Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain ...Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.展开更多
Direct,site-specific modification of native membrane proteins without genetic manipulation remains a significant challenge.Here,we have introduced an aptamer-directed dual-site photoaffinity labeling(ADPAL)strategy th...Direct,site-specific modification of native membrane proteins without genetic manipulation remains a significant challenge.Here,we have introduced an aptamer-directed dual-site photoaffinity labeling(ADPAL)strategy that enabled precise and functional modification of membrane proteins in their native context.This approach integrated aptamer-based recognition with proximity-activated photoreactive chemistry to achieve dual-site covalent labeling with high specificity and efficiency.As a proof of concept,we applied the ADPAL strategy to both isolated proteins,lysozyme and thrombin,as well as to membrane proteins on living cells,including the mesenchymal-epithelial transition factor and protein tyrosine kinase 7 receptors.These dual-site modifications triggered protein self-assembly,reprogrammed protein-protein interactions,facilitated protein cluster formation,and modulated cellular functions.Compared to conventional noncovalent or single-site covalent labeling methods,ADPAL offers enhanced specificity and regulatory control.This work establishes ADPAL as a versatile and programmable platform for precise membrane protein labeling and functional manipulation,with broad potential for biological research and therapeutic development.展开更多
Atherosclerosis is a persistent inflammatory state,while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development.Although ligustilide(Lig) was shown to exert obvious antiatherogenic e...Atherosclerosis is a persistent inflammatory state,while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development.Although ligustilide(Lig) was shown to exert obvious antiatherogenic effects in previous studies,its precise mechanism has not been deeply discussed.In this paper,we designed a Lig-derived photoaffinity labelling(PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach.Mothers against decapentaplegic homologue 3(SMAD3),a signal transmitter of transforming growth factor-β(TGF-β) which promotes the development of vascular fibrosis,was identified as a potential target of Lig.Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1,thereby inhibiting the collagen synthesis process.Hence,developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.展开更多
Berberine(BBR) is the primary alkaloid compound of the heat-clearing traditional Chinese medicine Huanglian(Coptis chinensis) and exerts regulatory effects on energy metabolism. However, the specific targets and molec...Berberine(BBR) is the primary alkaloid compound of the heat-clearing traditional Chinese medicine Huanglian(Coptis chinensis) and exerts regulatory effects on energy metabolism. However, the specific targets and molecular mechanisms are not clear. In this paper, the BBR-affected energy metabolism pathway was screened by nontargeted metabolomics, and a BBR-derived photoaffinity labeled(PAL) probe was designed to identify potential targets via a chemical proteomics approach. NDUFV1, a subunit of complex Ⅰ on mitochondria, was identified as a potential target of BBR. In the respiratory chain, BBR suppressed the activity of complex Ⅰ, reduced the electrochemical potential in the mitochondrial intermembrane and inhibited the generation of ATP and heat via competitive binding with NDUFV1. The results illustrated the underlying mechanism of BBR in the downregulation of energy metabolism.展开更多
In order to characterize binding sites of insecticidal compounds on GABA gated chloride channel, new photoaffinity probe candidates based on 5e-t-butyl-2e-[4-（substituted-propynyl）phenyl]-1,3-dithiane for the noncom...In order to characterize binding sites of insecticidal compounds on GABA gated chloride channel, new photoaffinity probe candidates based on 5e-t-butyl-2e-[4-（substituted-propynyl）phenyl]-1,3-dithiane for the noncompetitive blocker （NCB） site of the γ-aminobutyric acid （GABA）-gated chloride channel were designed and synthesized, and their potency as an inhibitor on NCB was measured by 4＇-ethynyl-4-n-[2,3-^3H2]-propylbicycloorthobenzoate （^3H EBOB） assay. The synthesized compounds showed high inhibition activities with half maximum inhibition concentrations （IC50） of lower than 35 nmol/L and were very stable in binding conditions as well photoreacted quickly at 300 nm light. These new compounds are expected to be good photoaffinity labeling probes if radioisotope iodine is incorporated.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.:U21A20407 and 81973467)Beijing Natural Science Foundation,China(Grant No.:7222276).
文摘Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.
基金the National Key Research and Development Program of China(grant no.2020YFA090900)the National Natural Science Foundation of China(grant nos.22174161,21991080,and 22304179)+1 种基金the Zhejiang Leading Innovation and Entrepreneurship Team,China(grant no.2022R01006)the Pioneer R&D Program of Zhejiang,China(grant no.2024SDYXS0003).
文摘Direct,site-specific modification of native membrane proteins without genetic manipulation remains a significant challenge.Here,we have introduced an aptamer-directed dual-site photoaffinity labeling(ADPAL)strategy that enabled precise and functional modification of membrane proteins in their native context.This approach integrated aptamer-based recognition with proximity-activated photoreactive chemistry to achieve dual-site covalent labeling with high specificity and efficiency.As a proof of concept,we applied the ADPAL strategy to both isolated proteins,lysozyme and thrombin,as well as to membrane proteins on living cells,including the mesenchymal-epithelial transition factor and protein tyrosine kinase 7 receptors.These dual-site modifications triggered protein self-assembly,reprogrammed protein-protein interactions,facilitated protein cluster formation,and modulated cellular functions.Compared to conventional noncovalent or single-site covalent labeling methods,ADPAL offers enhanced specificity and regulatory control.This work establishes ADPAL as a versatile and programmable platform for precise membrane protein labeling and functional manipulation,with broad potential for biological research and therapeutic development.
基金financially supported by National Key Research and Development Program of China(Nos.2018YFC1704800,2018YFC1704805)National Natural Science Foundation of China(No.81673637)the Key R&D Program of Tianjin(No.18YFYZCG00060)。
文摘Atherosclerosis is a persistent inflammatory state,while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development.Although ligustilide(Lig) was shown to exert obvious antiatherogenic effects in previous studies,its precise mechanism has not been deeply discussed.In this paper,we designed a Lig-derived photoaffinity labelling(PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach.Mothers against decapentaplegic homologue 3(SMAD3),a signal transmitter of transforming growth factor-β(TGF-β) which promotes the development of vascular fibrosis,was identified as a potential target of Lig.Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1,thereby inhibiting the collagen synthesis process.Hence,developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.
基金supported by the National Natural Science Foundation of China(No.81973449)the China Postdoctoral Science Foundation(No.2020M680871)。
文摘Berberine(BBR) is the primary alkaloid compound of the heat-clearing traditional Chinese medicine Huanglian(Coptis chinensis) and exerts regulatory effects on energy metabolism. However, the specific targets and molecular mechanisms are not clear. In this paper, the BBR-affected energy metabolism pathway was screened by nontargeted metabolomics, and a BBR-derived photoaffinity labeled(PAL) probe was designed to identify potential targets via a chemical proteomics approach. NDUFV1, a subunit of complex Ⅰ on mitochondria, was identified as a potential target of BBR. In the respiratory chain, BBR suppressed the activity of complex Ⅰ, reduced the electrochemical potential in the mitochondrial intermembrane and inhibited the generation of ATP and heat via competitive binding with NDUFV1. The results illustrated the underlying mechanism of BBR in the downregulation of energy metabolism.
文摘In order to characterize binding sites of insecticidal compounds on GABA gated chloride channel, new photoaffinity probe candidates based on 5e-t-butyl-2e-[4-（substituted-propynyl）phenyl]-1,3-dithiane for the noncompetitive blocker （NCB） site of the γ-aminobutyric acid （GABA）-gated chloride channel were designed and synthesized, and their potency as an inhibitor on NCB was measured by 4＇-ethynyl-4-n-[2,3-^3H2]-propylbicycloorthobenzoate （^3H EBOB） assay. The synthesized compounds showed high inhibition activities with half maximum inhibition concentrations （IC50） of lower than 35 nmol/L and were very stable in binding conditions as well photoreacted quickly at 300 nm light. These new compounds are expected to be good photoaffinity labeling probes if radioisotope iodine is incorporated.
