Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome rema...Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome remains challenging owing to the scarcity of specific labeling methods.This work develops an aggregation-induced emission(AIE)luminogen-mediated photocatalytic proximity labeling strategy that allows profiling of the GA-associated secretome with high spatiotemporal precision.Leveraging an AIE luminogen-derived GA-targeting photocatalyst,this strategy achieves efficient labeling of proteins in minutes within the Golgi lumen upon light activation,which enables spatiotemporally resolved modification of histidine and tyrosine residues.We succeed in profiling secretome in both living HeLa cells and hard-to-transfect macrophage HMC3 cells,and a significant subset of GA-associated secretome with 80%specificity is determined,linking the distinct GA-associated secretory profiles to cellular characteristics.The method is further applied to proteome mapping of brain and bone metastatic lung cancer cells,which reveals the underlying roles the GA-associated secretome plays in extracellular matrix organization during metastasis.This work delivers a robust tool to break the dilemma of chemical labeling of GA-associated secretome in living cells,and provides mechanistic insights into secretion regulation at the subcellular level.展开更多
Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(R...Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(Reiter and Leroux,2017;Wallmeier et al.,2020).To date,over 180 ciliopathy-associated genes have been identified(Reiter and Leroux,2017),yet the underlying mechanisms remain poorly understood.展开更多
Acute lung injury(ALI)is a serious clinical condition with a high mortality rate.Oxidative stress and inflammatory responses play pivotal roles in the pathogenesis of ALI.ONOO^(−)is a key mediator that exacerbates oxi...Acute lung injury(ALI)is a serious clinical condition with a high mortality rate.Oxidative stress and inflammatory responses play pivotal roles in the pathogenesis of ALI.ONOO^(−)is a key mediator that exacerbates oxidative damage and microvascular permeability in ALI.Accurate detection of ONOO^(−)would facilitate early diagnosis and intervention in ALI.Near-infrared fluorescence(NIRF)probes offer new solutions due to their sensitivity,depth of tissue penetration,and imaging capabilities.However,the developed ONOO^(−)fluorescent probes face problems such as interference from other reactive oxygen species and easy intracellular diffusion.To address these issues,we introduced an innovative self-immobilizing NIRF probe,DCI2F-OTf,which was capable of monitoring ONOO^(−)in vitro and in vivo.Importantly,leveraging the high reactivity of the methylene quinone(QM)intermediate,DCI2F-OTf was able to covalently label proteins in the presence of ONOO^(−),enabling in situ imaging.In mice models of ALI,DCI2F-OTf enabled real-time imaging of ONOO^(−)levels and found that ONOO^(−)was tightly correlated with the progression of ALI.Our findings demonstrated that DCI2F-OTf was a promising chemical tool for the detection of ONOO^(−),which could help to gain insight into the pathogenesis of ALI and monitor treatment efficacy.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
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
Germ granules are biomolecular condensates composed of RNA and proteins that play crucial roles in RNA metabolism and post-transcriptional gene regulation.C.elegans germ granules consist of multiple distinct subcompar...Germ granules are biomolecular condensates composed of RNA and proteins that play crucial roles in RNA metabolism and post-transcriptional gene regulation.C.elegans germ granules consist of multiple distinct subcompartments,including P granules,Mutator foci,Z granules,SIMR foci,P-bodies,D granules,and E granules.Among these condensates,the E granule,which is nonrandomly positioned within the germ granule,is required for the production of a specialized class of small interfering RNAs(siRNAs).However,the mechanisms underlying E granule formation and its functional significance remain largely unexplored.In this study,via the use of TurboID-based proximity labeling technology combined with an RNAi-based reverse genetic screen,we identified two novel components of the E granule,EGC-2/C27B7.5 and EGC-3/F59G1.8,which initiate E granule assembly.The depletion of EGC-2 or EGC-3 disrupts the perinuclear localization of the EGO and PICS complexes,both of which are enriched in E granules and are required for E-class siRNA and piRNA biogenesis,respectively.Small RNAomic analyses revealed that both EGC-2 and EGC-3 promote the production of 5′E-class siRNA,whereas piRNA accumulation is inhibited by EGC-3.Taken together,our results elucidate the roles of EGC-2 and EGC-3 in maintaining E granule integrity and small RNA homeostasis.Additionally,the combination of proximity labeling technology and reverse genetic screening provides a robust strategy for studying the assembly of biomolecular condensates.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
Spatiotemporal profiling of nuclear-associated proteomes is crucial for elucidating disease mechanisms,identifying key therapeutic targets,and guiding the design of effective drugs.Currently,proximity labeling(PL)usin...Spatiotemporal profiling of nuclear-associated proteomes is crucial for elucidating disease mechanisms,identifying key therapeutic targets,and guiding the design of effective drugs.Currently,proximity labeling(PL)using genetically transfected enzymes or photocatalyst-based probes has emerged as a powerful tool for proteomic mapping.However,these approaches are limited by their incompatibility with hard-to-transfect cells and primary tissues,as well as by the lack of efficient nucleus-targeting strategies.In this study,we developed a photocatalytic PL strategy(Pc-PL)that enables efficient enrichment of nuclear-associated proteins by combining a nucleus-targeted photosensitizer(NCP)with photocatalysis-mediated reactive biotin labeling.Compared with traditional photocatalysts such as chlorin e6 and rose Bengal,NCP exhibited superior nuclear accumulation across various cell types.Cellular experiments confirmed that NCP-mediated photoactivation precisely localized biotin labeling within the nucleus,enabling selective enrichment of nuclear proteins via subsequent streptavidin-based magnetic capture.Coupling Pc-PL with quantitative mass spectrometry enabled highresolution mapping of nuclear proteomes and led to the discovery of previously unrecognized senescence-associated regulators,including TMPO.Collectively,these findings establish Pc-PL as an innovative and versatile tool for highresolution nuclear proteomics,offering broad potential for target discovery and drug development.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
Enzyme-and catalyst-generated reactive species have been leveraged in the past decade to covalently label biomolecules within a short range of a defined site or space inside cells or at the cell–cell interface.Due to...Enzyme-and catalyst-generated reactive species have been leveraged in the past decade to covalently label biomolecules within a short range of a defined site or space inside cells or at the cell–cell interface.Due to their high spatial resolution,such proximity labeling strategies have been coupled with various bioanalytical techniques for dissecting dynamic and complex biological processes.Here,we review the development of enzyme-and catalyst-triggered proximity chemistry and their applications to identifying protein interaction networks as well as cell–cell communications in living systems.展开更多
Efficient and selective functionalization of peptides and proteins is essential for advancing chemical biology and therapeutic applications.In this study,we present alkynyl aldehyde as a novel thiol-specific reagent t...Efficient and selective functionalization of peptides and proteins is essential for advancing chemical biology and therapeutic applications.In this study,we present alkynyl aldehyde as a novel thiol-specific reagent that enables precise and chemoselective targeting of cysteine(Cys)residues.This approach operates in aqueous buffers under mild conditions and is broadly compatible with diverse biomolecules,ranging from small peptides to large proteins,without compromising their structural integrity or function.The incorporation of a versatile aldehyde group within the conjugates opens avenues for further functionalization,including hydrazone and oxime formation,carbon-carbon bond formation,and site-specific biotinylation.Notably,this aldehyde also facilitates proximity-driven conjugation with amine groups,enabling the formation of cyclic biomolecules.Furthermore,the method achieves remarkable stability of the conjugates under biologically relevant conditions.By providing a robust and versatile platform for protein and peptide modification,this study significantly expands the bioconjugation toolkit,offering promising applications in drug discovery,biomaterials development,and therapeutic innovation.展开更多
The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,th...The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,the role of PGAM5 in ulcerative colitis and the mechanisms underlying PGAM5 regulating NLRP3 activity remain unknown.Here,we show that PGAM5 deficiency ameliorates dextran sodium sulfate(DSS)-induced colitis in mice via suppressing NLRP3 inflammasome activation.By combining APEX2-based proximity labeling focused on PGAM5 with quantitative proteomics,we identify NEK7 as the new binding partner of PGAM5 to promote NLRP3 inflammasome assembly and activation in a PGAM5 phosphatase activity-independent manner upon inflammasome induction.Interfering with PGAM5eNEK7 interaction by punicalagin inhibits the activation of the NLRP3 inflammasome in macrophages and ameliorates DSS-induced colitis in mice.Altogether,our data demonstrate the PGAM5eNEK7 interaction in macrophages for NLRP3 inflammasome activation and further provide a promising therapeutic strategy for ulcerative colitis by blocking the PGAM5eNEK7 interaction.展开更多
Over 40 amyloidogenic proteins have been identified to cause amyloidosis diseases in clinics.Tissue deposition of amyloid proteins entangled with interacting partners is a characteristic pathological hallmark of amylo...Over 40 amyloidogenic proteins have been identified to cause amyloidosis diseases in clinics.Tissue deposition of amyloid proteins entangled with interacting partners is a characteristic pathological hallmark of amyloidosis diseases.However,the proteomic complexity of co-aggregated amyloid deposits poses a clinical challenge to diagnose the exact disease-causing pathogenic proteins in patients’biopsied tissue.Herein,we present a photocatalytic proteomic method,named Amyloid-ID,as a promising approach to identify the composition of amyloid deposits for clinical proteotyping of amyloidosis diseases.Amyloid-ID is enabled by a photosensitized probe analogous to a pan-amyloid sensor,Thioflavin T.We show this probe photocatalyzes protein labeling via reactive oxygen species and demonstrate its applicability in both AD mouse models and human laryngeal samples.Next,we exemplify its utility by proteotyping the pathogenic protein underlying the rare laryngeal amyloidosis(LA).Using patients’biopsied tissue sections,we label,enrich,and profile the amyloid deposits.Proteomics analysis top-ranks fibrinogen as a potential pathogenic protein.Biochemical and biophysical characterizations confirm that fibrinogen can aggregate into amyloid fibrils.Intriguingly,we observe that fibrinogen’s fibrillation is sensitive to mechanical forces,particularly impacted by sonication.Such observation coincides with its primary larynx deposition,where frequent vocal cord friction occurs.Overall,given the photocatalytic properties,our Amyloid-ID serves as a promising clinical proteotyping method for amyloidosis diseases.展开更多
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
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
基金support from the National Key R&D Program of China(2023YFA0913902)the National Natural Science Foundation of China(32088101,22074140,and 22474136)the Dalian Institute of Chemical Physics,Chinese Academy of Sciences(DICP-I202316).
文摘Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome remains challenging owing to the scarcity of specific labeling methods.This work develops an aggregation-induced emission(AIE)luminogen-mediated photocatalytic proximity labeling strategy that allows profiling of the GA-associated secretome with high spatiotemporal precision.Leveraging an AIE luminogen-derived GA-targeting photocatalyst,this strategy achieves efficient labeling of proteins in minutes within the Golgi lumen upon light activation,which enables spatiotemporally resolved modification of histidine and tyrosine residues.We succeed in profiling secretome in both living HeLa cells and hard-to-transfect macrophage HMC3 cells,and a significant subset of GA-associated secretome with 80%specificity is determined,linking the distinct GA-associated secretory profiles to cellular characteristics.The method is further applied to proteome mapping of brain and bone metastatic lung cancer cells,which reveals the underlying roles the GA-associated secretome plays in extracellular matrix organization during metastasis.This work delivers a robust tool to break the dilemma of chemical labeling of GA-associated secretome in living cells,and provides mechanistic insights into secretion regulation at the subcellular level.
基金supported by grants from the National Key Research and Development Program of China(2019YFA0802704)the National Natural Science Foundation of China(31771620)+2 种基金the Natural Science Foundation of Chongqing,China(CSTB2022NSCQMSX1424)Research Startup Fund of Southwest University(SWU117064)Open Research Fund of National Health Commission Key Laboratory of Birth Defects Prevention&Henan Key Laboratory of Population Defects Prevention(ZD202302)。
文摘Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(Reiter and Leroux,2017;Wallmeier et al.,2020).To date,over 180 ciliopathy-associated genes have been identified(Reiter and Leroux,2017),yet the underlying mechanisms remain poorly understood.
基金supported by the National Natural Science Foundation of China(Nos.22264013,21961010)Hainan Province Science and Technology Special Fund(Nos.ZDYF2021SHFZ219,ZDYF2022SHFZ037)+4 种基金Special Funds of S&T Cooperation and Exchange Projects of Shanxi Province(No.202204041101040)Natural Science Research Talent Project of Hainan Medical University(No.JBGS202101)Postgraduate Innovative Research Project of Hainan(No.Qhys2021-384)Hainan Province Clinical Medical Center(2021)Project for Functional Materials and Molecular Imaging Science Innovation Group of Hainan Medical University.
文摘Acute lung injury(ALI)is a serious clinical condition with a high mortality rate.Oxidative stress and inflammatory responses play pivotal roles in the pathogenesis of ALI.ONOO^(−)is a key mediator that exacerbates oxidative damage and microvascular permeability in ALI.Accurate detection of ONOO^(−)would facilitate early diagnosis and intervention in ALI.Near-infrared fluorescence(NIRF)probes offer new solutions due to their sensitivity,depth of tissue penetration,and imaging capabilities.However,the developed ONOO^(−)fluorescent probes face problems such as interference from other reactive oxygen species and easy intracellular diffusion.To address these issues,we introduced an innovative self-immobilizing NIRF probe,DCI2F-OTf,which was capable of monitoring ONOO^(−)in vitro and in vivo.Importantly,leveraging the high reactivity of the methylene quinone(QM)intermediate,DCI2F-OTf was able to covalently label proteins in the presence of ONOO^(−),enabling in situ imaging.In mice models of ALI,DCI2F-OTf enabled real-time imaging of ONOO^(−)levels and found that ONOO^(−)was tightly correlated with the progression of ALI.Our findings demonstrated that DCI2F-OTf was a promising chemical tool for the detection of ONOO^(−),which could help to gain insight into the pathogenesis of ALI and monitor treatment efficacy.
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 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.
基金supported by the National Key Research and Development Program of China(2022YFA1302700)the National Natural Science Foundation of China(32230016,32270583,32470633,32400435,2023M733425,32300438)+2 种基金the Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM(QYPY20230021)the Fundamental Research Funds for the Central UniversitiesSome strains were provided by the CGC,which is funded by the NIH Office of Research Infrastructure Programs(P40 OD010440).
文摘Germ granules are biomolecular condensates composed of RNA and proteins that play crucial roles in RNA metabolism and post-transcriptional gene regulation.C.elegans germ granules consist of multiple distinct subcompartments,including P granules,Mutator foci,Z granules,SIMR foci,P-bodies,D granules,and E granules.Among these condensates,the E granule,which is nonrandomly positioned within the germ granule,is required for the production of a specialized class of small interfering RNAs(siRNAs).However,the mechanisms underlying E granule formation and its functional significance remain largely unexplored.In this study,via the use of TurboID-based proximity labeling technology combined with an RNAi-based reverse genetic screen,we identified two novel components of the E granule,EGC-2/C27B7.5 and EGC-3/F59G1.8,which initiate E granule assembly.The depletion of EGC-2 or EGC-3 disrupts the perinuclear localization of the EGO and PICS complexes,both of which are enriched in E granules and are required for E-class siRNA and piRNA biogenesis,respectively.Small RNAomic analyses revealed that both EGC-2 and EGC-3 promote the production of 5′E-class siRNA,whereas piRNA accumulation is inhibited by EGC-3.Taken together,our results elucidate the roles of EGC-2 and EGC-3 in maintaining E granule integrity and small RNA homeostasis.Additionally,the combination of proximity labeling technology and reverse genetic screening provides a robust strategy for studying the assembly of biomolecular condensates.
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financial support of the National Natural Science Foundation of China(32501182)the Guangdong Provincial Department of Education Natural Science Research Project(2025ZDZX4030).
文摘Spatiotemporal profiling of nuclear-associated proteomes is crucial for elucidating disease mechanisms,identifying key therapeutic targets,and guiding the design of effective drugs.Currently,proximity labeling(PL)using genetically transfected enzymes or photocatalyst-based probes has emerged as a powerful tool for proteomic mapping.However,these approaches are limited by their incompatibility with hard-to-transfect cells and primary tissues,as well as by the lack of efficient nucleus-targeting strategies.In this study,we developed a photocatalytic PL strategy(Pc-PL)that enables efficient enrichment of nuclear-associated proteins by combining a nucleus-targeted photosensitizer(NCP)with photocatalysis-mediated reactive biotin labeling.Compared with traditional photocatalysts such as chlorin e6 and rose Bengal,NCP exhibited superior nuclear accumulation across various cell types.Cellular experiments confirmed that NCP-mediated photoactivation precisely localized biotin labeling within the nucleus,enabling selective enrichment of nuclear proteins via subsequent streptavidin-based magnetic capture.Coupling Pc-PL with quantitative mass spectrometry enabled highresolution mapping of nuclear proteomes and led to the discovery of previously unrecognized senescence-associated regulators,including TMPO.Collectively,these findings establish Pc-PL as an innovative and versatile tool for highresolution nuclear proteomics,offering broad potential for target discovery and drug development.
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.
基金funding from the National Natural Science Foundation of China(grant nos.21937001,22222701,22137001,91957101,and 22077004)the Ministry of Science and Technology of China(grant nos.2022YFA1304700,2019YFA0904201,and 2021YFA1302603)Beijing Natural Science Foundation(grant no.Z200010).
文摘Enzyme-and catalyst-generated reactive species have been leveraged in the past decade to covalently label biomolecules within a short range of a defined site or space inside cells or at the cell–cell interface.Due to their high spatial resolution,such proximity labeling strategies have been coupled with various bioanalytical techniques for dissecting dynamic and complex biological processes.Here,we review the development of enzyme-and catalyst-triggered proximity chemistry and their applications to identifying protein interaction networks as well as cell–cell communications in living systems.
基金the financial support from the Distinguished University Professor grant(Nanyang Technological University),and the Agency for Science,Technology and Research(A*STAR)under its Manufacturing,Trade and Connectivity(MTC)Individual Research Grant(M21K2c0114)RIE2025 MTC Programmatic Fund(M22K9b0049).
文摘Efficient and selective functionalization of peptides and proteins is essential for advancing chemical biology and therapeutic applications.In this study,we present alkynyl aldehyde as a novel thiol-specific reagent that enables precise and chemoselective targeting of cysteine(Cys)residues.This approach operates in aqueous buffers under mild conditions and is broadly compatible with diverse biomolecules,ranging from small peptides to large proteins,without compromising their structural integrity or function.The incorporation of a versatile aldehyde group within the conjugates opens avenues for further functionalization,including hydrazone and oxime formation,carbon-carbon bond formation,and site-specific biotinylation.Notably,this aldehyde also facilitates proximity-driven conjugation with amine groups,enabling the formation of cyclic biomolecules.Furthermore,the method achieves remarkable stability of the conjugates under biologically relevant conditions.By providing a robust and versatile platform for protein and peptide modification,this study significantly expands the bioconjugation toolkit,offering promising applications in drug discovery,biomaterials development,and therapeutic innovation.
基金supported by the National Natural Science Foundation of China(82174010 and 81973512)the Open Fund of the State Key Laboratory of Pharmaceutical Biotechnology,Nanjing University(Grant No.KF-202206,China)the Double First-Class Project of China Pharmaceutical University(CPUQNJC22_02,China)。
文摘The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,the role of PGAM5 in ulcerative colitis and the mechanisms underlying PGAM5 regulating NLRP3 activity remain unknown.Here,we show that PGAM5 deficiency ameliorates dextran sodium sulfate(DSS)-induced colitis in mice via suppressing NLRP3 inflammasome activation.By combining APEX2-based proximity labeling focused on PGAM5 with quantitative proteomics,we identify NEK7 as the new binding partner of PGAM5 to promote NLRP3 inflammasome assembly and activation in a PGAM5 phosphatase activity-independent manner upon inflammasome induction.Interfering with PGAM5eNEK7 interaction by punicalagin inhibits the activation of the NLRP3 inflammasome in macrophages and ameliorates DSS-induced colitis in mice.Altogether,our data demonstrate the PGAM5eNEK7 interaction in macrophages for NLRP3 inflammasome activation and further provide a promising therapeutic strategy for ulcerative colitis by blocking the PGAM5eNEK7 interaction.
基金supported,in part,by funds from National Natural Science Foundation of China(22222410,22374148,22477102,22207106)Liaoning Province Science Foundation for Distinguished Young Schol-ars(2024JH3/50100009)+4 种基金International Partnership Program of Chinese Academy of Sciences for Future Network(028GJHZ2023079FN)Innovation Program of Science and Research from the DICP,CAS(DICP I202529,DICP I202458,DICP I202310)Dalian Science Foundation for Distin-guished Young Scholars(2022RJ04)Liaoning Provincial Department of Science and Technology Fund Project(2022-YGJC-43)the Natural Science Foundation of Liaoning(2024-MSBA-59).
文摘Over 40 amyloidogenic proteins have been identified to cause amyloidosis diseases in clinics.Tissue deposition of amyloid proteins entangled with interacting partners is a characteristic pathological hallmark of amyloidosis diseases.However,the proteomic complexity of co-aggregated amyloid deposits poses a clinical challenge to diagnose the exact disease-causing pathogenic proteins in patients’biopsied tissue.Herein,we present a photocatalytic proteomic method,named Amyloid-ID,as a promising approach to identify the composition of amyloid deposits for clinical proteotyping of amyloidosis diseases.Amyloid-ID is enabled by a photosensitized probe analogous to a pan-amyloid sensor,Thioflavin T.We show this probe photocatalyzes protein labeling via reactive oxygen species and demonstrate its applicability in both AD mouse models and human laryngeal samples.Next,we exemplify its utility by proteotyping the pathogenic protein underlying the rare laryngeal amyloidosis(LA).Using patients’biopsied tissue sections,we label,enrich,and profile the amyloid deposits.Proteomics analysis top-ranks fibrinogen as a potential pathogenic protein.Biochemical and biophysical characterizations confirm that fibrinogen can aggregate into amyloid fibrils.Intriguingly,we observe that fibrinogen’s fibrillation is sensitive to mechanical forces,particularly impacted by sonication.Such observation coincides with its primary larynx deposition,where frequent vocal cord friction occurs.Overall,given the photocatalytic properties,our Amyloid-ID serves as a promising clinical proteotyping method for amyloidosis diseases.
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 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.
