Hydrogen peroxide(H2O2)plays a significant role in regulating a variety of biological processes.Dysregulation of H2O2 can lead to various diseases.Although numerous fluorescent imaging probes for H2O2 have been report...Hydrogen peroxide(H2O2)plays a significant role in regulating a variety of biological processes.Dysregulation of H2O2 can lead to various diseases.Although numerous fluorescent imaging probes for H2O2 have been reported,the development of H2O2 ratiometric fluorescent probe with large Stokes shift remains rather limited.Such probes have shown distinct advantages,such as minimized interference from environment and improved signal-to noise ratio.In this work,we reported a new pyrene-based compound Py-VPB as H2O2 fluorescent probe in vitro.The probe demonstrated ratiometric detection behavior,large Stokes shift and large emission shift.In addition,the probe showed high sensitivity and selectivity towards H2O2 in vitro.Based on these excellent properties,we successfully applied Py-VPB to the visualization of exogenous and endogenous H2O2 in living cells.Cell imaging study also showed that our probe was localized in the mitochondria.We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H2O2 in living systems.展开更多
Lysine lipoylation plays vital roles in cell metabolism and redox processes.For example,removal of lipoylation will decrease pyruvate dehydrogenase activity and affect the citric acid cycle.Despite the important funct...Lysine lipoylation plays vital roles in cell metabolism and redox processes.For example,removal of lipoylation will decrease pyruvate dehydrogenase activity and affect the citric acid cycle.Despite the important functions of lysine lipoylation,the mechanisms for the addition and removal of this modification remain largely unexplored.Very few useful chemical tools are available to study the interactions of lysine lipoylation with its regulatory delipoylation proteins.For example,immunoaffinity purification-mass spectrometry is one of such tools,which highly relies on antibody efficiency and purification techniques.Single-step activity based fluorogenic probes developed by our groups and others is also an efficient method to study the deacylation activity.Affinitybased labeling probe using photo-cross-linker is a powerful platform to study the transient and dynamic interactions of peptide ligands with the interacting proteins.Herein,we have designed and synthesized a dual-function probe KTLlip for studying enzymatic delipoylation(eraser)activity and interaction of lysine lipoylation with the eraser at the same time.We show that KTLlip can be used as a useful tool to detect delipoylation as demonstrated by its ability to fluorescently label the eraser activity of recombinant Sirt2.We envision that the probe will help delineate the roles of delipoylation enzyme in biology.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
Lysine acetylation is one of the most prevalent and important posttranslational modifications(PTMs) in proteins. The process can be recognized by bromodomains(BRDs), which are a class of proteininteraction modules inv...Lysine acetylation is one of the most prevalent and important posttranslational modifications(PTMs) in proteins. The process can be recognized by bromodomains(BRDs), which are a class of proteininteraction modules involved in chromatin remodeling and transcriptional activation. The development of BRD fluorescent probes will be useful for monitoring the activity of BRDs in living cells as well as aiding inhibitor development. Herein we designed a peptide-based probe based on the proximity-induced protein conjugation reaction. The peptide-based probe is capable of covalently and selectively reacting with the unique cysteine residue in the bromodomain through proximity effect. Our experimental data showed that the probe displayed noticeable fluorescence response upon addition of BRD4(1). In-gel fluorescence scanning demonstrated that BRD4(1) can be covalently labelled by the probe. Moreover, the probe was shown to selectively detect BRD4(1) over other proteins. We envision that the probe developed in this study will provide a useful tool to further investigate the biological roles of BRDs.展开更多
Dear Editor,Retinitis pigmentosa(RP)is an inherited photoreceptor degeneration disease with high genetic heterogeneity(>90 disease-causing genes according to RetNet:https://web.sph.uth.edu/RetNet/sumdis.htm).Taking...Dear Editor,Retinitis pigmentosa(RP)is an inherited photoreceptor degeneration disease with high genetic heterogeneity(>90 disease-causing genes according to RetNet:https://web.sph.uth.edu/RetNet/sumdis.htm).Taking a single RP disease gene RHO as an example,there are more than two hundred loss-of-function and gain-of-function mutations identified.1 While gene supplementation therapy has emerged as the most promising treatment for autosomal recessive RP(arRP)and X-linked RP(ClinicalTrials identifier:NCT01482195,NCT03328130,NCT03116113,NCT03252847,NCT03316560),therapeutic approaches to treat autosomal dominant RP(adRP)fall behind due to the low efficiency to disrupt mutant alleles specifically and a broad spectrum of the gain-of-function mutations.展开更多
Dear Editor,Recent studies from us and others have deciphered the clinical relevance of a variety of oncohistones1 in different diseases.The H3K27M and H3K36M mutant histones exert dominant-negative effects on methyla...Dear Editor,Recent studies from us and others have deciphered the clinical relevance of a variety of oncohistones1 in different diseases.The H3K27M and H3K36M mutant histones exert dominant-negative effects on methylation levels of histone H3K27 and H3K36 on wildtype histone protein in pediatric brain cancers2–4 and chondroblastoma,5,6 respectively.In contrast to H3K27M and H3K36M that act in trans to cause global reduction of methylation at the respective residues,the H3G34 mutations,including H3G34V/R and H3G34W/L found in pediatric high-grade glioma and giant cell tumor of the bone,affect the H3K36 methylation in cis and alter the H3K36 methylation on the nucleosome containing the H3G34 mutation(s).展开更多
Microarray technology has been widely applied in biomedical research.The key to microarray study is to develop efficient immobilization method.In this study,we designed a new reversible microarray immobilization metho...Microarray technology has been widely applied in biomedical research.The key to microarray study is to develop efficient immobilization method.In this study,we designed a new reversible microarray immobilization method based on thiol-quinone reaction.A quinone-functionalized slide was fabricated through H_(2)O_(2)treatment of dopamine-coated slides.Various thiol-containing molecules can be anchored onto the quinone-functionalized slides via thioether linker,which could be cleaved under H_(2)O_(2) treatment to regenerate quinone groups on the surface.The highly versatile approach can be widely used for immobilization of various thiol-containing molecules.展开更多
Dear Editor,Histones are essential proteins in compacting genomic DNA and regulating gene expression.Previous studies on histone H3 oncohistones in pediatric brain cancers1,2 and chondroblastoma3,documented the transc...Dear Editor,Histones are essential proteins in compacting genomic DNA and regulating gene expression.Previous studies on histone H3 oncohistones in pediatric brain cancers1,2 and chondroblastoma3,documented the transcriptomic reprogramming through the alterations of histone modifications.We recently reported the identification of a novel cancer associated mutation,the H2BG53-to-D in pancreatic ductal adenocarcinoma(PDAC)4.We showed that the H2BG53D mutation weakens the interaction between nucleosomal DNA and histone octamer,subsequently enhances transcription in vitro.We further showed that cells expressing the G53D mutant H2B acquired oncogenic phenotypes in our CRISPRCas9 knock-in model.However,the mechanism by which H2BG53D mutation promotes PDAC remains unknown.展开更多
基金This work was supported by the Science Technology and Innovation Committee of Shenzhen Municipality(JCYJ20180507181654823)the National Natural Science Foundation of China(21778044)and Sichuan Science and Technology Program(2018JY0360).
文摘Hydrogen peroxide(H2O2)plays a significant role in regulating a variety of biological processes.Dysregulation of H2O2 can lead to various diseases.Although numerous fluorescent imaging probes for H2O2 have been reported,the development of H2O2 ratiometric fluorescent probe with large Stokes shift remains rather limited.Such probes have shown distinct advantages,such as minimized interference from environment and improved signal-to noise ratio.In this work,we reported a new pyrene-based compound Py-VPB as H2O2 fluorescent probe in vitro.The probe demonstrated ratiometric detection behavior,large Stokes shift and large emission shift.In addition,the probe showed high sensitivity and selectivity towards H2O2 in vitro.Based on these excellent properties,we successfully applied Py-VPB to the visualization of exogenous and endogenous H2O2 in living cells.Cell imaging study also showed that our probe was localized in the mitochondria.We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H2O2 in living systems.
基金supported by the Science Technology and Innovation Committee of Shenzhen Municipality(Grant Nos.JCYJ20180507181654823 and JCYJ20170413141047772)the National Natural Science Foundation of China(Grant No.21778044).
文摘Lysine lipoylation plays vital roles in cell metabolism and redox processes.For example,removal of lipoylation will decrease pyruvate dehydrogenase activity and affect the citric acid cycle.Despite the important functions of lysine lipoylation,the mechanisms for the addition and removal of this modification remain largely unexplored.Very few useful chemical tools are available to study the interactions of lysine lipoylation with its regulatory delipoylation proteins.For example,immunoaffinity purification-mass spectrometry is one of such tools,which highly relies on antibody efficiency and purification techniques.Single-step activity based fluorogenic probes developed by our groups and others is also an efficient method to study the deacylation activity.Affinitybased labeling probe using photo-cross-linker is a powerful platform to study the transient and dynamic interactions of peptide ligands with the interacting proteins.Herein,we have designed and synthesized a dual-function probe KTLlip for studying enzymatic delipoylation(eraser)activity and interaction of lysine lipoylation with the eraser at the same time.We show that KTLlip can be used as a useful tool to detect delipoylation as demonstrated by its ability to fluorescently label the eraser activity of recombinant Sirt2.We envision that the probe will help delineate the roles of delipoylation enzyme in biology.
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the financial support from the National Natural Science Foundation of China (No. 21572190)the Hong Kong Early Career Scheme Grant (No. 21300714)the City University of Hong Kong Grant (No. 9667147)
文摘Lysine acetylation is one of the most prevalent and important posttranslational modifications(PTMs) in proteins. The process can be recognized by bromodomains(BRDs), which are a class of proteininteraction modules involved in chromatin remodeling and transcriptional activation. The development of BRD fluorescent probes will be useful for monitoring the activity of BRDs in living cells as well as aiding inhibitor development. Herein we designed a peptide-based probe based on the proximity-induced protein conjugation reaction. The peptide-based probe is capable of covalently and selectively reacting with the unique cysteine residue in the bromodomain through proximity effect. Our experimental data showed that the probe displayed noticeable fluorescence response upon addition of BRD4(1). In-gel fluorescence scanning demonstrated that BRD4(1) can be covalently labelled by the probe. Moreover, the probe was shown to selectively detect BRD4(1) over other proteins. We envision that the probe developed in this study will provide a useful tool to further investigate the biological roles of BRDs.
基金(Shanghai Institute of Nutrition and Health,Chinese Academy of Sciences)for their help and reagents on in vitro CRISPR/CAS9 screening.This research was funded by Research Grants Council Hong Kong Project 11103819,Hong Kong Health and Medical Research Fund Project 06172466,TUNG Biomedical Sciences Foundation,and Ming Wai Lau Center(MWLC)Associate Member Programme to W.X.
文摘Dear Editor,Retinitis pigmentosa(RP)is an inherited photoreceptor degeneration disease with high genetic heterogeneity(>90 disease-causing genes according to RetNet:https://web.sph.uth.edu/RetNet/sumdis.htm).Taking a single RP disease gene RHO as an example,there are more than two hundred loss-of-function and gain-of-function mutations identified.1 While gene supplementation therapy has emerged as the most promising treatment for autosomal recessive RP(arRP)and X-linked RP(ClinicalTrials identifier:NCT01482195,NCT03328130,NCT03116113,NCT03252847,NCT03316560),therapeutic approaches to treat autosomal dominant RP(adRP)fall behind due to the low efficiency to disrupt mutant alleles specifically and a broad spectrum of the gain-of-function mutations.
基金supported by grants from Research Grants Council Hong Kong[Project Nos.17101814,21100615,11102118,11101919(to K.M.C.),11102317(to X.W.),26100214(to T.I.)and C7007-17GF(to M.S.Y.H.,K.M.C.,and T.I.)]the Shenzhen Science and Technology Fund Program Project Nos.JCYJ20170818104203065,JCYJ20180307124019360(to K.M.C.)and JCYJ20170307091256048(to X.W.)+1 种基金National Natural Science Foundation of China[Project No.81802384(to X.W.)]supported by the Hong Kong Epigenomics Project of the EpiHK(to K.M.C.)and grants from the National Cancer Institute,National Institute of Health[CA72851,CA187956,CA202797,CA214254(to A.G.)].
文摘Dear Editor,Recent studies from us and others have deciphered the clinical relevance of a variety of oncohistones1 in different diseases.The H3K27M and H3K36M mutant histones exert dominant-negative effects on methylation levels of histone H3K27 and H3K36 on wildtype histone protein in pediatric brain cancers2–4 and chondroblastoma,5,6 respectively.In contrast to H3K27M and H3K36M that act in trans to cause global reduction of methylation at the respective residues,the H3G34 mutations,including H3G34V/R and H3G34W/L found in pediatric high-grade glioma and giant cell tumor of the bone,affect the H3K36 methylation in cis and alter the H3K36 methylation on the nucleosome containing the H3G34 mutation(s).
基金the financial support from the Research Grants Council of Hong Kong(Nos.11102719,11304118 and 14306317)Shenzhen Basic Research Project(No.JCYJ20160601173218804)+1 种基金National Natural Science Foundation of China(No.21778044)the Shandong University of Technology Ph.D.Startup Foundation(No.420033)。
文摘Microarray technology has been widely applied in biomedical research.The key to microarray study is to develop efficient immobilization method.In this study,we designed a new reversible microarray immobilization method based on thiol-quinone reaction.A quinone-functionalized slide was fabricated through H_(2)O_(2)treatment of dopamine-coated slides.Various thiol-containing molecules can be anchored onto the quinone-functionalized slides via thioether linker,which could be cleaved under H_(2)O_(2) treatment to regenerate quinone groups on the surface.The highly versatile approach can be widely used for immobilization of various thiol-containing molecules.
基金supported by grants from Research Grants Council Hong Kong[Project No.17101814,21100615,11102118,11101919(K.M.C.),11102317(X.W.),and C7007-17GF(K.M.C.)]the Shenzhen Science and Technology Fund Program Project No.JCYJ20170818104203065,JCYJ20180307124019360(K.M.C.),JCYJ20170307091256048(X.W.)National Natural Science Foundation of China[Project No.81802384(X.W.)].This work was also supported by the Hong Kong Epigenomics Project of the EpiHK(K.M.C.).
文摘Dear Editor,Histones are essential proteins in compacting genomic DNA and regulating gene expression.Previous studies on histone H3 oncohistones in pediatric brain cancers1,2 and chondroblastoma3,documented the transcriptomic reprogramming through the alterations of histone modifications.We recently reported the identification of a novel cancer associated mutation,the H2BG53-to-D in pancreatic ductal adenocarcinoma(PDAC)4.We showed that the H2BG53D mutation weakens the interaction between nucleosomal DNA and histone octamer,subsequently enhances transcription in vitro.We further showed that cells expressing the G53D mutant H2B acquired oncogenic phenotypes in our CRISPRCas9 knock-in model.However,the mechanism by which H2BG53D mutation promotes PDAC remains unknown.