Title: Selective modulation of alkali metal ions on acetylcholinesterase Mu X, Yuan S, Zhang D, Lai R, Liao C, Li G Ref: Phys Chem Chem Phys, :, 2023 : PubMed
Acetylcholinesterase (AChE) is an important hydrolase in cholinergic synapses and a candidate target in the treatment of Alzheimer's disease. The lithium treatment widely used in neurological disorders can alter the AChE activity, yet the underlying mechanism of how the ion species regulate the enzymatic activity remains unclear. In this work, we performed combined quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations and well-tempered metadynamics to understand the modulation of human AChE (hAChE) activity using three alkali metal ions (Li(+), Na(+), and K(+)) in different concentrations. Our simulations show that the binding affinity and catalytic activity are affected by different ion species through allosteric ion coordination geometries on the hAChE complex and distant electrostatic screening effect. A Li(+) cluster involving D330, E393, and D397 residues and three Li(+) ions was found to be highly conserved and can be critical to the enzyme activity. Binding energy calculations indicate that the electrostatic screening from allosterically bound cations can affect the key residues at the catalytic site and active-site gorge, including E199. Furthermore, an increase in ion concentration can lead to lower reactivity, especially for Li(+) ions, which exhibit more cation-hAChE contacts than Na(+) and K(+). The selective ion binding and their preferred modulation on hAChE are highly related to ion species. This work provides a molecular perspective on selective modulation by different ion species of the enzyme catalytic processes.
Title: Two New Picoline-Derived Meroterpenoids with Anti-Acetylcholinesterase Activity from Ascidian-Derived Fungus Amphichorda felina Jiang M, Guo H, Wu Q, Yuan S, Liu L Ref: Molecules, 27:, 2022 : PubMed
Amphichoterpenoids D (1) and E (2), two new picoline-derived meroterpenoids with a rare 6/6/6 tricyclic pyrano[3,2-c]pyridinyl-gamma-pyranone scaffold, were isolated from the ascidian-derived fungus Amphichorda felina SYSU-MS7908. Their structures, including the absolute configurations, were established by extensive spectroscopic methods (1D and 2D NMR and high-resolution mass spectrometry) and ECD calculations. Compounds 1 and 2 showed anti-acetylcholinesterase (anti-AChE) activities with IC(50) values of 12.5 microM and 11.6 microM, respectively. The binding interactions between 1, 2, and AChE were investigated using molecular docking analyses.
Shoot branching is regulated by multiple signals. Previous studies have indicated that sucrose may promote shoot branching through suppressing the inhibitory effect of the hormone strigolactone (SL). However, the molecular mechanisms underlying this effect are unknown. Here, we used molecular and genetic tools to identify the molecular targets underlying the antagonistic interaction between sucrose and SL. We showed that sucrose antagonizes the suppressive action of SL on tillering in rice and on the degradation of D53, a major target of SL signalling. Sucrose inhibits the gene expression of D3, the orthologue of the Arabidopsis F-box MAX2 required for SL signalling. Overexpression of D3 antagonizes sucrose inhibition of D53 degradation and enables the SL inhibition of tillering under high sucrose. Sucrose prevents SL-induced degradation of D14, the SL receptor involved in D53 degradation. In contrast to D3, D14 overexpression enhances D53 protein levels and sucrose-induced tillering, even in the presence of SL. Our results show that sucrose inhibits SL response by affecting key components of SL signalling and, together with previous studies reporting the inhibition of SL synthesis by nitrate and phosphate, demonstrate the central role played by SLs in the regulation of plant architecture by nutrients.
Title: Structure-Guided Rational Design of a Mono- and Diacylglycerol Lipase from Aspergillus oryzae: A Single Residue Mutant Increases the Hydrolysis Ability Lan D, Zhao G, Holzmann N, Yuan S, Wang J, Wang Y Ref: Journal of Agricultural and Food Chemistry, 69:5344, 2021 : PubMed
Engineering of enzymes on the basis of protein structures are rational and efficient approaches to acquire biocatalysts of desired performances. In this study, we focused on a special mono- and diacylglycerol lipase (MDGL) isolated from the lipolytic enzyme-enriched fungus Aspergillus oryzae and discovered improved variants based on its crystal structure. We first solved the crystal structure of Aspergillus oryzae lipase (AOL) at 1.7 A resolution. Structure analysis and sequence alignment of AOL and other MDGLs revealed that the residue V269 is of vital importance for catalysis. Replacement of the V269 in AOL with the corresponding residues in other MDGLs has led to noticeable changes in hydrolysis without sacrificing the thermostability and substrate specificity. Among the investigated variants, V269D exhibited about a six-fold higher hydrolysis activity compared to the wild type. Molecular dynamics simulations and protein-ligand interaction frequency analyses revealed that the Asp substitution enhanced the substrate affinity of AOL. Our work sheds light on understanding the catalytic process of AOL and helps tailoring MDGLs with desired catalytic performance to fulfill the demand for biotechnological applications.
Traditionally, it is believed that the substrate and products of a monoacylglycerol lipase (MGL) share the same path to enter and exit the catalytic site. Glycerol (a product of MGL), however, was recently hypothesized to be released through a different path. In order to improve the catalytic efficacy and thermo-stability of MGL, it is important to articulate the pathways of a MGL products releasing. In this study, with structure biological approaches, biochemical experiments, and in silico methods, we prove that glycerol is released from a different path in the catalytic site indeed. The fatty acid (another product of MGL) does share the same binding path with the substrate. This discovery paves a new road to design MGL inhibitors or optimize MGL catalytic efficacy.
Title: Structure-guided engineering of a Thermobifida fusca cutinase for enhanced hydrolysis on natural polyester substrate Dong Q, Yuan S, Wu L, Su L, Zhao Q, Wu J, Huang W, Zhou J Ref: Bioresour. Bioprocess, 7:37, 2020 : PubMed
Cutinases could degrade insoluble polyester, including natural cutin and synthetic plastic. However, their turnover efficiency for polyester remains too low for industrial application. Herein, we report the 1.54-A resolution X-ray crystal structure of a cutinase from Thermobifida fusca and modeling structure in complex with a cutin mimic oligo-polyester C24H42O8. These efforts subsequently guided our design of cutinase variants with less bulky residues in the vicinity of the substrate binding site. The L90A and I213A variants exhibit increased hydrolysis activity (5- and 2.4-fold, respectively) toward cutin and also showed enhanced cotton scouring efficiency compared with the wild-type enzyme.
Title: Aryl-phosphorus-containing flame retardants induce oxidative stress, the p53-dependent DNA damage response and mitochondrial impairment in A549 cells Yuan S, Han Y, Ma M, Rao K, Wang Z, Yang R, Liu Y, Zhou X Ref: Environ Pollut, 250:58, 2019 : PubMed
Aryl phosphorus-containing flame retardants (aryl-PFRs) have been frequently detected with increasingly used worldwide as one of alternatives for brominated flame retardants. However, information on their adverse effects on human health and ecosystem is insufficient, with limited study on their molecular mode of action insvitro. In this study, the cytotoxicity, DNA damage, mitochondrial impairment and the involved molecular mechanisms of certain frequently detectable aryl-PFRs, including 2-ethylhexyldiphenyl phosphate (EHDPP), methyl diphenyl phosphate (MDPP), bisphenol-A bis (diphenyl phosphate) (BDP), isodecyl diphenyl phosphate (IDPP), cresyl diphenyl phosphate (CDP) and the structurally similar and widely used organophosphorus pesticide chlorpyrifos (CPF), were evaluated in A549 cells using high-content screening (HCS) system. Aryl-PFRs showed different lethal concentration 50 (LC50) values ranging from 97.94 to 546.85 microM in A549 cells using CCK-8 assay. EHDPP, IDPP, CDP, MDPP and CPF demonstrated an ability to induce DNA damage, evidenced by increased DNA content and S phase-reducing cell cycle arrest effect using fluorophore dye cocktail assay. Additionally, the selected aryl-PFRs induced mitochondrial impairment by the increasing mitochondrial mass and decreasing mitochondrial membrane potential. Moreover, BDP, MDPP, and CDP, which contain short alkyl chains showed their potential oxidative stress with intracellular ROS and mitochondrial superoxide overproduction from an initially relatively low concentration. Additionally, based on the promotion of firefly luminescence in p53-transfected A549 cells, p53 activation was found to be involved in aryl-PFRs-induced DNA damage. Further real-time PCR results showed that all selected aryl-PFRs triggered p53/p21/gadd45beta-, and p53/p21/mdm2-mediated cell cycle pathways, and the p53/bax mediated apoptosis pathway to induce DNA damage and cytotoxic effects. These results suggest that aryl-PFRs (e.g., BDP, MDPP, CDP) cause oxidative stress-mediated DNA damage and mitochondrial impairment, and p53-dependent pathway was involved in the aryl-PFRs-induced DNA damage and cell cycle arrest. In conclusion, this study improves the understanding of PFRs-induced adverse outcomes and the involved molecular mechanism.
BACKGROUND: Biomarker studies revealed important clinical significance of exosome for cancer patients. However, there is currently no consensus on exosome quantification methods. METHODS: Bicinchoninic acid (BCA) method, acetylcholinesterase (AChE) method and nanoparticle tracking analysis (NTA) were utilized to quantify 20 plasma exosome samples, and interrelations between these three methods were explored. Associations of plasma exosome levels with characteristics and prognosis of 208 non-small-cell lung cancer (NSCLC) patients were investigated. RESULTS: Results of the three methods for exosome quantification were significantly correlated with each other. Correlation coefficient between AChE and NTA (r= 0.79, P< 0.001) was greater than that between BCA and NTA (r= 0.64, P= 0.003). Plasma exosome levels of 208 NSCLC patients were then quantified with AChE method. Exosome level was significantly associated with tumour stage (P< 0.001) and the history of chronic obstructive pulmonary disease (P= 0.023). Cox proportional hazard analysis demonstrated that higher exosome level was independently associated with poorer overall survival (P= 0.033; hazard ratio = 1.72, 95% confidence interval: 1.05-2.83). CONCLUSIONS: Plasma exosome level correlates with tumor stage and the history of chronic obstructive pulmonary disease, and may serve as a prognostic factor for NSCLC.
Title: Cigarette Smoke-Induced Pulmonary Inflammation and Autophagy Are Attenuated in Ephx2-Deficient Mice Li Y, Yu G, Yuan S, Tan C, Lian P, Fu L, Hou Q, Xu B, Wang H Ref: Inflammation, 40:497, 2017 : PubMed
Cigarette smoke (CS) increases the risk of chronic obstructive pulmonary disease (COPD) by causing inflammation, emphysema, and reduced lung function. Additionally, CS can induce autophagy which contributes to COPD. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) have promising anti-inflammatory properties that may protect the heart and liver by regulating autophagy. For this reason, the effect of decreased soluble epoxide hydrolase (sEH, Ephx2)-mediated EET hydrolysis on inflammation, emphysema, lung function, and autophagy was here studied in CS-induced COPD in vivo. Adult male wild-type (WT) C57BL/6J and Ephx2-/- mice were exposed to air or CS for 12 weeks, and lung inflammatory responses, air space enlargement (emphysema), lung function, and autophagy were assessed. Lungs of Ephx2-/- mice had a less pronounced inflammatory response and less autophagy with mild distal airspace enlargement accompanied by restored lung function and steady weight gain. These findings support the idea that Ephx2 may hold promise as a therapeutic target for COPD induced by CS, and it may be protective property by inhibiting autophagy.
Title: Chlorogenic acid protects against aluminium-induced cytotoxicity through chelation and antioxidant actions in primary hippocampal neuronal cells Wang X, Fan X, Yuan S, Jiao W, Liu B, Cao J, Jiang W Ref: Food Funct, 8:2924, 2017 : PubMed
Chlorogenic acid (CGA), a major polyphenolic component of many plants, displays antioxidant and neuroprotective properties in neurodegenerative diseases. To investigate whether CGA may influence aluminium (Al) induced cytotoxicity, aluminium chloride (50 muM Al) was administered in primary hippocampal neuronal cells presupplemented with CGA (10, 50 and 100 muM). Our study shows that the exposure to Al caused cell death, Al(3+) accumulation, reactive oxygen species generation and mitochondrial damage in cells. The administration of CGA (50 muM) increased cell viability by 37.5%, decreased the levels of Al(3+) by 26.0%, together with significantly weakening the oxidative damage compared with Al treatment alone. CGA protected neurons against Al-induced oxidative stress by increasing the expression of nuclear factor-E2-related factor 2 and its target phase 2 enzymes. The administration of CGA remarkably promoted the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, creatine kinase and acetylcholinesterase and attenuated the rate of ATP hydrolysis. Our finding shows that CGA has neuroprotective effects against Al-induced cytotoxicity by chelation and antioxidant activation.
Title: Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates Kong XD, Yuan S, Li L, Chen S, Xu JH, Zhou J Ref: Proc Natl Acad Sci U S A, 111:15717, 2014 : PubMed
Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other beta-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward alpha-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward alpha-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.
Strigolactones (SLs) are a novel class of plant hormones that inhibit shoot branching. Currently, two proteins in rice are thought to play crucial roles in SL signal transduction. DWARF14 (D14), an alpha/beta hydrolase, is responsible for SL perception, while DWARF3 (D3), an F-box protein with leucine-rich repeats, is essential for SL signal transduction. However, how these two proteins transmit SL signals to downstream factors remains unclear. Here, we characterized a high-tillering dwarf rice mutant, gsor300097, which is insensitive to GR24, a synthetic analog of SL. Mapping and sequencing analysis showed that gsor300097 is a novel allelic mutant of D3, in which a nonsense mutation truncates the protein from 720 to 527 amino acids. The D3 gene was strongly expressed in root, leaf, shoot base and panicle. Nuclear-localized F-box protein D3 played a role in the SCF complex by interacting with OSK1, OSK5 or OSK20 and OsCullin1. In addition, D3 associated with D14 in a GR24-dependent manner in vivo. Taken together, our findings suggested that D3 assembled into an SCF(D3) complex and associated with D14 to suppress rice shoot branching.
Title: Cloning, expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T Wei X, Jiang X, Ye L, Yuan S, Chen Z, Wu M, Yu H Ref: J Mol Catal B Enzym, 97:270, 2013 : PubMed
An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p-nitrophenyl esters (C2-C6), exhibited maximum activity toward p-nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts.
Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.
