Biomedical subjects

Junmei Wang

Publications and source records attributed to Junmei Wang.

At least 19 recordsLinked to original sources

PubMed2026

Analysis of deep-resequencing data of 984 soybean accessions reveals structural variations underlying agronomic traits.

Genomic structural variants (SVs) are major sources of genetic variation and have profound impacts on phenotypic traits. However, their functional effects remain largely unexplored in soybean. Here, we resequence 940 soybean accessions. Together with 44 publicly available datasets, we identify 602,281 SVs. Using a graph-based genome, we detect an additional 58,760 presence/absence variations (PAVs) that broadly affect gene expression. Population genomic analyses reveal that SVs serve as a core driving force for soybean domestication and improvement. Integrating SVs with QTLs for oil and protein content, and performing GWAS on 27 traits, we identify key functional SVs. These include transposable element insertions altering seed coat color, multiple insertions within a cytochrome P450 gene modifying flower and hypocotyl color, and a GmMATE1 deletion enhancing seed size. Together, our study establishes a comprehensive SV map of soybean, offering a valuable resource for dissecting the genetic basis of complex traits to accelerate molecular breeding.

Glycine max↗

PubMed2025

EZH2 variants derived from cryptic splice sites govern distinct epigenetic patterns during embryonic development.

EZH2 catalyzes H3K27me3 and is essential for embryonic development. Although multiple EZH2 variants have been identified, the functional implications and physiological significance of its heterogeneity remain unclear. Here, we revealed that conserved cryptic splice sites generated two EZH2 variants with (EZH2A) or without (EZH2B) a 27-nt region, coding for a 9-aa segment. Structural modeling showed that splice-in or splice-off of the 9-aa segment caused a topological change in EZH2 structure. The 9-aa surplus in EZH2A strengthened its interaction with other PRC2 components, particularly in PRC2.2 holocomplex. We developed point-mutation mouse lines specifically depleting EZH2A or EZH2B (Ezh2amut or Ezh2bmut). Biallelic deletion of Ezh2a caused developmental defects and embryonic lethality between E12.5 and E15.5, while the Ezh2bmut mice were fertile and developed normally. Combined RNA-seq and CUT&Tag analyses in mouse embryonic fibroblasts revealed that EZH2A and EZH2B bound to different genomic loci and affected H3K27me3 deposition in different subsets of genes related to development or the innate immune system, respectively. EZH2A depletion specifically suppressed the expression of genes involved in the development-related Hippo-Yap1 pathway, which might be attributable to a compensatory process mediated by JARID2. Our findings demonstrate that EZH2 heterogeneity from the 9-aa splicing event plays a crucial role in development.

Animals↗

PubMed2006

New-generation amber united-atom force field.

We have developed a new-generation Amber united-atom force field for simulations involving highly demanding conformational sampling such as protein folding and protein-protein binding. In the new united-atom force field, all hydrogens on aliphatic carbons in all amino acids are united with carbons except those on Calpha. Our choice of explicit representation of all protein backbone atoms aims at minimizing perturbation to protein backbone conformational distributions and to simplify development of backbone torsion terms. Tests with dipeptides and solvated proteins show that our goal is achieved quite successfully. The new united-atom force field uses the same new RESP charging scheme based on B3LYP/cc-pVTZ//HF/6-31g** quantum mechanical calculations in the PCM continuum solvent as that in the Duan et al. force field. van der Waals parameters are empirically refitted starting from published values with respect to experimental solvation free energies of amino acid side-chain analogues. The suitability of mixing new point charges and van der Waals parameters with existing Amber covalent terms is tested on alanine dipeptide and is found to be reasonable. Parameters for all new torsion terms are refitted based on the new point charges and the van der Waals parameters. Molecular dynamics simulations of three small globular proteins in the explicit TIP3P solvent are performed to test the overall stability and accuracy of the new united-atom force field. Good agreements between the united-atom force field and the Duan et al. all-atom force field for both backbone and side-chain conformations are observed. In addition, the per-step efficiency of the new united-atom force field is demonstrated for simulations in the implicit generalized Born solvent. A speedup around two is observed over the Duan et al. all-atom force field for the three tested small proteins. Finally, the efficiency gain of the new united-atom force field in conformational sampling is further demonstrated with a well-known toy protein folding system, an 18 residue polyalanine in distance-dependent dielectric. The new united-atom force field is at least a factor of 200 more efficient than the Duan et al. all-atom force field for ab initio folding of the tested peptide.

Models, Molecular↗

PubMed2006

Automatic atom type and bond type perception in molecular mechanical calculations.

In molecular mechanics (MM) studies, atom types and/or bond types of molecules are needed to determine prior to energy calculations. We present here an automatic algorithm of perceiving atom types that are defined in a description table, and an automatic algorithm of assigning bond types just based on atomic connectivity. The algorithms have been implemented in a new module of the AMBER packages. This auxiliary module, antechamber (roughly meaning "before AMBER"), can be applied to generate necessary inputs of leap-the AMBER program to generate topologies for minimization, molecular dynamics, etc., for most organic molecules. The algorithms behind the manipulations may be useful for other molecular mechanical packages as well as applications that need to designate atom types and bond types.

Algorithms↗

PubMed2006

3D-QSAR studies of arylpyrazole antagonists of cannabinoid receptor subtypes CB1 and CB2. A combined NMR and CoMFA approach.

The present work focuses on the study of the three-dimensional (3D) structural requirements for selective antagonist activity of arylpyrazole compounds at the cannabinoid CB1 and CB2 receptors. Initially, a combined high-resolution two-dimensional (2D) NMR and computer modeling approach was carried out to study the solution structure of the key pyrazole derivative N-(piperidin-1-yl)-5-phenyl-1-(n-pentyl)-4-methyl-1H-pyrazole-3-carboxamide (AM263). By using the NMR-determined molecular conformers as templates, the 3D quantitative structure-activity relationship (QSAR) studies were performed with the comparative molecular field analysis (CoMFA) approach on a set of arylpyrazole cannabinoid receptor antagonists. Molecular alignments suitable for deriving valuable pharmacophoric features for this series of compounds were determined. Such systematic 3D-QSAR/CoMFA analyses of 29 molecules and their receptor affinities gave guidance for understanding the binding affinities of arylpyrazoles at the CB1 and CB2 binding sites, respectively. Comparison of CoMFA steric and potential contour maps for affinity at the two cannabinoid receptor subtypes helps to differentiate structural requirements for each subtype and serves as a basis for the design of later-generation analogues.

Amino Acid Motifs↗

PubMed2006

Recent advances in computational prediction of drug absorption and permeability in drug discovery.

Approximately 40%-60% of developing drugs failed during the clinical trials because of ADME/Tox deficiencies. Virtual screening should not be restricted to optimize binding affinity and improve selectivity; and the pharmacokinetic properties should also be included as important filters in virtual screening. Here, the current development in theoretical models to predict drug absorption-related properties, such as intestinal absorption, Caco-2 permeability, and blood-brain partitioning are reviewed. The important physicochemical properties used in the prediction of drug absorption, and the relevance of predictive models in the evaluation of passive drug absorption are discussed. Recent developments in the prediction of drug absorption, especially with the application of new machine learning methods and newly developed software are also discussed. Future directions for research are outlined.

Computer Simulation↗

PubMed2005

Identification of a specific inhibitor of the dishevelled PDZ domain.

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Hierarchical database screenings for HIV-1 reverse transcriptase using a pharmacophore model, rigid docking, solvation docking, and MM-PB/SA.

In this work, an efficient strategy was presented to search drug leads for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) using hierarchical database screenings, which included a pharmacophore model, multiple-conformation rigid docking, solvation docking, and molecular mechanics-Poisson-Boltzmann/surface area (MM-PB/SA) sequentially. Encouraging results were achieved in searching a refined available chemical directory (ACD) database: the enrichment factor after the first three filters was estimated to be 25-fold; the hit rate for all the four filters was predicted to be 41% in a control test using 37 known HIV-1 non-nucleoside reverse transcriptase inhibitors; 10 out of 30 promising solvation-docking hits had MM-PB/SA binding free energies better than -6.8 kcal/mol and the best one, HIT15, had -17.0 kcal/mol. In conclusion, the hierarchical multiple-filter database searching strategy is an attractive strategy in drug lead exploration.

Allosteric Site↗

PubMed2005

Characterization of flavonoids by aluminum complexation and collisionally activated dissociation.

Aluminum complexes of the type [Al(III) (flavonoid-H)2]+ are generated by electrospray ionization in order to allow differentiation of isomeric flavonoids by tandem mass spectrometry. The dominant species observed from the aluminum complexation reaction has a 1:2 aluminum(III):flavonoid stoichiometry. Differentiation of 18 flavonoids constituting seven isomeric series was achieved based on the collisionally activated dissociation patterns of the aluminum complexes. Characteristic fragmentation pathways allow identification of the site of glycosylation, the type of saccharide (rutinose versus neohesperidose) and the type of bond between the C-2 and C-3 atoms (thus distinguishing flavanones from flavonols and flavones). Two stable coordination geometries of the aluminum complex of apigenin were identified. The non-planar structure with a plane-angle of nearly 90 degrees is 25.3 kcal mol-1 more favorable than the planar structure. The conformations of the complexes, which involve multiple interactions between the aglycone and disaccharide portions of the flavonoid with the metal ion, are significantly different for the isomeric flavonoids.

Aluminum↗

PubMed2005

Threshold dissociation and molecular modeling of transition metal complexes of flavonoids.

The relative threshold dissociation energies of a series of flavonoid/transition metal/auxiliary ligand complexes of the type [MII (flavonoid - H) auxiliary ligand]+ formed by electrospray ionization (ESI) were measured by energy-variable collisionally activated dissociation (CAD) in a quadrupole ion trap (QIT). For each of the isomeric flavonoid diglycoside pairs, the rutinoside (with a 1-6 inter-saccharide linkage) requires a greater CAD energy and thus has a higher dissociation threshold than its neohesperidoside (with a 1-2 inter-saccharide linkage) isomer. Likewise, the threshold energies of complexes containing flavones are higher than those containing flavanones. The monoglycoside isomers also have characteristic threshold energies. The flavonoids that are glycosylated at the 3-O- position tend to have lower threshold energies than those glycosylated at the 7-O- or 4'-O- position, and those that are C- bonded have lower threshold energies than the O- bonded isomers. The structural features that substantially influence the threshold energies include the aglycon type (flavanone versus flavone), the type of disaccharide (rutinose versus neohesperidose), and the linkage type (O- bonded versus C- bonded). Various computational means were applied to probe the structures and conformations of the complexes and to rationalize the differences in threshold energies of isomeric flavonoids. The most favorable coordination geometry of the complexes has a plane-angle of about 62 degrees , which means that the deprotonated flavonoid and 2,2'-bipyridine within a complex do not reside on the same plane. Stable conformations of five cobalt complexes and five deprotonated flavonoids were identified. The conformations were combined with the point charges and helium accessible surface areas to explain qualitatively the differences in threshold energies for isomeric flavonoids.

Electrochemistry↗

PubMed2004

Development and testing of a general amber force field.

We describe here a general Amber force field (GAFF) for organic molecules. GAFF is designed to be compatible with existing Amber force fields for proteins and nucleic acids, and has parameters for most organic and pharmaceutical molecules that are composed of H, C, N, O, S, P, and halogens. It uses a simple functional form and a limited number of atom types, but incorporates both empirical and heuristic models to estimate force constants and partial atomic charges. The performance of GAFF in test cases is encouraging. In test I, 74 crystallographic structures were compared to GAFF minimized structures, with a root-mean-square displacement of 0.26 A, which is comparable to that of the Tripos 5.2 force field (0.25 A) and better than those of MMFF 94 and CHARMm (0.47 and 0.44 A, respectively). In test II, gas phase minimizations were performed on 22 nucleic acid base pairs, and the minimized structures and intermolecular energies were compared to MP2/6-31G* results. The RMS of displacements and relative energies were 0.25 A and 1.2 kcal/mol, respectively. These data are comparable to results from Parm99/RESP (0.16 A and 1.18 kcal/mol, respectively), which were parameterized to these base pairs. Test III looked at the relative energies of 71 conformational pairs that were used in development of the Parm99 force field. The RMS error in relative energies (compared to experiment) is about 0.5 kcal/mol. GAFF can be applied to wide range of molecules in an automatic fashion, making it suitable for rational drug design and database searching.

Nucleic Acids↗

PubMed2004

Discovery, modeling, and human pharmacokinetics of N-(2-acetyl-4,6-dimethylphenyl)-3-(3,4-dimethylisoxazol-5-ylsulfamoyl)thiophene-2-carboxamide (TBC3711), a second generation, ETA selective, and orally bioavailable endothelin antagonist.

Sitaxsentan (1) (Wu et al. J. Med. Chem. 1997, 40, 1690) is our first endothelin antagonist being evaluated in clinical trials. It has demonstrated biological effects in an acute hemodynamic study in CHF (Givertz et al. Circulation 2000, 101, 2922), an open-label 20-patient pulmonary hypertension trial (Barst et al. Chest 2002, 121, 1860-1868), and a 31-patient trial in essential hypertension (Calhoun et al. AHA Scientific Sessions 2000). In a phase 2b/3 pulmonary arterial hypertension trial, once a day treatment of 100 mg of sitaxsentan statistically significantly improved 6-min walk distance and NYHA class at 12 weeks (Barst et al. Am. J. Respir. Crit. Care Med. 2004, 169, 441). We have since reported on our efforts in generating follow-up compounds (Wu et al. J. Med. Chem. 1999, 42, 4485) and recently communicated that an ortho acyl group on the anilino ring enhanced oral absorption in this category of compounds (Wu et al. J. Med. Chem. 2001, 44, 1211). Here we report an expansion of this work by substituting a variety of electron-withdrawing groups at the ortho position and evaluating their effects on oral bioavailability as well as structure-activity relationships. As a result, TBC3711 (7z) was identified as our second endothelin antagonist to enter the clinic due to its good oral bioavailability (approximately 100%) in rats, high potency (ET(A) IC(50) = 0.08 nM), and optimal ET(A)/ET(B) selectivity (441 000-fold). Compound 7z has completed phase-I clinical development and was well tolerated with desirable pharmacokinetics in humans (t(1/2) = 6-7 h, oral availability > 80%).

Administration, Oral↗

PubMed2004

Development of single-domain recombinant antibodies to reverse transcriptase domain of human hTERT.

This paper describes the development of single-domain recombinant antibodies against human telomerase core protein. A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E. coli and used to immunize BALB/c mice. The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library. All VHs were cloned into phagemid vectors and displayed to generate 4 x 10(10) phage libraries. The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot. Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay. DNA sequencing proved their mouse VH origin. The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3. The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition.

Amino Acid Sequence↗

PubMed2003

Regulation of acetylcholine receptor clustering by the tumor suppressor APC.

At the developing neuromuscular junction, motor neuron-derived agrin triggers the differentiation of postsynaptic membrane into a highly specialized structure, where the nicotinic acetylcholine receptors (AChRs) are aggregated into high-density clusters. Agrin acts by activating the muscle-specific kinase MuSK and inducing coaggregation of the 43-kDa protein rapsyn with AChRs on muscle cell membrane. The signaling mechanism downstream of MuSK is poorly defined. We report here that the mouse tumor suppressor protein adenomatous polyposis coli (APC) has a role in AChR clustering and that the Wnt/beta-catenin pathway may crosstalk with agrin signaling cascade during synapse formation.

Adenomatous Polyposis Coli Protein↗

PubMed2003

A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations.

Molecular mechanics models have been applied extensively to study the dynamics of proteins and nucleic acids. Here we report the development of a third-generation point-charge all-atom force field for proteins. Following the earlier approach of Cornell et al., the charge set was obtained by fitting to the electrostatic potentials of dipeptides calculated using B3LYP/cc-pVTZ//HF/6-31G** quantum mechanical methods. The main-chain torsion parameters were obtained by fitting to the energy profiles of Ace-Ala-Nme and Ace-Gly-Nme di-peptides calculated using MP2/cc-pVTZ//HF/6-31G** quantum mechanical methods. All other parameters were taken from the existing AMBER data base. The major departure from previous force fields is that all quantum mechanical calculations were done in the condensed phase with continuum solvent models and an effective dielectric constant of epsilon = 4. We anticipate that this force field parameter set will address certain critical short comings of previous force fields in condensed-phase simulations of proteins. Initial tests on peptides demonstrated a high-degree of similarity between the calculated and the statistically measured Ramanchandran maps for both Ace-Gly-Nme and Ace-Ala-Nme di-peptides. Some highlights of our results include (1) well-preserved balance between the extended and helical region distributions, and (2) favorable type-II poly-proline helical region in agreement with recent experiments. Backward compatibility between the new and Cornell et al. charge sets, as judged by overall agreement between dipole moments, allows a smooth transition to the new force field in the area of ligand-binding calculations. Test simulations on a large set of proteins are also discussed.

Algorithms↗

PubMed2002

Molecular dynamics and free energy analyses of cathepsin D-inhibitor interactions: insight into structure-based ligand design.

In this study, we compare the calculated and experimental binding free energies for a combinatorial library of inhibitors of cathepsin D (CatD), an aspartyl protease. Using a molecular dynamics (MD)-based, continuum solvent method (MM-PBSA), we are able to reproduce the experimental binding affinity for a set of seven inhibitors with an average error of ca. 1 kcal/mol and a correlation coefficient of 0.98. By comparing the dynamical conformations of the inhibitors complexed with CatD with the initial conformations generated by CombiBuild (University of California, San Francisco, CA, 1995), we have found that the docking conformation observed in an X-ray structure of one peptide inhibitor bound to CatD (Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6796-6800) is in good agreement with our MD simulation. However, the DOCK scoring function, based on intermolecular van der Waals and electrostatics, using a distance-dependent dielectric constant (J. Comput. Chem. 1992, 13, 505-524), poorly reproduces the trend of experimental binding affinity for these inhibitors. Finally, the use of PROFEC (J. Comput.-Aided Mol. Des. 1998, 12, 215-227) analysis allowed us to identify two possible substitutions to improve the binding of one of the better inhibitors to CatD. This study offers hope that current methods of estimating the free energy of binding are accurate enough to be used in a multistep virtual screening protocol.

Algorithms↗

PubMed2002

Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently (15)N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.

Amino Acid Sequence↗

PubMed2002

Intrastriatal grafting of glomus cells ameliorates behavioral defects of Parkinsonian rats.

Parkinson's disease is associated with severe motor dysfunctions due to a progressive loss of dopaminergic neurons in substantia nigra. Transplantation of midbrain neurons from human fetuses to the striatum of patients provides effective treatment for the disease. This type of approach, however, could not be adopted widely due to insufficient supply of fetal materials and the controversial ethical and legal issues. The carotid body is a chemoreceptive organ containing chromaffin-like glomus cells that secrete dopamine (DA) as the neurotransmitter. Here, we report the generation of a clonal dopaminergic cell line of the carotid body using the H-2K(b)-tsA58 transgenic mouse. Cells from the carotid body were immortalized at the permissive temperatures and in the presence of gamma-interferon. The glomus cells were isolated by flow cytometry, and purified to homogeneity by a limited dilution procedure. Upon switching the culture to a nonpermissive condition, the immortal cells ceased to divide, became terminally differentiated and secreted high levels of DA. In rats rendered hemi-Parkinsonian by injection of 6-hydroxydopamine (6-OHDA) into the substantial nigra, intrastriatal grafting of the glomus cells resulted in significant recovery of motor asymmetries and sensorimotor dysfunction. The effects were apparent approximately 10 days after transplantation and remained throughout the 4 months of the study. The recovery of behavioral defects was correlated with the ability of cell grafts to release DA in the brain. As none of the existing treatments for Parkinson's disease is completely satisfactory, establishment of a clonal cell line that secretes DA opens a new avenue for the effective control of this neurological disorder.

Amphetamine↗