Junping Fan, Linghan Hu, Zongwei Yue, Daohong Liao, Fusheng Guo, Han Ke, Daohua Jiang, Yong Yang & Xiaoguang Lei Nature Chemical Biology (2023) https://doi.org/10.1038/s41589-022-01166-5
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Publication Date:October 20, 2022 https://doi.org/10.1021/acs.analchem.2c02116 Subcellular protein−protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical crosslinking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.
Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BAinteracting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the twocomponent systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.
The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process.
Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart.
Jianyong Du, Lixia Zheng,Peng Gao, Hang Yang, Wan-Jie Yang, Fusheng Guo,Ruqi Liang,Mengying Feng, Zihao Wang,Zongwang Zhang, Linlu Bai,Ye Bu, Shijia Xing, Wen Zheng, Xuelian Wang, Li Quan, Xinli Hu, Haosen Wu,Zhixing Chen, Liangyi Chen, Ke Wei, Zhe Zhang, Xiaojun Zhu, Xiaolin Zhang, Qiang Tu, Shi-Min Zhao,* Xiaoguang Lei,* and Jing-Wei Xiong.*
Cell Stem Cell 2022, 29, 545.
Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart
Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao* Angew. Chem. Int. Ed. 2022, 61, e202201908.
Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.
Zongwei Yue, Fan Wu, Fusheng Guo, Jiyeong Park, Jin Wang, Liyun Zhang, Daohong Liao, Wenyang Li, Orlando D Schärer, Xiaoguang Lei
National Science Review, nwac046, https://doi.org/10.1093/nsr/nwac046 Published: 11 March 2022 To maintain genomic integrity and avoid diseases, the DNA damage response (DDR) not only detects and repairs DNA lesions, but also contributes to the resistance to DNA-damaging chemotherapeutics. Targeting the DDR plays a significant role in drug discovery using the principle of synthetic lethality. The incomplete current knowledge of the DDR encouraged us to develop new strategies to identify and study its components and pathways. Polycarcin V, belonging to the C-aryl glycoside natural products, is a light-activatable DNA intercalating agent which causes DNA damage by forming a covalent [2+2] cycloadduct with thymine residue under 365–450 nm light irradiation in a DNA sequence independent manner. Taking advantage of the light-activatable feature and temporal control of DDR, we designed and synthesized polycarcin V-based bifunctional chemical probes, including one that crosslinks DNA to DNA-binding protein to explore the DDR induced by polycarcin V and uncover novel DNA-protein interactions. Utilizing this chemical probe and ABPP-SILAC, we identified 311 DNA-binding proteins, including known DDR factors and additional proteins that may be of interest in discovering new biology. We validated our approach by showing that our probe could specifically crosslink proteins involved in nucleotide excision repair (NER) that repair bulky DNA adducts. Our studies showed that the [2+2] cycloadduct formed by polycarcin V could indeed be repaired by NER in vivo. As a DNA damaging agent, polycarcin V or its drug-like derivative plus blue light showed promising properties for psoriasis treatment, suggesting that it may itself hold promise for clinic applications.
Plant metabolites are dynamically modified and distributed in response to environmental changes. However, it is poorly understood how metabolic change functions in plant stress responses. Maintaining ion homeostasis under salt stress requires coordinated activation of two types of central regulators: plasma membrane (PM) H+-ATPase and Na+/H+ antiporter. In this study, we used a bioassay-guided isolation approach to identify endogenous small molecules that affect PM H+-ATPase and Na+/H+ antiporter activities and identified phosphatidylinositol (PI), which inhibits PM H+-ATPase activity under non-stress conditions in Arabidopsis by directly binding to the C terminus of the PM H+-ATPase AHA2. Under salt stress, the phosphatidylinositol 4-phosphate-to-phosphatidylinositol (PI4P-to-PI) ratio increased, and PI4P bound and activated the PM Na+/H+ antiporter. PI prefers binding to the inactive form of PM H+-ATPase, while PI4P tends to bind to the active form of the Na+/H+ antiporter. Consistent with this, pis1 mutants, with reduced levels of PI, displayed increased PM H+-ATPase activity and salt stress tolerance, while the pi4kβ1 mutant, with reduced levels of PI4P, displayed reduced PM Na+/H+ antiporter activity and salt stress tolerance. Collectively, our results reveal that the dynamic change between PI and PI4P in response to salt stress in Arabidopsis is crucial for maintaining ion homeostasis to protect plants from unfavorable environmental conditions
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Structural basis of TRPV3 inhibition by an antagonist
Junping Fan, Linghan Hu, Zongwei Yue, Daohong Liao, Fusheng Guo, Han Ke, Daohua Jiang, Yong Yang & Xiaoguang Lei
Nature Chemical Biology (2023) https://doi.org/10.1038/s41589-022-01166-5
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Subcellular Interactomes Revealed by Merging APEX with Cross- Linking Mass Spectrometry
Mengze Sun, Feng Yuan, Yuliang Tang, Peng Zou,* and Xiaoguang Lei*
Anal. Chem. 2022, 94, 14878-14888.
Publication Date:October 20, 2022
https://doi.org/10.1021/acs.analchem.2c02116
Subcellular protein−protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical crosslinking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.
Chemoproteomic Profiling Reveals the Mechanism of Bile Acid Tolerance in Bacteria
Biwei Liu, Shentian Zhuang, Runze Tian, Yuan Liu, Yanqi Wang, Xiaoguang Lei,* and Chu Wang* ACS Chemical Biology Article ASAP
DOI: 10.1021/acschembio.2c00286
Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BAinteracting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the twocomponent systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic
experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.
Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry
Tiantian Wei, Jue Wang, Ruqi Liang, Wendong Chen, Yilan Chen, Mingzhe Ma, An He, Yifei Du, Wenjing Zhou, Zhiying Zhang, Xin Zeng, Chu Wang, Jin Lu, Xing Guo, Xiao-Wei Chen, Youjun Wang, Ruijun Tian, Junyu Xiao, Xiaoguang Lei
eLife 2022, 11 :e77696. https://doi.org/10.7554/eLife.77696
The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process.
Hesperadin Is a Novel CaMKII-δ Inhibitor Hesperadin Exerts Dual Functions to Ameliorate Cardiac Ischemia/Reperfusion Injury and Inhibit Tumor Growth
Junxia Zhang; Ruqi Liang; Kai Wang; Wenjia Zhang; Mao Zhang; Li Jin; Peng Xie; Wen Zheng; Haibao Shang; Qingmei Hu; Jiayi Li; Gengjia Chen; Fujian Wu; Feng Lan; Lipeng Wang; Shi-Qiang Wang; Yongfeng Li; Yong Zhang; Jinghao Liu; Fengxiang Lv; Xinli Hu; Rui-Ping Xiao; Xiaoguang Lei; Yan Zhang.
Circulation 2022, 145, 1154-1168
Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart.
A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration
Jianyong Du, Lixia Zheng,Peng Gao, Hang Yang, Wan-Jie Yang, Fusheng Guo,Ruqi Liang,Mengying Feng, Zihao Wang,Zongwang Zhang, Linlu Bai,Ye Bu, Shijia Xing, Wen Zheng, Xuelian Wang, Li Quan, Xinli Hu, Haosen Wu,Zhixing Chen, Liangyi Chen, Ke Wei, Zhe Zhang, Xiaojun Zhu, Xiaolin Zhang, Qiang Tu, Shi-Min Zhao,* Xiaoguang Lei,* and Jing-Wei Xiong.*
Cell Stem Cell 2022, 29, 545.
Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart
Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering
Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao*
Angew. Chem. Int. Ed. 2022, 61, e202201908.
Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.
Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers
Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers，
Jian-Hua Wang ， Yu-Liang Tang, Zhou Gong, Rohit Jain , Fan Xiao, Yu Zhou , Dan Tan,Qiang Li, Niu Huang , Shu-Qun Liu, Keqiong Ye, Chun Tang *, Meng-Qiu Dong * &Xiaoguang Lei *
Nature Communications, 2022, 13(1), 1468
Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2. Cross-linking of proteins with DOPA2 is 60–120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links show better agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages when working at low pH, low temperature, or in the presence of denaturants. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of progressively denatured forms of these proteins. Furthermore, DOPA2 cross-linking allows time-course analysis of protein conformational changes during denaturant-induced unfolding. © 2022, The Author(s).
Polycarcin V induces DNA damage response and enables the profiling of DNA-binding proteins
Zongwei Yue, Fan Wu, Fusheng Guo, Jiyeong Park, Jin Wang, Liyun Zhang, Daohong Liao, Wenyang Li, Orlando D Schärer, Xiaoguang Lei
National Science Review, nwac046, https://doi.org/10.1093/nsr/nwac046
Published:
11 March 2022
To maintain genomic integrity and avoid diseases, the DNA damage response (DDR) not only detects and repairs DNA lesions, but also contributes to the resistance to DNA-damaging chemotherapeutics. Targeting the DDR plays a significant role in drug discovery using the principle of synthetic lethality. The incomplete current knowledge of the DDR encouraged us to develop new strategies to identify and study its components and pathways. Polycarcin V, belonging to the C-aryl glycoside natural products, is a light-activatable DNA intercalating agent which causes DNA damage by forming a covalent [2+2] cycloadduct with thymine residue under 365–450 nm light irradiation in a DNA sequence independent manner. Taking advantage of the light-activatable feature and temporal control of DDR, we designed and synthesized polycarcin V-based bifunctional chemical probes, including one that crosslinks DNA to DNA-binding protein to explore the DDR induced by polycarcin V and uncover novel DNA-protein interactions. Utilizing this chemical probe and ABPP-SILAC, we identified 311 DNA-binding proteins, including known DDR factors and additional proteins that may be of interest in discovering new biology. We validated our approach by showing that our probe could specifically crosslink proteins involved in nucleotide excision repair (NER) that repair bulky DNA adducts. Our studies showed that the [2+2] cycloadduct formed by polycarcin V could indeed be repaired by NER in vivo. As a DNA damaging agent, polycarcin V or its drug-like derivative plus blue light showed promising properties for psoriasis treatment, suggesting that it may itself hold promise for clinic applications.
Dynamic changes of phosphatidylinositol and phosphatidylinositol 4-phosphate levels modulate H+-ATPase and Na+/H+ antiporter activities to maintain ion homeostasis in Arabidopsis under salt stress
Molecular Plant, 2021, 14(12), pp. 2000–2014
Yongqing Yang, Xiuli Han, Liang Ma, Yujiao Wu, Xiao Liu, Haiqi Fu, Guoyong Liu,
Xiaoguang Lei*, Yan Guo*
Plant metabolites are dynamically modified and distributed in response to environmental changes. However, it is poorly understood how metabolic change functions in plant stress responses. Maintaining ion homeostasis under salt stress requires coordinated activation of two types of central regulators: plasma membrane (PM) H+-ATPase and Na+/H+ antiporter. In this study, we used a bioassay-guided isolation approach to identify endogenous small molecules that affect PM H+-ATPase and Na+/H+ antiporter activities and identified phosphatidylinositol (PI), which inhibits PM H+-ATPase activity under non-stress conditions in Arabidopsis by directly binding to the C terminus of the PM H+-ATPase AHA2. Under salt stress, the phosphatidylinositol 4-phosphate-to-phosphatidylinositol (PI4P-to-PI) ratio increased, and PI4P bound and activated the PM Na+/H+ antiporter. PI prefers binding to the inactive form of PM H+-ATPase, while PI4P tends to bind to the active form of the Na+/H+ antiporter. Consistent with this, pis1 mutants, with reduced levels of PI, displayed increased PM H+-ATPase activity and salt stress tolerance, while the pi4kβ1 mutant, with reduced levels of PI4P, displayed reduced PM Na+/H+ antiporter activity and salt stress tolerance. Collectively, our results reveal that the dynamic change between PI and PI4P in response to salt stress in Arabidopsis is crucial for maintaining ion homeostasis to protect plants from unfavorable environmental conditions