INTRODUCTION: Heat-related illnesses can lead to morbidity, which are anticipated to increase frequency with predictions of increased global surface temperatures and extreme weather events. Although heat acclimation training (HAT) could prevent heat-related diseases, the mechanisms underlying HAT-promoting beneficial changes in organ function, immunity, and gut microbes remain unclear. METHODS: In the current study, we recruited 32 healthy young soldiers and randomly divided them into 4 teams to conduct HATs for 10 days: the equipment-assisted training team at high temperature (HE); the equipment-assisted training team under normal hot weather (NE); the high-intensity interval training team at high temperature (HIIT), and the control team without training. A standard heat tolerance test (HTT) was conducted before (HTT-1st) and after (HTT-2nd) the training to judge whether the participants met the heat acclimation (HA) criteria. RESULTS: We found that the participants in both HE and NE teams had significantly higher acclimation rates (HA/total population) than whom in the HIIT team. The effects of HAT on the participants of the HE team outperformed that of the NE team. In the HA group, the differences of physiological indicators and plasma organ damage biomarkers (ALT, ALP, creatinine, LDH, alpha-HBDH and cholinesterase) before and after HTT-2nd were significantly reduced to those during HTT-1st, but the differences of immune factors (IL-10, IL-6, CXCL2, CCL4, CCL5, and CCL11) elevated. The composition, metabolism, and pathogenicity of gut microbes changed significantly, with a decreased proportion of potentially pathogenic bacteria (Escherichia-Shigella and Lactococcus) and increased probiotics (Dorea, Blautia, and Lactobacillus) in the HA group. Training for a longer time in a high temperature and humidity showed beneficial effects for intestinal probiotics. CONCLUSION: These findings revealed that pathogenic gut bacteria decrease while probiotics increase following HA, with elevated immune factors and reduced organ damage during heat stress, thereby improving the body's heat adaption.
Angiopoietin-like protein 3 (ANGPTL3) is an important regulator of lipoproteins by inhibiting both lipoprotein and endothelial lipases. It has been intensively investigated as a drug target for the treatment of dyslipidemia. In the present study, a modified small interfering RNA (siRNA) conjugated with GalNAc ANGsiR10 was characterized by insvivo and insvitro studies for its effect on ANGPTL3 silencing, the reduction of plasma triglycerides (TGs), and cholesterol levels in disease models. The results showed that ANGsiR10 displayed a significant and long-lasting efficacy in reducing blood TG and cholesterol levels in both mice and monkeys. Remarkably, the maximal reductions of plasma TG levels in the hApoC3-Tg mice, a model with high TG levels, and the spontaneous dyslipidemia model of rhesus monkey were 96.3% and 67.7%, respectively, after a single dose of ANGsiR10, with long-lasting effects up to 15sweeks. The cholesterol levels were also reduced in response to treatment, especially the non-HDL-c level, without altering the ApoA/ApoB ratio. This study showed that ANGsiR10 is effective in treating dyslipidemia and is worth further development.
The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.
Title: m(6) A transferase METTL3-induced lncRNA ABHD11-AS1 promotes the Warburg effect of non-small-cell lung cancer Xue L, Li J, Lin Y, Liu D, Yang Q, Jian J, Peng J Ref: Journal of Cellular Physiology, 236:2649, 2021 : PubMed
N(6) -methyladenosine (m(6) A) and long noncoding RNAs (lncRNAs) are both crucial regulators in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the pathological roles of m(6) A and lncRNAs in NSCLC progression are still limited and undefined. Here, lncRNA ABHD11-AS1 was upregulated in NSCLC tissue specimens and cells and the ectopic overexpression was closely correlated with unfavorable prognosis of NSCLC patients. Functionally, ABHD11-AS1 promoted the proliferation and Warburg effect of NSCLC. Mechanistically, m(6) A profile was analyzed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). MeRIP-Seq presented that there was m(6) A modification site in ABHD11-AS1. m(6) A methyltransferase-like 3 (METTL3) installed the m(6) A modification and enhanced ABHD11-AS1 transcript stability to increase its expression. In conclusion, our findings highlight the function and mechanism of METTL3-induced ABHD11-AS1 in NSCLC and inspire the understanding of m(6) A and lncRNA in cancer biology.
Title: Construction and application of a high-content analysis for identifying human carboxylesterase 2 inhibitors in living cell system Xue L, Qian X, Jin Q, Zhu Y, Wang X, Wang D, Ge G, Yang L Ref: Anal Bioanal Chem, :, 2020 : PubMed
Human carboxylesterase 2 (hCE2), one of the most principal drug-metabolizing enzymes, catalyzes the hydrolysis of a variety of endogenous esters, anticancer agents, and environmental toxicants. The significant roles of hCE2 in both endobiotic and xenobiotic metabolism sparked great interest in the discovery and development of efficacious and selective inhibitors. However, the safe and effective inhibitors of hCE2 are scarce, due to the lack of efficient screening and evaluation systems for complex biological systems. To offer a solution to this problem, a high-content analysis (HCA)-based cell imaging and multiparametric assay method was constructed for evaluating the inhibitory effect and safety of hCE2 inhibitors in living cell system. In this study, we first established a cell imaging-based method for identifying hCE2 inhibitors at the living cell level with hCE2 fluorescent probe NCEN. Meanwhile, two nuclear probes, Hoechst 33342 and PI, were integrated to evaluate the potential cytotoxicity of compounds simultaneously. Then, the accuracy of the HCA-based method was verified by the LC-FD-based method with a positive inhibitor BNPP, and the results showed that the HCA-based method exhibited excellent precision, robustness, and reliability. Finally, the newly established HCA-based multiparametric assay panel was successfully applied to re-evaluate a series of reported hCE2 inhibitors in living cells. In summary, the HCA-based multiparametric method could serve as an efficient tool for the accuracy measurement inhibitory effect and cytotoxicity of compounds against hCE2 in living cell system. Graphical abstract.
Title: Effects of fasting on the activities and mRNA expression levels of lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and fatty acid synthetase (FAS) in spotted seabass Lateolabrax maculatus Huang H, Zhang Y, Cao M, Xue L, Shen W Ref: Fish Physiol Biochem, 44:387, 2018 : PubMed
To investigate the effects of fasting on lipid metabolism in spotted seabass muscle and liver tissues, we analyzed mRNA levels and enzyme activities of lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and fatty acid synthetase (FAS), and the relationship among fat content, mRNA level, and enzyme activity during fasting of 35 days. The results showed that expressions of all the three genes were ubiquitous. During the fasting experiment, the hepatosomatic index (HSI) and fat content of muscle and liver tissues significantly decreased before 5 days of fasting (P < 0.05). mRNA levels of LPL increased significantly after 5 days of fasting in liver and 7 days in muscle. Abundance of HSL transcripts increased significantly after 14 days of fasting in both muscle and liver. The activities of LPL and HSL presented a trend that increased firstly, decreased subsequently, and then raised again with the prolonged fasting experiment (P < 0.05). However, activities and mRNA levels of FAS decreased significantly after 1 day of fasting in both muscle and liver. Moreover, activities and mRNA levels of FAS showed a moderate correlation in muscle. These results suggested that FAS had a sooner response to fasting than LPL and HSL in both muscle and liver tissues. LPL and HSL played important roles in lipolysis mainly by increasing enzyme activities in the early stage of fasting and mRNA levels in the later stage of fasting in both muscle and liver. Our results also provided useful information on regulating muscle fat content by fasting.
We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.
Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (kcat/KM) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.
Title: Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase Fang L, Hou S, Xue L, Zheng F, Zhan CG Ref: Chemico-Biological Interactions, 214C:18, 2014 : PubMed
Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.
Title: Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification Fang L, Chow KM, Hou S, Xue L, Chen X, Rodgers DW, Zheng F, Zhan CG Ref: ACS Chemical Biology, 9:1764, 2014 : PubMed
Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only approximately 12 min at 37 degrees C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of approximately 6 h at 37 degrees C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 degrees C to >100 days, but also significantly improved the catalytic efficiency against cocaine by approximately 150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of approximately 94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.
Compared with naturally occurring enzymes, computationally designed enzymes are usually much less efficient, with their catalytic activities being more than six orders of magnitude below the diffusion limit. Here we use a two-step computational design approach, combined with experimental work, to design a highly efficient cocaine hydrolysing enzyme. We engineer E30-6 from human butyrylcholinesterase (BChE), which is specific for cocaine hydrolysis, and obtain a much higher catalytic efficiency for cocaine conversion than for conversion of the natural BChE substrate, acetylcholine (ACh). The catalytic efficiency of E30-6 for cocaine hydrolysis is comparable to that of the most efficient known naturally occurring hydrolytic enzyme, acetylcholinesterase, the catalytic activity of which approaches the diffusion limit. We further show that E30-6 can protect mice from a subsequently administered lethal dose of cocaine, suggesting the enzyme may have therapeutic potential in the setting of cocaine detoxification or cocaine abuse.
Title: Substrate selectivity of high-activity mutants of human butyrylcholinesterase Hou S, Xue L, Yang W, Fang L, Zheng F, Zhan CG Ref: Org Biomol Chem, 11:7477, 2013 : PubMed
Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.
Title: Choline acetate enhanced the catalytic performance of Candida rogusa lipase in AOT reverse micelles Xue L, Zhao Y, Yu L, Sun Y, Yan K, Li Y, Huang X, Qu Y Ref: Colloids Surf B Biointerfaces, 105C:81, 2013 : PubMed
Choline acetate is an ionic liquid composed of a kosmotropic anion and a chaotropic cation. According to Hofmeister series, a kosmotropic anion and/or a chaotropic cation could stabilize an enzyme, thereby facilitating the retention of the catalytic activity of the enzyme. In this work, we first report the influence of choline acetate on the activity and stability of lipase in AOT/water/isooctane reverse micelles. The indicator reaction is the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate. The results show that a low level of choline acetate does not affect the microstructure of the AOT reverse micelles, but the ionic liquid can improve the catalytic efficiency of lipase. Fluorescence spectra show that a high level of choline acetate has an impact on the conformation of lipase, so the activation is mainly due to the influence of choline acetate on the nucleophilicity of water. Infrared spectra demonstrate that choline acetate can form stronger hydrogen bonds with water surrounding lipase, and therefore enhance the nucleophilicity of the water, which makes it easier to attack the acyl enzyme intermediate, thereby increasing the activity of the lipase-catalyzed hydrolysis of the ester. A study on the stability of lipase in AOT reverse micelles indicates that the ionic liquid is able to maintain the activity of lipase to a certain extent. The effect of choline acetate is consistent with that predicted based on Hofmeister series.
Title: Catalytic activities of a cocaine hydrolase engineered from human butyrylcholinesterase against (+)- and (-)-cocaine Xue L, Hou S, Yang W, Fang L, Zheng F, Zhan CG Ref: Chemico-Biological Interactions, 203:57, 2013 : PubMed
It can be argued that an ideal anti-cocaine medication would be one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, wild-type BChE has a low catalytic efficiency against naturally occurring (-)-cocaine. Interestingly, wild-type BChE has a much higher catalytic activity against unnatural (+)-cocaine. According to available positron emission tomography (PET) imaging analysis using [(11)C](-)-cocaine and [(11)C](+)-cocaine tracers in human subjects, only [(11)C](-)-cocaine was observed in the brain, whereas no significant [(11)C](+)-cocaine signal was observed in the brain. The available PET data imply that an effective therapeutic enzyme for treatment of cocaine abuse could be an exogenous cocaine-metabolizing enzyme with a catalytic activity against (-)-cocaine comparable to that of wild-type BChE against (+)-cocaine. Our recently designed A199S/F227A/S287G/A328 W/Y332G mutant of human BChE has a considerably improved catalytic efficiency against (-)-cocaine and has been proven active in vivo. In the present study, we have characterized the catalytic activities of wild-type BChE and the A199S/F227A/S287G/A328 W/Y332G mutant against both (+)- and (-)-cocaine at the same time under the same experimental conditions. Based on the obtained kinetic data, the A199S/F227A/S287G/A328 W/Y332G mutant has a similarly high catalytic efficiency (kcat/KM) against (+)- and (-)-cocaine, and indeed has a catalytic efficiency (kcat/KM=1.84x10(9)M(-1)min(-1)) against (-)-cocaine comparable to that (kcat/KM=1.37x10(9)M(-1)min(-1)) of wild-type BChE against (+)-cocaine. Thus, the mutant may be used to effectively prevent (-)-cocaine from entering brain and producing physiological effects in the enzyme-based treatment of cocaine abuse.
Title: Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine Xue L, Hou S, Tong M, Fang L, Chen X, Jin Z, Tai HH, Zheng F, Zhan CG Ref: Biochemical Journal, 453:447, 2013 : PubMed
Cocaine is a widely abused drug without an FDA (Food and Drug Administration)-approved medication. It has been recognized that an ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. human BChE (butyrylcholinesterase)-catalysed hydrolysis. However, the native human BChE has a low catalytic activity against cocaine. We recently designed and discovered a BChE mutant (A199S/F227A/S287G/A328W/Y332G) with a high catalytic activity (kcat=5700 min-1, Km=3.1 muM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180 mg/kg of body weight, LD100). Further characterization in animal models requires establishment of a high-efficiency stable cell line for the BChE mutant production at a relatively larger scale. It has been extremely challenging to develop a high-efficiency stable cell line expressing BChE or its mutant. In the present study, we successfully developed a stable cell line efficiently expressing the BChE mutant by using a lentivirus-based repeated-transduction method. The scaled-up protein production enabled us to determine for the first time the in vivo catalytic activity and the biological half-life of this high-activity mutant of human BChE in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant (administered to mice 1 min prior to cocaine) can quickly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.
Title: [Construction of Aspergillus niger lipase mutants with oil-water interface independence] Chen D, Shu Z, Xue L, Lin R, Wu J, Jiang Y, Li X, Lin Y, Huang J Ref: Sheng Wu Gong Cheng Xue Bao, 27:860, 2011 : PubMed
Based on previous bioinformational analysis results, two Aspergillus niger lipase (ANL) mutants, ANL-Ser84Gly and ANL-Asp99Pro were constructed to screen ANL mutants with oil-water interface independence. ANL-Ser84Gly still displayed a pronounced interfacial activation, while ANL-Asp99Pro displayed no interfacial activation. The specific activity of ANL-Ser84Gly towards p-nitrophenyl palmitate (-myristate, -laurate and -decanoate) decreased by 29.8% (53.1, 60.1 and 77.1, respectively) than that of ANL, while the specific activity of ANL-Asp99Pro towards p-nitrophenyl palmitate increased by 2.2-fold. The mutation in the hinge region at both sides of the lid domain also destabilized various secondary structure factors of ANL-S84G and ANL-D99P, which resulted in a substantial decrease in thermostability. The achievement to construct oil-water interface-independent ANL mutants would help to further understand lipase interfacial activation mechanism.
Title: Construction of Aspergillus niger lipase mutants with oil-water interface independence Shu Z, Wu J, Xue L, Lin R, Jiang Y, Tang L, Li X, Huang J Ref: Enzyme Microb Technol, 48:129, 2011 : PubMed
Based on previous bioinformational analytical results [Shu ZY, et al. Biotechnol Prog 2009;25:409-16], four A. niger lipase (ANL) mutants, ANL-Ser84Gly, ANL-Asp99Pro, ANL-Lys108Glu and ANL-EalphaH (obtained by replacing the lid domain of ANL with the corresponding domain from A. niger feruloyl esterase), were constructed to screen out ANL mutants with oil-water interface independence. ANL-S84G displayed a pronounced interfacial activation, while ANL-D99P and ANL-K108E displayed no interfacial activation. The specific activity of ANL-S84G towards p-nitrophenyl esters decreased from 29.8% to 76.5% compared with that of ANL, while the specific activity of ANL-D99P towards p-nitrophenyl palmitate increased 2.2-fold. The thermostability of ANL-K108E was almost unchanged, while the thermostability of ANL-S84G and ANL-D99P significantly decreased compared with that of ANL. The construction of oil-water interface-independent ANL mutants would help to further understand the mechanism of lipase interfacial activation.
Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (-)-cocaine but also certain selectivity for (-)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (-)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (-)-cocaine but also the desirable selectivity for (-)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.
Title: Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine Yang W, Xue L, Fang L, Chen X, Zhan CG Ref: Chemico-Biological Interactions, 187:148, 2010 : PubMed
Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (k(cat)) and Michaelis-Menten constant (K(M)) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3,060 min(-1) and 3.1 microM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a approximately 1,080-fold improved catalytic efficiency (k(cat)/K(M)) against (-)-cocaine compared to the wild-type BChE. The approximately 1,080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the approximately 1,000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.
Title: Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency Zheng F, Yang W, Xue L, Hou S, Liu J, Zhan CG Ref: Biochemistry, 49:9113, 2010 : PubMed
The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least approximately 1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.
Title: Enzyme-catalyzed polycondensation of polyester macrodiols with divinyl adipate: a green method for the preparation of thermoplastic block copolyesters Dai S, Xue L, Zinn M, Li Z Ref: Biomacromolecules, 10:3176, 2009 : PubMed
Enzyme-catalyzed polycondensation for the synthesis of block copolymers was reported for the first time. Thermoplastic block copolyesters containing poly[(R)-3-hydroxybutyrate] (PHB) and poly[(R)-3-hydroxyoctanoate] (PHO) blocks were enzymatically prepared by one- or two-step lipase-catalyzed polycondensation. Novozym 435-catalyzed reaction of PHB-diol (M(n) of 3100 g/mol, GPC), PHO-diol (M(n) of 3200 g/mol, GPC), and divinyl adipate gave block poly(HB-co-HO) (M(n) of 9800 g/mol, GPC) with randomly arranged blocks in 55% yield. In two-step polycondensations, Novozym 435-catalyzed reaction of PHB-diol and divinyl adipate afforded 73% of PHB containing two vinyl ester end groups (M(n) of 2700 g/mol, GPC), which was further reacted with PHO-diol in the presence of Novozym 435 to give block poly(HB-co-HO)s (M(n) of 8800-14 200 g/mol, GPC) with A-B-type arranged blocks in 55-62% yield. The enzymatically prepared block copolyesters demonstrated T(m) of 136-142 degrees C and 142-153 degrees C and T(g) of -37 to -39 degrees C and were potentially useful thermoplastic biodegradable and biocompatible materials.
Asthma is a common disease in children and young adults. Four separate reports have linked asthma and related phenotypes to an ill-defined interval between 2q14 and 2q32 (refs. 1-4), and two mouse genome screens have linked bronchial hyper-responsiveness to the region homologous to 2q14 (refs. 5,6). We found and replicated association between asthma and the D2S308 microsatellite, 800 kb distal to the IL1 cluster on 2q14. We sequenced the surrounding region and constructed a comprehensive, high-density, single-nucleotide polymorphism (SNP) linkage disequilibrium (LD) map. SNP association was limited to the initial exons of a solitary gene of 3.6 kb (DPP10), which extends over 1 Mb of genomic DNA. DPP10 encodes a homolog of dipeptidyl peptidases (DPPs) that cleave terminal dipeptides from cytokines and chemokines, and it presents a potential new target for asthma therapy.
