Biology subjects

Yang, L.-W.

Publications and source records attributed to Yang, L.-W..

11 recordsLinked to original sources

bioRxiv2026

Drug Functional Site-Unknown Molecular Targets Exemplified by SARS-CoV-2 pseudoknot and ribosomal frameshifting-modulators

The -1 Programmed Ribosomal Frameshifting (-1 PRF) signal of SARS-CoV-2, driven by a conserved three-stemmed RNA pseudoknot (PK), is indispensable for viral replication and represents a structurally stable yet underexplored therapeutic target. Unlike rapidly mutating viral proteins, this RNA element offers an opportunity for durable intervention but has historically been considered "undruggable". We developed an integrative drug discovery and characterization pipeline that combines molecular docking, molecular dynamics simulations, and dual-luciferase assays to systematically identify and validate frameshifting-efficiency (Feff) modulators from FDA-approved compounds. To move beyond traditional similarity-based screening, we introduced a contact-distribution-matching method, which ranks candidate compounds by comparing their predicted RNA interaction fingerprints with those of reference modulators. This computational approach, paired with experimental validation, enabled us to expand the repertoire of Feff modulators and establish correlations between binding patterns and functional outcomes. To uncover the underlying mechanisms, we applied steered molecular dynamics simulations and single-molecule optical tweezers measurements, revealing that Feff-enhancing modulators preferentially stabilize the remote stem (stem 3) of the PK, promoting variety of intermediate force species with the beginning base pairs of stem 1 being refolded, even after those base pairs have been unwound by ribosome. The refold of the tips of the stem 1 in turn "push back" the ribosome on the slippery sequence to result in enhanced frameshifting. On the other hand, Feff-suppressing modulators rigidify early stem regions, increasing resistance to ribosomal progression and increase the drop-off rate, eventually leading to a reduced -1 frame to 0 frame ratio in translation. Together, these findings provide the first integrated demonstration of how small molecules can modulate -1 PRF by altering RNA PK folding dynamics. More broadly, our framework establishes a generalizable strategy for rationally targeting structured RNAs with repurposed drugs and offers new opportunities to expand the druggable genome to include noncoding RNA elements and other biomolecular targets lacking known functional sites.

biochemistry↗

bioRxiv2025

Core elements play distinct roles in promoter birth and transcriptional regulation

Gene expression shapes phenotypes and evolution. However, studies of gene regulation focus on transcription factors, overlooking core promoters. To investigate how promoters emerge and regulate transcription, we determined the sequence-function landscapes of core elements, -35 and -10, in constitutive and transcription factor-regulated promoters in Escherichia coli. Characterization of in vivo transcriptional landscapes and in vitro RNA polymerase-promoter interactions showed the -10 element as essential for promoter evolution from random sequences. In contrast, the -35 element, though broadly conserved, is dispensable for promoter birth. Instead, it exerts greater impact on gene regulation via coordinated interactions with transcription activators and RNA polymerase. We further showed that evolution fine-tunes the -35 and -10 sequences of transcription factor-regulated promoters to achieve near-maximal fold changes by lowering basal while elevating induced expression. A notable exception is PluxI, whose leaky expression provides a crucial baseline for initiating quorum sensing. These findings elucidate promoter design principles and underscore the interdependence and coevolution of core elements, RNA polymerase, and transcription factors.

systems biology↗

bioRxiv2022

Computer simulations reveal pathogenicity and inheritance modes of hearing loss-causing germinal variants

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In the present study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal "plugs" aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homomeric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable heteromeric channels did not. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus cells and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

biophysics↗

bioRxiv2021

Actin filaments act as a size-dependent diffusion barrier around centrosomes

The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that the centrosome may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we found that centrosomes were highly accessible to soluble molecules with a Stokes radius of [≤] 5.1 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of barriers was tightly regulated by branched actin filaments outside of centrosomes. Such barrier gated the microtubule nucleation. We propose that actin filaments spatiotemporally constrain the distribution of molecules at centrosomes in a size-dependent manner. Significance StatementCentrosome maintains its microenvironment without membrane. Whether the dense protein complexes outside centrosomes including pericentriolar matrix, microtubules, and branched actin filaments provide physical obstruction is unclear yet. We here established a series of new tools for quantitative evaluation of the diffusion rates of varisized soluble proteins in different sub-compartments of centrosomes. Our results demonstrated that branched actin filaments, but not pericentriolar matrix or microtubules, around centrosome have acted as a size-dependent diffusion barrier and physically constrain centrosome microtubule nucleation.

cell biology↗

bioRxiv2021

ZHX2 Promotes HIF1α Oncogenic Signaling in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers And Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia inducible factor (HIF) family members and positively regulated HIF1 activity in TNBC. Integrated ChIP-Seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1 on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Furthermore, multiple residues (R491, R581 and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling HIF1 activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1 signaling, therefore serving as a potential therapeutic target for TNBC.

cancer biology↗

bioRxiv2021

DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme

MotivationDrug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed. ResultsHere we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2'-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks. Availability and implementationDRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)

bioinformatics↗

bioRxiv2019

Molecular dynamics simulations and linear response theories jointly describe biphasic responses of myoglobin relaxation and reveal evolutionarily conserved frequent communicators

In this study, we provide a time-dependent (td-) mechanical model, taking advantage of molecular dynamics (MD) simulations, quasiharmonic analysis of MD trajectories and td-linear response theories (td-LRT) to describe vibrational energy redistribution within the protein matrix. The theoretical description explains the observed biphasic responses of specific residues in myoglobin to CO-photolysis and photoexcitation on heme. The fast responses are found triggered by impulsive forces and propagated mainly by principal modes <40 cm-1. The predicted fast responses for individual atoms are then used to study signal propagation within protein matrix and signals are found to propagate [~] 8 times faster across helices (4076 m/s) than within the helices, suggesting the importance of tertiary packing in proteins sensitivity to external perturbations. We further develop a method to integrate multiple intramolecular signal pathways and discover frequent \"communicators\". These communicators are found evolutionarily conserved including those distant from the heme.

biophysics↗

bioRxiv2019

Intramolecular Communication and Allosteric Sites in Enzymes Unraveled by Time-Dependent Linear Response Theory

It has been an established idea in recent years that protein is a physiochemically connected network. Allostery, understood in this new context, is a manifestation of residue communicating between remote sites in this network, and hence a rising interest to identify functionally relevant communication pathways and the frequent communicators within. However, there have been limited computationally trackable general methods to discover proteins allosteric sites in atomistic resolution with good accuracy. In this study, we devised a time-dependent linear response theory (td-LRT) integrating intrinsic protein dynamics and perturbation forces that excite proteins temporary reconfiguration at the non-equilibrium state, to describe atom-specific time responses as the propagating mechanical signals and discover that the most frequent remote communicators can be important allosteric sites, mutation of which could deteriorate the hydride transfer rate in DHFR by 3 orders. The preferred directionality of the signal propagation can be inferred from the asymmetric connection matrix (CM), where the coupling strength between a pair of residues is suggested by their communication score (CS) in the CM, which is found consistent with experimentally characterized nonadditivity of double mutants. Also, the intramolecular communication centers (ICCs), having high CSs, are found evolutionarily conserved, suggesting their biological importance. We also identify spatially clustered top ICCs as the newly found allosteric site in ATG4B. Among 2016 FDA-approved drugs screened to target the site, two interacting with the site most favorably, confirmed by MD simulations, are found to inhibit ATG4B biochemically and be tumor suppressive in colorectal, pancreatic and breast cancer cell lines with an observed additive therapeutic effect when co-used with an active-site inhibitor.

biophysics↗

bioRxiv2019

Data-Driven Design of Diagnostic Kits and Therapeutic Peptides

To ensure a physicochemically desired sequence motif to adapt a specific type of secondary structures, we compile an -helix database allowing complicate search patterns to facilitate a data-driven design of therapeutic peptides. Nearly 1.7 million helical peptides in >130 thousand proteins are extracted along with their interacting partners from the protein data bank (PDB). The sequences of the peptides are indexed with patterns and gaps and deposited in our Therapeutic Peptide Design dataBase (TP-DB). We here demonstrate its utility in three medicinal design cases. By our pattern-based search engine but not PHI-BLAST, we can identify a pathogenic protein, Helicobacter pylori neutrophil-activating protein (HP-NAP), a virulence factor of H. pylori, which contains a motif DYKYLE that belongs to the affinity determinant motif DYKXX[DE] of the FLAG-tag and can be recognized by the anti-FLAG M2 antibody. By doing so, the known purification-tag-specific antibody is repurposed into a diagnostic kit for H. pylori. Also by leveraging TP-DB, we discovered a stretch of helical peptide matching the potent membrane-insertion pattern WXXWXXW, elucidated by MD simulations. The newly synthesized peptide has a better minimal inhibitory concentration (MIC) and much lower cytotoxicity against Candida albicans (fungus) than that of previously characterized homologous antimicrobial peptides. In a similar vein, taking the discontinued anchoring residues in the helix-helix interaction interface as the search pattern, TP-DB returns several helical peptides as potential tumor suppressors of hepatocellular carcinoma (HCC) whose helicity and binding affinity were examined by MD simulations. Taken together, we believe that TP-DB and its pattern-based search engine provide a new opportunity for a (secondary-)structure-based design of peptide drugs and diagnostic kits for pathogens without inferring evolutionary homology between sequences sharing the same pattern. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/.

bioinformatics↗

bioRxiv2018

DR-SIP: Protocols for Higher Order Structure Modeling with Distance Restraints- and Cyclic Symmetry-Imposed Packing

MotivationQuaternary structure determination for proteins is difficult especially for transmembrane proteins. Even if the monomeric constituents of complexes have been experimentally resolved, computational prediction of quaternary structures is a challenging task particularly for higher order complexes. It is essential to have a reliable computational protocol to predict quaternary structures of both transmembrane and soluble proteins leveraging experimentally determined distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins.\n\nResultsWe survey 115 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) to discover that 90% of them are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) filter and the Symmetry-Imposed Packing (SIP) filter which takes advantage of the statistically derived tilt angle cutoff and the Cn symmetry of HoTPs without prior knowledge of the number (\"n\") of monomers. Using only the geometrical filter, SIP, near-native poses of the 115 HoTPs can be correctly identified in the top-5 for 52% of all cases, or 49% among the HoTPs having an n >2 (~60% of the dataset), while ZDOCK alone returns 41% and 24%, respectively. Applying only SIP to three HoTPs with distance restraints, the near-native poses for two HoTPs are ranked 1st and the other 7th among 54,000 possible decoys. With both filters, the two remain 1st while the other improved to 2nd. While a soluble system with distance restraints is recovered at the 1st-ranked pose by applying only DR.\n\nAvailability and Implementationhttps://github.com/capslockwizard/drsip\n\nSupplementary informationSupplementary methods and results are available.

bioinformatics↗

bioRxiv2018

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in the ribosome

The clock of life ticks as fast as how efficiently proteins could perform their functional dynamics. Protein complexes execute functions via several large-scale intrinsic motions across multiple conformational states, which occur at a timescale of nano-to milliseconds for well-folded proteins. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to time and size these motions, though barely to their slowest ends. Here, we convert a simple elastic network model (ENM), which takes a few seconds (ubiquitin) to hours (ribosome) for the analysis, into a molecular timer and sizer to gauge the slowest functional motions of proteins. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener-Khintchine theorem (WKT) are used to define the \"time-periods\", t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective \"time-periods\" are mapped to the eigenvalues ({lambda}ENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 86.9{lambda}ENM-1.9 and {sigma}2([A]2) = 46.1 {lambda}ENM-2.5 are established allowing the characterization of the time scales of Nuclear Magnetic Resonance (NMR)-solved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.\n\nAuthor SummaryThe time scale of biological processes is governed by protein functional dynamics that often corresponds to the largest conformational spread and the longest time scales among all possible motions. Current simulation methodologies cannot reach the slowest, often functional, motions especially for supramolecular machineries. Borrowing the spring-bead model used in polymer physics since 60s, the efficient elastic network model (ENM), introduced in 90s, captured all modes of protein motions but largely underestimated the time scales of slowest modes due to its harmonic approximation.\n\nHere we map water-damped modes sampled by MD simulations to corresponding ones in ENM and thereby establish 2 power laws that describe the authentic time scales and sizes for the slowest anharmonic modes as functions of ENM eigenvalues. With that, we adequately describe the sizes and time scales for three proteins, confirmed by NMR spectroscopy, and ribosome that contains ~0.2 million heavy atoms (~ 20 thousands coarse-grained nodes).

biophysics↗