Suppressor of G2 allele of skp1(SGT1)is a highly conserved eukaryotic protein that plays a vital role in growth,development,and immunity in both animals and plants.Although some SGT1 interactors have been identified,t...Suppressor of G2 allele of skp1(SGT1)is a highly conserved eukaryotic protein that plays a vital role in growth,development,and immunity in both animals and plants.Although some SGT1 interactors have been identified,the molecular regulatory network of SGT1 remains unclear.SGT1 serves as a co-chaperone to stabilize protein complexes such as the nucleotide-binding leucine-rich repeat(NLR)class of immune receptors,thereby positively regulating plant immunity.SGT1 has also been found to be asso-ciated with the SKP1-Cullin-F-box(SCF)E3 ubiquitin ligase complex.However,whether SGT1 targets im-mune repressors to coordinate plant immune activation remains elusive.In this study,we constructed a toolbox for TurbolD-and split-TurbolD-based proximity labeling(PL)assays in Nicotiana benthamiana and used the PL toolbox to explore the SGT1 interactome during pre-and post-immune activation.The comprehensive SGT1 interactome network we identified highlights a dynamic shift from proteins associ-ated with plant development to those linked with plant immune responses.We found that SGT1 interacts with Necrotic Spotted Lesion1(NSL1),which negatively regulates salicylic acid-mediated defenseby inter-fering with the nucleocytoplasmic trafficking of non-expressor of pathogenesis-related genes 1(NPR1)during N NLR-mediated response to tobacco mosaic virus.SGT1 promotes the SCF-dependent degrada-tion of NSL1 to facilitate immune activation,while salicylate-induced protein kinase-mediated phosphory-lation of SGT1further potentiates this process.Besides NNLR,NSL1also functions in several other NLR-mediated immunity.Collectively,our study unveils the regulatory landscape of SGT1 and reveals a novel SGT1-NSL1 signaling module that orchestrates plant innate immunity.展开更多
As a cornerstone of the central dogma of molecular biology,RNA plays vital roles in living organisms.Over the past few decades,many RNA labeling technologies have been developed to elucidate the biological function of...As a cornerstone of the central dogma of molecular biology,RNA plays vital roles in living organisms.Over the past few decades,many RNA labeling technologies have been developed to elucidate the biological function of RNA.These technologies have signifi-cantly advanced our understanding of RNA secondary structure,localization,and turnover.Additionally,taking advantage of these innovative RNA labeling approaches,plenty of tool kits have been devised for the regulation of RNA-related biological process,such as gene expression and gene editing.In this review,we primarily focus on an array of intracellular RNA labeling methods,encom-passing chemical probes-based labeling,metabolic labeling,and proximity-dependent labeling.We also provide a brief overview of their applications in the research of RNA biology.Finally,the perspectives of RNA labeling are also discussed.展开更多
Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and...Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.展开更多
The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the de...The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the development of the neuron synapse. Hence, research on its protein interactome is key to understanding its functionality and specific properties. A newly discovered biotin ligase, TurboID, is a proximity labeler that is designed to be able to label and observe transmembrane proteins, something that previous methods struggled with. The TurboID method is verified in HEK293T cells and primary cultured mouse cortical neurons. Results have proven the validity of the TurboID method in observing PCDH9-interacting proteins.展开更多
Protein–protein interaction(PPI)networks are key to nearly all aspects of cellular activity.Therefore,the identification of PPIs is important for understanding a specific biological process in an organism.Compared wi...Protein–protein interaction(PPI)networks are key to nearly all aspects of cellular activity.Therefore,the identification of PPIs is important for understanding a specific biological process in an organism.Compared with conventional methods for probing PPIs,the recently described proximity labeling(PL)approach combined with mass spectrometry(MS)-based quantitative proteomics hasemerged as apowerful approach for characterizing PPIs.However,the application of PL in planta remains in its infancy.Here,we summarize recent progress in PL and its potential utilization in plant biology.We specifically summarize advances in PL,including the development and comparison of different PL enzymes and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems.展开更多
Protein-biomolecule interactions play pivotal roles in almost all biological processes.For a biomolecule of interest,the identification of the interacting protein(s)is essential.For this need,although many assays are ...Protein-biomolecule interactions play pivotal roles in almost all biological processes.For a biomolecule of interest,the identification of the interacting protein(s)is essential.For this need,although many assays are available,highly robust and reliable methods are always desired.By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin(SA)-biotin system,we developed the Specific Pupylation as IDEntity Reporter(SPIDER)method for identifying protein-biomolecule interactions.Using SPIDER,we validated the interactions between the known binding proteins of protein,DNA,RNA,and small molecule.We successfully applied SPIDER to construct the global protein interactome for m^(6)A and m RNA,identified a variety of uncharacterized m^(6)A binding proteins,and validated SRSF7 as a potential m^(6)A reader.We globally identified the binding proteins for lenalidomide and Cob B.Moreover,we identified SARS-CoV-2-specific receptors on the cell membrane.Overall,SPIDER is powerful and highly accessible for the study of proteinbiomolecule interactions.展开更多
Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions.However,current methods depend on the expres...Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions.However,current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins.To address this limitation,we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2(pA-APEX2) labeling(AMAPEX).In this method,a modified protein is bound in situ by a specific antibody,which then tethers a pA-APEX2 fusion protein.Activation of APEX2 labels the nearby proteins with biotin;the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry.We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3 K27 me3,H3 K9 me3,H3 K4 me3,H4 K5 ac,and H4 K12 ac,as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.展开更多
基金supported by grants from the National Natural Science Foundation of China(32320103003 and 32122070)the“High-end Foreign Experts Recruitment Plan”of Ministry of Science and Technology(G2023108007L)+3 种基金Chinese Universities Scientific Fund(2023TC074)Pinduoduo-China Agricultural University Research Fund(PC2023B02012)China National Postdoctoral Program for Innovative Talents(BX20240421)2115 Talent Development Program of China Agricultural University.
文摘Suppressor of G2 allele of skp1(SGT1)is a highly conserved eukaryotic protein that plays a vital role in growth,development,and immunity in both animals and plants.Although some SGT1 interactors have been identified,the molecular regulatory network of SGT1 remains unclear.SGT1 serves as a co-chaperone to stabilize protein complexes such as the nucleotide-binding leucine-rich repeat(NLR)class of immune receptors,thereby positively regulating plant immunity.SGT1 has also been found to be asso-ciated with the SKP1-Cullin-F-box(SCF)E3 ubiquitin ligase complex.However,whether SGT1 targets im-mune repressors to coordinate plant immune activation remains elusive.In this study,we constructed a toolbox for TurbolD-and split-TurbolD-based proximity labeling(PL)assays in Nicotiana benthamiana and used the PL toolbox to explore the SGT1 interactome during pre-and post-immune activation.The comprehensive SGT1 interactome network we identified highlights a dynamic shift from proteins associ-ated with plant development to those linked with plant immune responses.We found that SGT1 interacts with Necrotic Spotted Lesion1(NSL1),which negatively regulates salicylic acid-mediated defenseby inter-fering with the nucleocytoplasmic trafficking of non-expressor of pathogenesis-related genes 1(NPR1)during N NLR-mediated response to tobacco mosaic virus.SGT1 promotes the SCF-dependent degrada-tion of NSL1 to facilitate immune activation,while salicylate-induced protein kinase-mediated phosphory-lation of SGT1further potentiates this process.Besides NNLR,NSL1also functions in several other NLR-mediated immunity.Collectively,our study unveils the regulatory landscape of SGT1 and reveals a novel SGT1-NSL1 signaling module that orchestrates plant innate immunity.
基金supported by grants from the National Natural Science Foundation of China (92253202 and 22177087 to X.W.)the Ministry of Science and Technology (2023YFC3402200)the Fundamental Research Funds for the Central Universities (2042023kfyq05).
文摘As a cornerstone of the central dogma of molecular biology,RNA plays vital roles in living organisms.Over the past few decades,many RNA labeling technologies have been developed to elucidate the biological function of RNA.These technologies have signifi-cantly advanced our understanding of RNA secondary structure,localization,and turnover.Additionally,taking advantage of these innovative RNA labeling approaches,plenty of tool kits have been devised for the regulation of RNA-related biological process,such as gene expression and gene editing.In this review,we primarily focus on an array of intracellular RNA labeling methods,encom-passing chemical probes-based labeling,metabolic labeling,and proximity-dependent labeling.We also provide a brief overview of their applications in the research of RNA biology.Finally,the perspectives of RNA labeling are also discussed.
基金supported by Natural Science Foundation of Beijing Municipality(L212013)National Key Research and Development Program of China(No.2022YFA1206104)+2 种基金AI+Health Collaborative Innovation Cultivation Project(Z211100003521002)National Natural Science Foundation of China(81971718,82073786,81872809,U20A20412,81821004)Beijing Natural Science Foundation(7222020).
文摘Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.
文摘The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the development of the neuron synapse. Hence, research on its protein interactome is key to understanding its functionality and specific properties. A newly discovered biotin ligase, TurboID, is a proximity labeler that is designed to be able to label and observe transmembrane proteins, something that previous methods struggled with. The TurboID method is verified in HEK293T cells and primary cultured mouse cortical neurons. Results have proven the validity of the TurboID method in observing PCDH9-interacting proteins.
基金supported by grants from the National Natural Science Foundation of China(31872637 to Y.Z.and 31830106 to D.L.)NSF-IOS-1354434+1 种基金NSF-IOS-1339185NIH-GM132582 to S.P.D.-K.
文摘Protein–protein interaction(PPI)networks are key to nearly all aspects of cellular activity.Therefore,the identification of PPIs is important for understanding a specific biological process in an organism.Compared with conventional methods for probing PPIs,the recently described proximity labeling(PL)approach combined with mass spectrometry(MS)-based quantitative proteomics hasemerged as apowerful approach for characterizing PPIs.However,the application of PL in planta remains in its infancy.Here,we summarize recent progress in PL and its potential utilization in plant biology.We specifically summarize advances in PL,including the development and comparison of different PL enzymes and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems.
基金supported by the National Key Research and Development Program of China(2020YFE0202200)the National Natural Science Foundation of China(31900112,21907065,31970130 and 31670831)。
文摘Protein-biomolecule interactions play pivotal roles in almost all biological processes.For a biomolecule of interest,the identification of the interacting protein(s)is essential.For this need,although many assays are available,highly robust and reliable methods are always desired.By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin(SA)-biotin system,we developed the Specific Pupylation as IDEntity Reporter(SPIDER)method for identifying protein-biomolecule interactions.Using SPIDER,we validated the interactions between the known binding proteins of protein,DNA,RNA,and small molecule.We successfully applied SPIDER to construct the global protein interactome for m^(6)A and m RNA,identified a variety of uncharacterized m^(6)A binding proteins,and validated SRSF7 as a potential m^(6)A reader.We globally identified the binding proteins for lenalidomide and Cob B.Moreover,we identified SARS-CoV-2-specific receptors on the cell membrane.Overall,SPIDER is powerful and highly accessible for the study of proteinbiomolecule interactions.
基金supported by the National Key R&D Program of China(Grant No.2019YFA0903803)the Major Program of National Natural Science Foundation of China(Grant No.32090031)+10 种基金the General Program of National Natural Science Foundation of China(Grant Nos.31971354 and 32070610)the National Natural Science Foundation of China for Young Scholars(Grant No.32000580)the Guangdong Province Fund for Distinguished Young Scholars,China(Grant No.2021B1515020109)the Key Project from Natural Science Foundation of Guangdong Province,China(Grant No.2020B1515120034)the Guangdong Provincial Key Laboratory of Synthetic Genomics,China(Grant No.2019B030301006)the Shenzhen Key Laboratory of Synthetic Genomics,China(Grant No.ZDSYS201802061806209)the Project from Shenzhen Science and Technology Innovation Committee,China(Grant No.JCYJ20170818164014753)the Mayo Clinic Cancer Center Eagles Cancer Fund awarded to ZWthe Mayo Clinic Cancer Center Hematologic Malignancies Program awarded to ZWthe Mayo Clinic division of Hematology awarded to ZWthe Mayo Clinic Center for Biomedical Discovery awarded to SMO,United States。
文摘Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions.However,current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins.To address this limitation,we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2(pA-APEX2) labeling(AMAPEX).In this method,a modified protein is bound in situ by a specific antibody,which then tethers a pA-APEX2 fusion protein.Activation of APEX2 labels the nearby proteins with biotin;the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry.We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3 K27 me3,H3 K9 me3,H3 K4 me3,H4 K5 ac,and H4 K12 ac,as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
