrology the First Affiliated HospitalSun Yat-Sen UniversityGuangzhou 510000China Research Center of Translational Medicine the First Affiliated HospitalSun Yat-Sen UniversityGuangzhou 510000China State Key Laboratory of Plant Genomics National Centre for Plant Gene ResearchInstitute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China Animal Center Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China

Detection of protein-protein interaction can provide valuable information for investigating the biological function of *** current methods that applied in protein-protein interaction,such as co-immunoprecipitation and pull down etc.,often cause plenty of working time due to the burdensome cloning and puri-fication *** we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within *** *** modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease,two cleavage site F and two multiple cloning sites that flanking cleavage *** target proteins(for example:protein A and protein B)were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His6 *** NIa protease carried out intracellular cleavage along expression,then led to the separation of polyprotein components,therefore,the interaction between protein A-protein B can be detected through one-step purification and *** control for protein B was brought into this system for monitoring interaction *** successfully employed this system to prove two cases of reported protien-protein interaction:RHA2a/ANAC and FTA/*** conclusion,a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

关键词： PPV NIa protease protein-protein interaction in vivo cleavage fusion protein

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mTERF5 Acts as a Transcriptional Pausing Factor to Positively Regulate Transcription of Chloroplast psbEFLJ

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Molecular Plant 2019年第9期12卷 1259-1277页

作者： Shunhua Ding Yi Zhang Zhi Hu xiahe huang Bohan Zhang Qingtao Lu Xiaogang Wen Yingchun Wang Congming Lu Key Laboratory of Photobiology Institute of BotanyChinese Academy of SciencesBeijing 100093China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China State Key Laboratory of Crop Biology College of Life SciencesShandong Agricultural UniversityTaianShandong 271018China

RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans,but it is unknown whether this phenomenon occurs in plant ***,we report that transcriptional pausing occurs in *** found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem *** found that mTERF5 causes the plastid-encoded RNA polymerase(PEP)complex to pause at psbEFLJ by binding to the+30 to+51 region of double-stranded ***,we revealed that mTERF5 interacts with pTAC6,an essential subunit of the PEP complex,although pTAC6 is not involved in the transcriptional pausing at *** showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ,and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ *** together,our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.

关键词： mTERF5 transcriptional pausing transcription chloroplast psbEFLJ

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Non-transcriptional regulatory activity of SMAX1 and SMXL2 mediates karrikin-regulated seedling response to red light in Arabidopsis

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Molecular Plant 2024年第7期17卷 1054-1072页

作者： Wenwen Chang Qiao Qiao Qingtian Li Xin Li Yanyan Li xiahe huang Yingchun Wang Jiayang Li Bing Wang Lei Wang Key Laboratory of Seed Innovation Center for Agricultural Resources ResearchInstitute of Genetics and Developmental BiologyChinese Academy of SciencesShijiazhuangHebei050021China Key Laboratory of Seed Innovation Institute of Genetics and Developmental BiologyThe Innovative Academy for Seed DesignChinese Academy of SciencesBeijing100101China University of Chinese Academy of Sciences Beijing 100049China Yazhouwan National Laboratory SanyaHainan 572024China

Karrikins and strigolactones govern plant development and environmental responses through closely related signaling *** transcriptional repressor proteins SUPPRESSOR OF MAX21(SMAX1),SMAX1-like2(SMXL2),and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or byinhibiting the activities of transcription *** this study,we characterized the non-transcriptional regulatory activities of SMXL proteins in *** discovered that SMAX1 and SMXL2 with mutations in their ethylene-responsefactor-associated amphiphilic repression(EAR)motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 ***1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR4(PIF4)and PIF5 to enhance their protein stability by interacting with phytochrome B(phyB)and suppressing the association of phyB with PIF4 and *** karrikin-responsive genes were then identified by treatment with GR24ent-ssa,GR24 analog showing karrikin ***,INDOLE-3-ACETIC ACID INDUCIBLE 29(IAA29)expression was repressed by GR24^(ent-5D)streatment in a PIF4-and PIF5-dependent and EARindependent manner,whereas KARRIKIN UPREGULATED F-BOX 1(KUF1)expression was induced in a PIF4-and PIF5-independent and EAR-dependent ***,the non-transcriptional regulatory activity of SMAX1,which is independent of the EAR motif,had a global effect on gene *** together,these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.

关键词： karrikin signaling SMAX1 and SMXL2 non-transcriptional repression activity PIF4 and PIF5 gene expression red light

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GreenPhos,a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility

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Molecular Plant 2024年第1期17卷 199-213页

作者： Xiaoxiao Duan Yuanya Zhang xiahe huang Xiao Ma Hui Gao Yan Wang Zhen Xiao Chengcheng huang Zhongshu Wang Bolong Li Wenqiang Yang Yingchun Wang State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyChinese Academy of SciencesBejing 100101China University of Chinese Academy of Sciences Bejing 100049China Key Laboratory of Photobiology Instute of BotanyChinese Academy of SciencesBejing 100093China

Protein phosphorylation regulates a variety of important cellular and physiological processes in ***-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal *** 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 *** 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)*** 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 *** 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 *** 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.

关键词： plant phosphoproteomics phosphopeptides LC–MS Arabidopsis salt stress

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A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions

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Molecular Plant 2020年第8期13卷 1162-1177页

作者： Zechen Bai Jing Zhang Xin Ning Hailong Guo Xiumei Xu xiahe huang Yingchun Wang Zhubing Hu Congming Lu Lixin Zhang Wei Chi Photosynthesis Research Center Key Laboratory of PhotobiologyInstitute of BotanyChinese Academy of SciencesNanxincun 20XiangshanBeijing 100093China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China State Key Laboratory of Crop Stress Adaptation and Improvement School of Life SciencesHenan University85 Minglun StreetKaifeng 475001China State Key Laboratory of Crop Biology College of Life SciencesShandong Agricultural UniversityTaianShandong 271018China The Innovative Academy of Seed Design Chinese Academy of SciencesBeijing 100101China

Adventitious roots form from non-root tissues as part of normal development or in response to stress or *** root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely ***,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis ***4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR *** by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ***3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR *** propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.

关键词： ABI4 MPK3/MPK6 PP2C12 adventitious root root-hypocotyl junction PCD

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OsHYPK-mediated protein N-terminal acetylation coordinates plant development and abiotic stress responses in rice

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Molecular Plant 2022年第4期15卷 740-754页

作者： Xiaodi Gong Yaqian huang Yan Liang Yundong Yuan Yuhao Liu Tongwen Han Shujia Li Hengbin Gao Bo Lv xiahe huang Eric Linster Yingchun Wang Markus Wirtz Yonghong Wang Institute of Genetics and Developmental Biology Chinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Crop Biology College of Life SciencesShandong Agriculture UniversityTaianShandong 271018China State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China Centre for Organismal Studies Heidelberg University69120 HeidelbergGermany

N-terminal acetylation is one of the most common protein modifications in eukaryotes,and approximately 40%of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A(NatA)in a co-translational ***,the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain ***,we identify Huntingtin Yeast partner K(HYPK),an evolutionarily conserved chaperone-like protein,as a positive regulator of NatA activity in *** found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses,attributable to perturbation of the N-terminal acetylome and accelerated global protein ***,we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation,probably through the Ac/N-degron pathway,which could be induced by abiotic *** together,our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover,which are essential for maintaining adaptive phenotypic plasticity in rice.

关键词： rice OsHYPK N-terminal acetylation protein turnover plant architecture stress response

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OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice

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Journal of Genetics and Genomics 2022年第8期49卷 766-775页

作者： Meiru Jia Nan Luo Xiangbing Meng Xiaoguang Song Yanhui Jing Liquan Kou Guifu Liu xiahe huang Yingchun Wang Jiayang Li Bing Wang Hong Yu State Key Laboratory of Plant Genomics and National Center for Plant Gene Research Institute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing 100101China

Salt stress adversely affects plant growth,development,and crop ***(Oryza sativa L.)is one of the most salt-sensitive cereal crops,especially at the early seedling ***-activated protein kinase(MAPK/MPK)cascades have been shown to play critical roles in salt response in ***,the roles of the MPK cascade signaling in rice salt response and substrates of Os MPK remain largely ***,we report that the salt-induced Os MPK4-Ideal Plant Architecture 1(IPA1)signaling pathway regulates the salt tolerance in *** salt stress,Os MPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180,leading to degradation of *** evidence shows that IPA1 is a negative regulator of salt tolerance in rice,whereas Os MPK4 promotes salt response in an IPA1-dependent *** together,our results uncover an Os MPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.

关键词： Oryza sativa MPK OsMPK4 IPA1 Salt tolerance

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Endogenous ceramide phosphoethanolamine modulates circadian rhythm via neural-glial coupling in Drosophila

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National Science Review 2022年第12期9卷 104-123页

作者： Xiupeng Chen Jie Li Zhongbao Gao Yang Yang Wenqing Kuang Yue Dong Gek Huey Chua xiahe huang Binhua Jiang He Tian Yingchun Wang Xun huang Yan Li Sin Man Lam Guanghou Shui State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental Biology Chinese Academy of Sciences University of Chinese Academy of Sciences State Key Laboratory of Brain and Cognitive Science Institute of Biophysics Chinese Academy of Sciences Lipid ALL Technologies Company Limited

While endogenous lipids are known to exhibit rhythmic oscillations,less is known about how specific lipids modulate circadian *** a series of loss-of-function and gain-of-function experiments on cer amide phosphoethanolamine(CPE) synthase of Drosophila,we demonstrated that pan-glial-specific deficiency in membrane CPE,the structural analog of mammalian sphingomyelin(SM),leads to arrhythmic locomotor behavior and shortens lifespan,while the reverse is true for increasing *** proteomics uncovered dysregulated synaptic glutamate utilization and transport in CPE-deficient *** extensive genetic screen was conducted to verify the role of differentially expressed proteins in circadian *** locomotion under cpes1mutant background was rescued only by restoring endogenous CPE or SM through expressing their respective *** results underscore the essential role of CPE in maintaining synaptic glutamate homeostasis and modulating circadian behavior in *** findings suggest that region-specific elevations of functional membrane lipids can benefit circadian regulation.

关键词： ceramide phosphoethanolamine sphingomyelin circadian longevity glutamate homeostasis

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The quantitative proteome atlas of a model cyanobacterium

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Journal of Genetics and Genomics 2022年第2期49卷 96-108页

作者： Jinlong Wang xiahe huang Haitao Ge Yan Wang Weiyang Chen Limin Zheng Chengcheng huang Haomeng Yang Lingyu Li Na Sui Yu Wang Yuanya Zhang Dandan Lu Longfa Fang Wu Xu Yuqiang Jiang Fang huang Yingchun Wang State Key Laboratory of Molecular Developmental Biology The Innovative Academy of Seed DesignInstitute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China School of Life Science Hangzhou Institute for Advanced StudyUniversity of Chinese Academy of SciencesHangzhouZhejiang 310024China University of Chinese Academy of Sciences Beijing 100049China Key Laboratory of Photobiology Institute of BotanyChinese Academy of SciencesBejing 100093China Shandong Provincial Key Laboratory of Plant Stress College of Life ScienceShandong Normal UniversityJinanShandong 250014China State Key Laboratory of Crop Stress Adaptation and Improvement School of Life SciencesHenan UniversityKaifengHenan 475004China Department of Chemistry University of Louisiana at LafayetteLafayetteLA 70504USA

Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority of proteins in any cyanobacteria remain undetermined, representing a major challenge in using cyanobacteria for both basic and industrial researches. Here, using label-free quantitative proteomics, we map 2027 proteins of Synechocystis sp. PCC6803, a model cyanobacterium, to different subcellular compartments and generate a proteome atlas with such information. The atlas leads to numerous unexpected but important findings, including the predominant localization of the histidine kinases Hik33 and Hik27 on the thylakoid but not the plasma membrane. Such information completely changes the concept regarding how the two kinases are activated. Together, the atlas provides subcellular localization information for nearly 60% proteome of a model cyanobacterium, and will serve as an important resource for the cyanobacterial research community.

关键词： Proteome atlas Cyanobacteria Proteomics Membranes Synechocystis

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An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro

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Molecular Plant 2022年第8期15卷 1285-1299页

作者： Huihui Liu Simiao Liu Hong Yu xiahe huang Yingchun Wang Liang Jiang Xiangbing Meng Guifu Liu Mingjiang Chen Yanhui Jing Feifei Yui Bing Wang Jiayang Li State Key Laboratory of Plant Genomics and National Center for Plant Gene Research(Beijing) Institute of Genetics and Developmental BiologyThe Innovative Academy for Seed DesignChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in *** E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ***,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 *** this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in *** further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone ***,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 *** work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

关键词： rice SCF E3 ligase ubiquitination strigolactones DWARF53 Sic1

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