1.Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining.
Wenqing SHUI ; Song LIN ; Allen ZHANG ; Yan CHEN ; Yingying HUANG ; Mark SANDERS
Protein & Cell 2011;2(8):680-688
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.
Chromatography, High Pressure Liquid
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methods
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Data Mining
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Humans
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Mass Spectrometry
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methods
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Microsomes, Liver
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metabolism
2.Identification and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1.
Jinhua XU ; Xiaochao WEI ; Limin YAN ; Dan LIU ; Yuanyuan MA ; Yu GUO ; Chune PENG ; Honggang ZHOU ; Cheng YANG ; Zhiyong LOU ; Wenqing SHUI
Protein & Cell 2013;4(10):771-781
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-associated molecular patterns (PAMPs). Although site-specific 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 autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time. The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spectrometry (MS). Thr-237, Thr-242 and Tyr-250 were found to most significantly affect BIK1 activity in autophosphorylation and phosphorylation of BAK1 in vitro. A structural model of BIK1 was built to further illustrate the molecular functions of specific 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 mediates downstream signaling specificity.
Amino Acids
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chemistry
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Arabidopsis
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enzymology
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Arabidopsis Proteins
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chemistry
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genetics
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isolation & purification
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Gene Expression Regulation, Plant
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Immunity, Innate
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Mutation
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Phosphorylation
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Protein-Serine-Threonine Kinases
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chemistry
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genetics
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isolation & purification
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Signal Transduction
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Threonine
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genetics
3.Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.
Linlin ZHANG ; Shanshan LIU ; Ningning LIU ; Yong ZHANG ; Min LIU ; Dengwen LI ; Edward SETO ; Tso-Pang YAO ; Wenqing SHUI ; Jun ZHOU
Protein & Cell 2015;6(1):42-54
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot 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 protein 70 (Hsc70), 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 protein acetylation with the capability of coordinating various cellular functions.
Acetylation
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Actins
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chemistry
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metabolism
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Animals
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Cell Line
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Chromatography, High Pressure Liquid
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HSC70 Heat-Shock Proteins
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metabolism
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HSP40 Heat-Shock Proteins
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metabolism
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Histone Deacetylase 6
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Histone Deacetylases
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metabolism
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Isotope Labeling
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Liver
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metabolism
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Lysine
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metabolism
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Microscopy, Confocal
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Nonmuscle Myosin Type IIA
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metabolism
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Protein Binding
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Proteomics
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Substrate Specificity
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Tandem Mass Spectrometry
4.Phosphoregulation of the dimerization and functions of end-binding protein 1.
Jie CHEN ; Youguang LUO ; Lixin LI ; Jie RAN ; Xincheng WANG ; Siqi GAO ; Min LIU ; Dengwen LI ; Wenqing SHUI ; Jun ZHOU
Protein & Cell 2014;5(10):795-799
Adenomatous Polyposis Coli Protein
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chemistry
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metabolism
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Amino Acid Sequence
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Chromatography, High Pressure Liquid
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HeLa Cells
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Humans
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Kinesin
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chemistry
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metabolism
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Microtubule-Associated Proteins
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chemistry
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metabolism
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Microtubules
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metabolism
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Molecular Sequence Data
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Phosphopeptides
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analysis
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Phosphorylation
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Protein Multimerization
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Tandem Mass Spectrometry
5.A structural view of the antibiotic degradation enzyme NDM-1 from a superbug.
Yu GUO ; Jing WANG ; Guojun NIU ; Wenqing SHUI ; Yuna SUN ; Honggang ZHOU ; Yaozhou ZHANG ; Cheng YANG ; Zhiyong LOU ; Zihe RAO
Protein & Cell 2011;2(5):384-394
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 three-dimensional 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.
Amino Acid Sequence
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Anti-Bacterial Agents
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metabolism
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Binding Sites
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Captopril
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chemistry
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pharmacology
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Catalytic Domain
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Crystallography, X-Ray
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Drug Resistance, Bacterial
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Enterobacteriaceae
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enzymology
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Molecular Sequence Data
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Sequence Alignment
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Sequence Homology, Amino Acid
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beta-Lactamases
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chemistry
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metabolism