Title: Integrative Analysis of Transcriptome and Metabolome Reveals Molecular Responses in Eriocheir sinensis with Hepatopancreatic Necrosis Disease Zhan M, Wen L, Zhu M, Gong J, Xi C, Wen H, Xu G, Shen H Ref: Biology (Basel), 11:, 2022 : PubMed
Hepatopancreatic necrosis disease (HPND) is a highly lethal disease that first emerged in 2015 in Jiangsu Province, China. So far, most researchers believe that this disease is caused by abiotic factors. However, its true pathogenic mechanism remains unknown. In this study, the effects of HPND on the metabolism and other biological indicators of the Chinese mitten crab (Eriocheir sinensis) were evaluated by integrating transcriptomics and metabolomics. Our findings demonstrate that the innate immunity, antioxidant activity, detoxification ability, and nervous system of the diseased crabs were affected. Additionally, metabolic pathways such as lipid metabolism, nucleotide metabolism, and protein metabolism were dysregulated, and energy production was slightly increased. Moreover, the IL-17 signaling pathway was activated and high levels of autophagy and apoptosis occurred in diseased crabs, which may be related to hepatopancreas damage. The abnormal mitochondrial function and possible anaerobic metabolism observed in our study suggested that functional hypoxia may be involved in HPND progression. Furthermore, the activities of carboxylesterase and acetylcholinesterase were significantly inhibited, indicating that the diseased crabs were likely stressed by pesticides such as pyrethroids. Collectively, our findings provide new insights into the molecular mechanisms altered in diseased crabs, as well as the etiology and pathogenic mechanisms of HPND.
        
Title: Catalytic activities of cocaine hydrolases against the most toxic cocaine metabolite norcocaethylene Zheng X, Chen X, Zhang T, Zhan M, Zhan CG, Zheng F Ref: Org Biomol Chem, :, 2020 : PubMed
A majority of cocaine users also consume alcohol. The concurrent use of cocaine and alcohol produces the pharmacologically active metabolites cocaethylene and norcocaethylene, in addition to norcocaine. Both cocaethylene and norcocaethylene are more toxic than cocaine itself. Hence, a truly valuable cocaine-metabolizing enzyme for cocaine abuse/overdose treatment should be effective for the hydrolysis of not only cocaine, but also its metabolites norcocaine, cocaethylene, and norcocaethylene. However, there has been no report on enzymes capable of hydrolyzing norcocaethylene (the most toxic metabolite of cocaine). The catalytic efficiency parameters (kcat and KM) of human butyrylcholinesterase (BChE) and two mutants (known as cocaine hydrolases E14-3 and E12-7) against norcocaethylene have been characterized in the present study for the first time, and they are compared with those against cocaine. According to the obtained kinetic data, wild-type human BChE showed a similar catalytic efficiency against norcocaethylene (kcat = 9.5 min-1, KM = 11.7 muM, and kcat/KM = 8.12 x 105 M-1 min-1) to that against (-)-cocaine (kcat = 4.1 min-1, KM = 4.5 muM, and kcat/KM = 9.1 x 105 M-1 min-1). E14-3 and E12-7 showed an improved catalytic activity against norcocaethylene compared to wild-type BChE. E12-7 showed a 39-fold improved catalytic efficiency against norcocaethylene (kcat = 210 min-1, KM = 6.6 muM, and kcat/KM = 3.18 x 107 M-1 min-1). It has been demonstrated that E12-7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.
Cocaine is one of the most addictive drugs without a U.S. Food and Drug Administration (FDA)-approved medication. Enzyme therapy using an efficient cocaine-metabolizing enzyme is recognized as the most promising approach to cocaine overdose treatment. The actual enzyme, known as RBP-8000, under current clinical development for cocaine overdose treatment is our previously designed T172R/G173Q mutant of bacterial cocaine esterase (CocE). The T172R/G173Q mutant is effective in hydrolyzing cocaine but inactive against benzoylecgonine (a major, biologically active metabolite of cocaine). Unlike cocaine itself, benzoylecgonine has an unusually stable zwitterion structure resistant to further hydrolysis in the body and environment. In fact, benzoylecgonine can last in the body for a very long time (a few days) and, thus, is responsible for the long-term toxicity of cocaine and a commonly used marker for drug addiction diagnosis in pre-employment drug tests. Because CocE and its mutants are all active against cocaine and inactive against benzoylecgonine, one might simply assume that other enzymes that are active against cocaine are also inactive against benzoylecgonine. Here, through combined computational modeling and experimental studies, we demonstrate for the first time that human butyrylcholinesterase (BChE) is actually active against benzoylecgonine, and that a rationally designed BChE mutant can not only more efficiently accelerate cocaine hydrolysis but also significantly hydrolyze benzoylecgonine in vitro and in vivo. This sets the stage for advanced studies to design more efficient mutant enzymes valuable for the development of an ideal cocaine overdose enzyme therapy and for benzoylecgonine detoxification in the environment.
        
Title: Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene Hou S, Zhan M, Zheng X, Zhan CG, Zheng F Ref: Biochemical Journal, 460:447, 2014 : PubMed
It is known that the majority of cocaine users also consume alcohol. Alcohol can react with cocaine to produce a significantly more cytotoxic compound, cocaethylene. Hence a truly valuable cocaine-metabolizing enzyme as treatment for cocaine abuse/overdose should be efficient for not only cocaine itself, but also cocaethylene. The catalytic parameters (kcat and KM) of human BChE (butyrylcholinesterase) and two mutants (known as cocaine hydrolases E14-3 and E12-7) for cocaethylene are characterized in the present study, for the first time, in comparison with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a lower catalytic activity for cocaethylene (kcat=3.3 min-1, KM=7.5 muM and kcat/KM=4.40x105 M-1.min-1) compared with its catalytic activity for (-)-cocaine. E14-3 and E12-7 have a considerably improved catalytic activity against cocaethylene compared with the wild-type BChE. E12-7 is identified as the most efficient enzyme for hydrolysing cocaethylene in addition to its high activity for (-)-cocaine. E12-7 has an 861-fold improved catalytic efficiency for cocaethylene (kcat=3600 min-1, KM=9.5 muM and kcat/KM=3.79x108 M-1.min-1). It has been demonstrated that E12-7 as an exogenous enzyme can indeed rapidly metabolize cocaethylene in rats. Further kinetic modelling has suggested that E12-7 with an identical concentration as that of the endogenous BChE in human plasma can effectively eliminate (-)-cocaine, cocaethylene and norcocaine in simplified kinetic models of cocaine abuse and overdose associated with the concurrent use of cocaine and alcohol.
        
Title: Kinetic characterization of high-activity mutants of human butyrylcholinesterase for the cocaine metabolite norcocaine Zhan M, Hou S, Zhan CG, Zheng F Ref: Biochemical Journal, 457:197, 2014 : PubMed
It has been known that cocaine produces its toxic and physiological effects through not only cocaine itself, but also norcocaine formed from cocaine oxidation catalysed by microsomal CYP (cytochrome P450) 3A4 in the human liver. The catalytic parameters (kcat and Km) of human BChE (butyrylcholinesterase) and its three mutants (i.e. A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/E441D and A199S/F227A/S287G/A328W/Y332G) for norcocaine have been characterized in the present study for the first time and compared with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a significantly lower catalytic activity for norcocaine (kcat=2.8 min-1, Km=15 muM and kcat/Km=1.87x105 M-1.min-1) compared with its catalytic activity for (-)-cocaine. The BChE mutants examined in the present study have considerably improved catalytic activities against both cocaine and norcocaine compared with the wild-type enzyme. Within the enzymes examined in the present study, the A199S/F227A/S287G/A328W/Y332G mutant (CocH3) is identified as the most efficient enzyme for hydrolysing both cocaine and norcocaine. CocH3 has a 1080-fold improved catalytic efficiency for norcocaine (kcat=2610 min-1, Km=13 muM and kcat/Km=2.01x108 M-1.min-1) and a 2020-fold improved catalytic efficiency for cocaine. It has been demonstrated that CocH3 as an exogenous enzyme can rapidly metabolize norcocaine, in addition to cocaine, in rats. Further kinetic modelling has suggested that CocH3 with an identical concentration with that of the endogenous BChE in human plasma can effectively eliminate both cocaine and norcocaine in a simplified kinetic model of cocaine abuse.
        
Title: Modeling in vitro inhibition of butyrylcholinesterase using molecular docking, multi-linear regression and artificial neural network approaches Zheng F, Zhan M, Huang X, Abdul Hameed MD, Zhan CG Ref: Bioorganic & Medicinal Chemistry, 22:538, 2014 : PubMed
Butyrylcholinesterase (BChE) has been an important protein used for development of anti-cocaine medication. Through computational design, BChE mutants with approximately 2000-fold improved catalytic efficiency against cocaine have been discovered in our lab. To study drug-enzyme interaction it is important to build mathematical model to predict molecular inhibitory activity against BChE. This report presents a neural network (NN) QSAR study, compared with multi-linear regression (MLR) and molecular docking, on a set of 93 small molecules that act as inhibitors of BChE by use of the inhibitory activities (pIC50 values) of the molecules as target values. The statistical results for the linear model built from docking generated energy descriptors were: r(2)=0.67, rmsd=0.87, q(2)=0.65 and loormsd=0.90; the statistical results for the ligand-based MLR model were: r(2)=0.89, rmsd=0.51, q(2)=0.85 and loormsd=0.58; the statistical results for the ligand-based NN model were the best: r(2)=0.95, rmsd=0.33, q(2)=0.90 and loormsd=0.48, demonstrating that the NN is powerful in analysis of a set of complicated data. As BChE is also an established drug target to develop new treatment for Alzheimer's disease (AD). The developed QSAR models provide tools for rationalizing identification of potential BChE inhibitors or selection of compounds for synthesis in the discovery of novel effective inhibitors of BChE in the future.
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.
Reductive cyclization of 5-hydroxy-3-methyl-3-methoxycarbonylmethylenebenzofuran-2(3H)-one (4) gave 5-hydroxy-3a-methyl-2,3,3a,8a-tatrahydrofuro[2,3-b]benzofuran (5) and the rearrangement product 7-hydroxy-4,5-dihydro-2,5-methano-1,3-benzodioxepine (6). Reaction of compounds 5 and 6 with different isocyanates provided two series novel carbamates (7-12) whose structures were confirmed by X-ray crystallography. These were assessed for anticholinesterase action against freshly prepared human enzyme and proved to be potent inhibitors of either acetyl- (AChE) or butyrylcholinesterase (BChE) with specific compounds exhibiting remarkable selectivity. Because the two series of carbamates (7-12) differ in their phenolic moieties, their respective potency and selectivity for AChE versus BChE was governed by their N-substituted groups. This same characteristic was also present in a series of physovenine analogues (1, 13, 15, 17) and physostigmine analogues (2, 14, 16, 18). These structure-activity relations proved valuable in elucidating the mechanisms underpinning the interaction between carbamate-based cholinesterase inhibitors and their enzyme target. In addition, because physostigmine analogues have demonstrated activity in lowering the Alzheimer's disease protein, amyloid precursor protein (APP), examples of the two new series of carbamates were characterized in culture assays of quantifying cell viability and synthesis of APP.
        
Title: Genetic determinants of low high-density lipoprotein cholesterol Miller M, Zhan M Ref: Curr Opin Cardiol, 19:380, 2004 : PubMed
PURPOSE OF REVIEW: High-density lipoprotein cholesterol (HDL-C) has been well established as an inverse predictor of coronary heart disease (CHD), and in recent years, investigations have focused on the genetic regulation of high-density lipoprotein. Although numerous candidate genes contribute to the low HDL-C phenotype, their impact on CHD is heterogeneous, reflecting diverse gene-gene interactions and gene-environmental relationships. This review summarizes recent data involving HDL regulatory genes and their role in atherothrombosis. RECENT FINDINGS: The primary genetic determinants associated with relative HDL-C deficiency states are the ATP binding cassette protein, ABCA1; apolipoprotein (APO) A1; and lecithin cholesteryl acyl transferase. Other potentially important candidates invoked in low HDL-C syndromes in humans include APOC3, lipoprotein lipase, sphingomyelin phosphodiesterase 1, and glucocerebrosidase. Molecular variation in ABCAI and APOAI and, in selected cases, lecithin cholesteryl acyl transferase deficiency have been associated with increased CHD, whereas two notable variants, APOAIMilano and APOAIParis, are associated with reduced risk. SUMMARY: Low HDL-C syndromes have generally been correlated with an increased risk of CHD. However, single-gene abnormalities responsible for HDL-C deficiency states may have variable effects on atherothrombotic risk.
A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.
Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.