Splicing factor TRA2A contributes to esophageal cancer progression via a noncanonical role in lncRNA m6 A methylation.

Transformer 2 alpha homolog (TRA2A), a member of the serine/arginine-rich splicing factor family, has been shown to control mRNA splicing in development and cancers. However, it remains unclear whether TRA2A is involved in lncRNA regulation. In the present study, we found that TRA2A was upregulated and correlated with poor prognosis in esophageal cancer. Downregulation of TRA2A suppressed the tumor growth in xenograft nude mice. Epitranscriptomic microarray showed that depletion of TRA2A affected global lncRNA methylation similarly to the key m6 A methyltransferase, METTL3, by silencing. MeRIP-qPCR, RNA pull-down, CLIP analyses, and stability assays indicated that ablation of TRA2A reduced m6 A-modification of the oncogenic lncRNA MALAT1, thus inducing structural alterations and reduced stability. Furthermore, Co-IP experiments showed TRA2A directly interacted with METTL3 and RBMX, which also affected the writer KIAA1429 expression. Knockdown of TRA2A inhibited cell proliferation in a manner restored by RBMX/KIAA1429 overexpression. Clinically, MALAT1, RBMX, and KIAA1429 were prognostic factors of worse survival in ESCA patients. Structural similarity-based virtual screening in FDA-approved drugs repurposed nebivolol, a ß1 -adrenergic receptor antagonist, as a potent compound to suppress the proliferation of esophageal cancer cells. Cellular thermal shift and RIP assay indicated that nebivolol may compete with MALAT1 to bind TRA2A. In conclusion, our study revealed the noncanonical function of TRA2A, which coordinates with multiple methylation proteins to promote oncogenic MALAT1 during ESCA carcinogenesis.

fRNC: Uncovering the dynamic and condition-specific RBP-ncRNA circuits from multi-omics data.

The RNA binding protein (RBP) and non-coding RNA (ncRNA) interacting networks are increasingly recognized as the main mechanism in gene regulation, and are tightly associated with cellular malfunction and disease. Here, we present fRNC, a systems biology tool to uncover the dynamic spectrum of RBP-ncRNA circuits (RNC) by integrating transcriptomics, interactomics and proteomics data. fRNC constructs the RBP-ncRNA network derived from CLIP-seq or PARE experiments. Given scoring on nodes and edges according to differential analysis of expression data, it finds an RNC containing global maximum significant RBPs and ncRNAs. Alternatively, it can also capture the locally maximum scoring RNC according to user-defined starting nodes with the greedy search. When compared with existing tools, fRNC can detect more accurate and robust sub-network with scalability. As shown in the cases of esophageal carcinoma, breast cancer and Alzheimer's disease, fRNC enables users to analyze the collective behaviors between RBP and the interacting ncRNAs, and reveal novel insights into the disease-associated processes. The fRNC R package is available at https://github.com/BioinformaticsSTU/fRNC.

Redox Potentials of Disulfide Bonds in LOXL2 Studied by Nonequilibrium Alchemical Simulation.

Lysyl oxidase-like 2 (LOXL2) is a metalloenzyme that catalyzes the oxidative deamination ε-amino group of lysine. It is found that LOXL2 is a promotor for the metastasis and invasion of cancer cells. Disulfide bonds are important components in LOXL2, and they play a stabilizing role for protein structure or a functional role for regulating protein bioactivity. The redox potential of disulfide bond is one important property to determine the functional role of disulfide bond. In this study, we have calculated the reduction potential of all the disulfide bonds in LOXL2 by non-equilibrium alchemical simulations. Our results show that seven of seventeen disulfide bonds have high redox potentials between -182 and -298 mV and could have a functional role, viz., Cys573-Cys625, Cys579-Cys695, Cys657-Cys673, and Cys663-Cys685 in the catalytic domain, Cys351-Cys414, Cys464-Cys530, and Cys477-Cys543 in the scavenger receptor cysteine-rich (SRCR) domains. The disulfide bond of Cys351-Cys414 is predicted to play an allosteric function role, which could affect the metastasis and invasion of cancer cells. Other functional bonds have a catalytic role related to enzyme activity. The rest of disulfide bonds are predicted to play a structural role. Our study provides an important insight for the classification of disulfide bonds in LOXL2 and can be utilized for the drug design that targets the cysteine residues in LOXL2.

New insights into the function of Fascin in actin bundling: A combined theoretical and experimental study.

Fascin, one of actin bundling proteins, plays an important role in the cross-linking of actin filaments (F-actin). Phosphorylation of Fascin is an important posttranslational modification to affect its structure and function. For example, a phosphomimetic mutation of Fascin-S39D decrease its bundling ability with F-actin significantly. In this paper, we studied the actin-bundling activity of Fascin by using molecular dynamics (MD) simulations and biochemical methods. All single-site mutations from serine/threonine to aspartic acid were mimicked by MD simulations. For five mutants (S146D, S156D, S218D, T239D and S259D), the mutated residues in domain 2 of Fascin were found to form salt-bridge interactions with an adjacent residue, indicating that mutations of these residues could potentially reduce actin-bundling activity. Further, F-actin-bundling assays and immunofluorescence technique showed S146D and T239D to have a strong effect on Fascin bundling with F-actin. Finally, we show that single-site mutations do not change the general shape of Fascin, but local structures near the mutated residues in Fascin-S146D and T239D become unstable, thereby affecting the ability of Fascin to bind with F-actin. These findings suggest that targeting domain 2 of Fascin would be very useful for the drug design. In addition, our study indicates that MD simulation is a useful method to screening which residues on Fascin are important.

Potential Inhibitors of Fascin From A Database of Marine Natural Products: A Virtual Screening and Molecular Dynamics Study.

Marine nature products are unique compounds that are produced by the marine environment including plants, animals, and microorganisms. The wide diversity of marine natural products have great potential and are versatile in terms of drug discovery. In this paper, we use state-of-the-art computational methods to discover inhibitors from marine natural products to block the function of Fascin, an overexpressed protein in various cancers. First, virtual screening (pharmacophore model and molecular docking) was carried out based on a marine natural products database (12015 molecules) and provided eighteen molecules that could potentially inhibit the function of Fascin. Next, molecular mechanics generalized Born surface area (MM/GBSA) calculations were conducted and indicated that four molecules have higher binding affinities than the inhibitor NP-G2-029, which was validated experimentally. ADMET analyses of pharmacokinetics demonstrated that one of the four molecules does not match the criterion. Finally, ligand Gaussian accelerated molecular dynamics (LiGaMD) simulations were carried out to validate the three inhibitors binding to Fascin stably. In addition, dynamic interactions between protein and ligands were analyzed systematically. Our study will accelerate the development of the cancer drugs targeting Fascin.

Computational Prediction of Hot Spots and Binding Site of Inhibitor NSC23766 on Rac1 Binding With Tiam1.

Rac1 is a small signaling protein, which belongs to the Rho subfamily of Ras superfamily. It is activated by binding GTP and inactivated by exchanging GDP for GTP. The ability of nucleotide exchange depends on guanine nucleotide exchange factors (GEFs) family proteins. T-lymphoma invasion and metastasis factor 1 (Tiam1) is a member of GEFs. Rac1 participates in multiple signaling pathways and regulates various cellular events by interacting with GEFs. Particularly, it is involved in the development and progression of various kinds of tumors. In this paper, we have studied the detailed interaction between Rac1 and Tiam1. Seven residues on Rac1 are predicted to be important for the interaction with Tiam1, i.e. E31, Y32, D38, N39, Y64, D65 and W56. All these residues are located on the switch 1 and 2 domains which are the interface between Rac1 and Tiam1, except W56. In addition, we analyzed how inhibitor NSC23766 interacts with Rac1. Our docking results show that NSC23766 binds to the same region as Tiam1. Several residues, i.e. F37, D38, N39, W56, Y64, L67, L70 and S71, contribute much to binding free energy. These findings are very useful for the structure-based design of inhibitors toward Rac1.

Reaction mechanism of lysyl oxidase-like 2 (LOXL2) studied by computational methods.

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent amine oxidase that catalyzes the oxidative deamination of the ε-amino group of lysines/hydroxylysines on substrate proteins (collagen and elastin) to form aldehyde groups. The generated aldehyde groups are of significance in crosslinking with the adjacent aldehyde or ε-amino group on proteins in extracellular matrix. In this paper, we have studied the reaction mechanism of LOXL2 by means of quantum mechanics (QM) and combined QM and molecular mechanics (QM/MM) methods. This study is divided into two parts, i.e. the biosynthesis of lysine tyrosylquinone (LTQ) cofactor and oxidative deamination of ε-amino group of lysine by LTQ. For the former part, the reaction is driven by a large exothermicity of about 284 kJ/mol. Dopaquinone radical (DPQr) is suggested to be an intermediate state in this reaction. In addition, His652 residue is predicted to serve as proton acceptor. The rate-determining step for the biosynthesis of LTQ is found to be hydrogen-atom abstraction from the benzene ring on substrate by Cu2+-hydroxide, which is a proton-coupled electron transfer (PCET) process with an energy barrier of 84 kJ/mol. For the latter part, the reaction is exothermic by about 145 kJ/mol, and the copper ion is proposed to play a role of redox catalyst in the last step to generate the product of aldehyde. However, the copper ion might not be indispensable for the latter part, which is consistent with the previous study.