Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1(NDM-1)are a type of newly discovered antibioticresistant bacteria.The rapid pandemic spread of NDM-1 bacteri...Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1(NDM-1)are a type of newly discovered antibioticresistant bacteria.The rapid pandemic spread of NDM-1 bacteria worldwide(spreading to India,Pakistan,Europe,America,and Chinese Taiwan)in less than 2 months characterizes these microbes as a potentially major global health problem.The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations.The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics,allowing the bacteria to escape the action of antibiotics.Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues,the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified.Here,we report the threedimensional structure of NDM-1 with two catalytic zinc ions in its active site.Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity.The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases(MBLs)and implicate its role in wide spectrum drug resistance.We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria.Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.展开更多
As one of the most successful therapeutic target families,G protein-coupled receptors(GPCRs)have experienced a transformation from random ligand screening to knowledge-driven drug design.We are eye-witnessing tremendo...As one of the most successful therapeutic target families,G protein-coupled receptors(GPCRs)have experienced a transformation from random ligand screening to knowledge-driven drug design.We are eye-witnessing tremendous progresses made recently in the understanding of their structure-function relationships that facilitated drug development at an unprecedented pace.This article intends to provide a comprehensive overview of this important field to a broader readership that shares some common interests in drug discovery.展开更多
Histone deacetylase 6 （HDAC6）, a predominantly cyto- plasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 can- not...Histone deacetylase 6 （HDAC6）, a predominantly cyto- plasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 can- not be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 （MYH9）, heat shock cognate pro- tein 70 （HscT0）, and dnaJ homolog subfamily A member 1 （DNAJA1）, were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6- mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of proteinacetylation with the capability of coordinating various cellular functions.展开更多
Arabidopsis BOTRYTIS-INDUCED KINASE1(BIK1)is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity.It is known to form a signaling complex with a...Arabidopsis BOTRYTIS-INDUCED KINASE1(BIK1)is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity.It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-asso-ciated molecular patterns(PAMPs).Although site-specifi c phosphorylation is speculated to mediate the activation and function of BIK1,few studies have been devoted to complete profiling of BIK1 phosphorylation residues.Here,we identified nineteen in vitro autophosphoryla-tion sites of BIK1 including three phosphotyrosine sites,thereby proving BIK1 is a dual-specifi city kinase for the fi rst time.The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spec-trometry(MS).Thr-237,Thr-242 and Tyr-250 were found to most signifi cantly affect BIK1 activity in autophosphoryla-tion and phosphorylation of BAK1 in vitro.A structural model of BIK1 was built to further illustrate the molecular functions of specifi c phosphorylation residues.We also mapped new sites of FLS2 phosphorylation by BIK1,which are different from those by BAK1.These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and medi-ates downstream signaling specifi city.展开更多
Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery.For drug candidate screening,it is common practice to use ultra-high performance liquid chromatograp...Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery.For drug candidate screening,it is common practice to use ultra-high performance liquid chromatography(U-HPLC)coupled with triple quadrupole mass spectrometry.This approach certainly results in short analytical run time;however,in assessing the true throughput,all aspects of the workflow needs to be considered,including instrument optimization and the necessity to re-run samples when information is missed.Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency.In addition,as the data is acquired in a non-biased manner,high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.展开更多
Dear Editor, End-binding protein 1 （EB1）, a member of microtubule plus- end tracking proteins （＋TIPs）, plays an important role in the regulation of microtubule dynamics and has been implicated in cancer developme...Dear Editor, End-binding protein 1 （EB1）, a member of microtubule plus- end tracking proteins （＋TIPs）, plays an important role in the regulation of microtubule dynamics and has been implicated in cancer development （Dong et al., 2010; Gouveia and Ak- hmanova, 2010; Wang et al., 2005）. However, it remains poorly understood how EB1 functions are regulated by phosphorylation in mammalian cells. To map the phosphory- lation pattern of EB1, we overexpressed GST-EB1 in HeLa cells and enriched GST-EB1 from cell lysates by GST pull- down. The pulldown preparations showed strong serine, threonine, and tyrosine phosphorylation signals in the immu- noblots （Fig. SlA）. GST-EB1 purified from cells was clearly visualized on the gel prior to in-gel digestion （Fig. S 1B）.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.30870486 and 30730022)the National Major Projects(Grant Nos.2009ZX10004-804,2009ZX09311-001 and 2009ZX10004-304)the National Basic Research Program(973 Program)(Grant Nos.2011CB915501 and 2011CB910304).
文摘Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1(NDM-1)are a type of newly discovered antibioticresistant bacteria.The rapid pandemic spread of NDM-1 bacteria worldwide(spreading to India,Pakistan,Europe,America,and Chinese Taiwan)in less than 2 months characterizes these microbes as a potentially major global health problem.The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations.The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics,allowing the bacteria to escape the action of antibiotics.Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues,the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified.Here,we report the threedimensional structure of NDM-1 with two catalytic zinc ions in its active site.Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity.The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases(MBLs)and implicate its role in wide spectrum drug resistance.We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria.Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.
基金The authors acknowledge funding support from the National Natural Science Foundation of China 81872915(to M.-W.W.),81773792(to D.Y.),81973373(to D.Y.),21704064(to Q.Z.),31971362(to W.S.),31971178(to S.Z.),and 31770796(to Y.J.)National Science&Technology Major Project of China-Key New Drug Creation and Manufacturing Program 2018ZX09735-001(to M.-W.W.),2018ZX09711002-002-005(to D.Y.),and 2018ZX09711002-002-002(to Y.J.)+4 种基金the National Key Basic Research Program of China 2018YFA0507000(to M.-W.W.,S.Z.,W.S.,and H.T.)Novo Nordisk-CAS Research Fund grant NNCAS-2017-1-CC(to D.Y.)The Belt and Road Master Fellowship program(to V.L.)UCAS Scholarship for International Students(to S.D.)and The CAS-TWAS President's Fellowship for International Doctoral Students(to E.Y.).
文摘As one of the most successful therapeutic target families,G protein-coupled receptors(GPCRs)have experienced a transformation from random ligand screening to knowledge-driven drug design.We are eye-witnessing tremendous progresses made recently in the understanding of their structure-function relationships that facilitated drug development at an unprecedented pace.This article intends to provide a comprehensive overview of this important field to a broader readership that shares some common interests in drug discovery.
基金This work was supported by grants from the National Natural Sci- ence Foundation of China （Grant Nos. 91313302, 31171334, and 31170782） and the Tianjin Natural Science Foundation （11JCYBJC25500）. L.Z. performed most of the experiments. S.L. and Y.Z. carried out proteomics experiments and bioinformatics analysis. L.Z., S.L., N.L., M.L., D.L., and W.S. analyzed data. E.S. gave important suggestions about the research strategy. T.-P.Y. provided HDAC6 knockout mice. L.Z., S.L., W.S., and J.Z. wrote the manuscript. W.S. and J.Z. con- ceived and designed experiments.
文摘Histone deacetylase 6 （HDAC6）, a predominantly cyto- plasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 can- not be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 （MYH9）, heat shock cognate pro- tein 70 （HscT0）, and dnaJ homolog subfamily A member 1 （DNAJA1）, were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6- mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of proteinacetylation with the capability of coordinating various cellular functions.
基金the National Natural Science Foundation of China(Grant Nos.31170782 and 31100208)Tianjin Natural Science Foundation(Grant No.11JCYBJC25500)Spe-cialized Research Fund for the Doctoral Program of Higher Education(Grant No.20110031120019).
文摘Arabidopsis BOTRYTIS-INDUCED KINASE1(BIK1)is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity.It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-asso-ciated molecular patterns(PAMPs).Although site-specifi c phosphorylation is speculated to mediate the activation and function of BIK1,few studies have been devoted to complete profiling of BIK1 phosphorylation residues.Here,we identified nineteen in vitro autophosphoryla-tion sites of BIK1 including three phosphotyrosine sites,thereby proving BIK1 is a dual-specifi city kinase for the fi rst time.The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spec-trometry(MS).Thr-237,Thr-242 and Tyr-250 were found to most signifi cantly affect BIK1 activity in autophosphoryla-tion and phosphorylation of BAK1 in vitro.A structural model of BIK1 was built to further illustrate the molecular functions of specifi c phosphorylation residues.We also mapped new sites of FLS2 phosphorylation by BIK1,which are different from those by BAK1.These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and medi-ates downstream signaling specifi city.
文摘Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery.For drug candidate screening,it is common practice to use ultra-high performance liquid chromatography(U-HPLC)coupled with triple quadrupole mass spectrometry.This approach certainly results in short analytical run time;however,in assessing the true throughput,all aspects of the workflow needs to be considered,including instrument optimization and the necessity to re-run samples when information is missed.Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency.In addition,as the data is acquired in a non-biased manner,high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.
文摘Dear Editor, End-binding protein 1 （EB1）, a member of microtubule plus- end tracking proteins （＋TIPs）, plays an important role in the regulation of microtubule dynamics and has been implicated in cancer development （Dong et al., 2010; Gouveia and Ak- hmanova, 2010; Wang et al., 2005）. However, it remains poorly understood how EB1 functions are regulated by phosphorylation in mammalian cells. To map the phosphory- lation pattern of EB1, we overexpressed GST-EB1 in HeLa cells and enriched GST-EB1 from cell lysates by GST pull- down. The pulldown preparations showed strong serine, threonine, and tyrosine phosphorylation signals in the immu- noblots （Fig. SlA）. GST-EB1 purified from cells was clearly visualized on the gel prior to in-gel digestion （Fig. S 1B）.
