Biology subjects

Wang, H.-C.

Publications and source records attributed to Wang, H.-C..

7 recordsLinked to original sources

bioRxiv2025

Structural Dissection of Vaccinia G9 Identifies Residues Essential for Membrane Fusion and Complex Assembly

Vaccinia virus, a prototypical poxvirus, utilizes a unique multi-protein Entry Fusion Complex (EFC), comprising 11 components, to mediate membrane fusion during host cell entry. Although the crystal structure of a truncated form of the G9 protein has been determined, the functional relevance of its structural features remains poorly understood. In this study, we systematically analyzed 47 G9 mutants to identify critical functional residues. Using trans-complementation assays, co-immunoprecipitation, membrane fusion assays, and structural analysis, we identified nine key mutants, which were categorized into three functional groups. Group 1 mutants failed to interact with A16 and other EFC components, highlighting their essential roles in G9-A16 subcomplex formation. Group 2 and Group 3 mutants retained A16 binding but disrupted interactions with other EFC proteins, suggesting their roles in broader complex assembly. Notably, Group 3 mutants targeted a conserved P(R/Y)XCW motif and a loop structure shared among vaccinia G9, A16, and J5 proteins. A similar motif was also identified in G9 homologs from Nucleocytoviricota, suggesting an evolutionarily conserved fusion mechanism. Collectively, our findings demonstrated that G9 function requires multiple domains, including A16-binding interfaces and conserved motifs not resolved in previous protein structure. These results establish G9 as a central EFC component and underscore its potential as a target for antiviral development. ImportanceUnderstanding how viruses enter host cells is critical for developing antiviral strategies. Vaccinia virus, a model poxvirus, uses a unique 11-protein entry fusion complex (EFC) to mediate membrane fusion--unlike other viruses that rely on a single fusion protein. In this study we identified specific residues in the G9 protein that are critical for maintaining EFC function. Notably, we discovered a conserved P(R/Y)XCW motif within G9 that is also present in orthologs from both poxviruses and members of the Nucleocytoviricota phylum, suggesting an evolutionarily conserved mechanism of membrane fusion. These conserved structural elements can serve as potential targets for antiviral intervention against pathogenic poxvirus infections in humans.

microbiology↗

bioRxiv2024

Evidence for Stepwise Disruption of E. coli RNA Polymerase-{lambda}R Promoter Contacts and Bubble Collapse in Transcription Initiation

In transcription initiation by E. coli RNA polymerase (RNAP), translocation of the RNA-DNA hybrid disrupts RNAP-promoter and {sigma}70-core RNAP contacts, releasing {sigma}70 and allowing RNAP to escape. Previously, to investigate whether RNAP-promoter contacts break step-by-step as the hybrid lengthens or concertedly at escape, we determined rate constants and activation energies of nucleotide-incorporation steps involving translocation at the {lambda}PR promoter. Trends in these quantities with hybrid length were inconsistent with concerted models and provided evidence for stepwise disruption of RNAP-promoter contacts and collapse of the upstream bubble (-1 to -11). Here we report kinetic m-values quantifying urea and glycine betaine (GB) effects on rate constants of individual nucleotide-incorporation steps and compare with m-values predicted from structural and model compound information. GB is predicted to favor and urea disfavor binding the initiating NTPs and trigger-helix formation in 2-mer (pppApU) synthesis, largely because of burial of phosphate oxygens in NTP binding and amide oxygens in trigger-helix formation. Consistent with these predictions, GB accelerates and urea retards steps synthesizing 3-mer and 4-mer. However, both solutes retard mid-initiation steps (5-mer to 9-mer synthesis) where -10 contacts are proposed to break, exposing DNA phosphates and allowing the upstream initiation bubble to collapse. Strikingly, urea greatly accelerates while GB retards the 11-mer synthesis step, where -35-contacts are proposed to break, {sigma}70 is released and RNAP escapes. Urea and GB kinetic m-values and activation energies of these steps are inconsistent with concerted models and support a stepwise model of contact disruption and bubble collapse in initiation.

biochemistry↗

bioRxiv2023

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex

Virus-mediated membrane fusion involves conformational changes of the viral fusion protein to fuse the opposing viral and host lipid bilayers. Unlike all other known viruses that contain a single fusion protein, poxviruses harbor a multimeric protein complex of 11 subunits, termed the entry fusion complex (EFC), to mediate fusion with host membranes. Yet, how the poxviral EFC mediates membrane fusion remains enigmatic. To establish the mechanism of EFC-triggered membrane fusion, we are deciphering the structure and function of individual EFC components. Here, we determined the crystal structure of the H2 ectodomain by X-ray diffraction, revealing a folded conformation comprising a central five-stranded {beta}-sheet and three cladding -helices. We reconstructed the full-length H2 by in silico prediction, revealing that the N-terminal region (aa 51-90) of H2 protein may fold as a long helix connecting the ectodomain and transmembrane region. Using alanine-mutagenesis screening in a transient complementation system, coimmunoprecipitation, isothermal titration calorimetry and MV-triggered membrane fusion assays, we concluded that the surface of the ectodomain of H2 protein, including two loop regions, 170LGYSG174 and 125RRGTGDAW132, constitutes a broad A28-binding region. Moreover, although not involved in A28 binding, the N-terminal helical region approximal to the transmembrane part, encompassing 64RIK66, 72W, and 83ESDRGR88, is also crucial for viral EFC formation and MV infectivity.

microbiology↗

bioRxiv2022

Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

E. coli RNA polymerase (2{beta}{beta}{omega}{sigma}70) forms stable open complexes (OC) at {lambda}PR and T7A1 promoters at 37 {degrees}C at similar rates but with very different lifetimes. To probe the origins of these differences, we determine OC lifetimes for full-length (FL) and downstream-truncated (DT) {lambda}PR and T7A1 promoter variants with eight combinations of discriminators and upstream elements. We find the discriminator is the major determinant of OC lifetime, while upstream elements modulate the discriminator effect. The very different lifetimes of these stable OC arise primarily from differences in [Formula], the free energy change for converting I2 (the initial open intermediate) to stable OC. [Formula] is twice as favorable for {lambda}PR-discriminator variants as for T7A1-discriminator variants. Truncation at +6 (DT+6) eliminates most differences in [Formula], destabilizing {lambda}PR-discriminator variants without affecting T7A1-discriminator variants. All DT+6 OC are much more stable and longer-lived than I2. Urea greatly destabilizes OC, primarily affecting RNAP interactions with +6 and upstream DNA. From these findings we deduce that stabilization of I2 involves closing the {beta}-clamp with extensive coupled folding to contact the region of promoter DNA from the transcription start-site to +6. Lifetime differences between DT+6, DT+12 and FL promoters allow dissection of contributions to OC stability from interactions of regions of the downstream duplex with downstream mobile elements (DME; {beta}-lobe, {beta}-clamp, {beta}-jaw). We propose an allosteric network by which differences in discriminator and -10 sequence are sensed by {sigma}1.2 and {beta}-gate loop and transmitted to {sigma}1.1 and {beta}-lobe to affect DME-duplex interactions that determine OC lifetime.

biophysics↗

bioRxiv2020

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by E. coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

FRET (fluorescence energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of {lambda}PR promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC). Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC}c of four intermediates (initial (RPC), early (I1E), mid-(I1M), late (I1L)), followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RPC advances to I1E, reducing -100/+14 distance to [~]75[A] and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper {beta}-clamp while downstream DNA contacts the top of the {beta}-pincer in I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14PIFE, interpreted as clamp-opening that moves far-upstream DNA (on {beta}) away from downstream DNA (on {beta}) to increase the -100/+14 distance by ~14[A]. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the -100/+14 distance by ~21[A]. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L converts to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream-binding, DNA-bending transcription activators are discussed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/932780v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1068460org.highwire.dtl.DTLVardef@dcd6d9org.highwire.dtl.DTLVardef@9d4727org.highwire.dtl.DTLVardef@1aa8e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Invasive plants facilitated by socioeconomic change shelter vectors of scrub typhus and spotted fever

BackgroundEcological determinants of most emerging vector-borne diseases are little studied, particularly for neglected tropical disease; meanwhile, although socioeconomic change can have significant downstream effect on human risks to vector-borne diseases via a change in land cover, particularly facilitating the invasion of exotic plants, related studies remain very scarce. Scrub typhus and spotted fever are neglected diseases emerging around the globe and are transmitted by chigger mites and ticks, respectively, with small mammals as the primary hosts of both vectors.\n\nMethodology/Principal findingsWe investigated how invasion of Leucaena leucocephala plant after extensive abandonment of farmlands driven by industrialization in Penghu Islands of Taiwan affected abundance of chiggers and ticks by trapping small mammals in three types of habitats (invasion site, agricultural field, human residence) every two months for a year. Invasion sites sheltered more chiggers and ticks than the other two habitats; moreover, both vectors maintained higher abundance in early winter and populations of chiggers were more stable across seasons in invasion sites, suggesting that the invasive sites could be a temporary refuge for both vectors and might help mitigate the negative influence of unfavorable climate. Infective rates of etiologic agents in chiggers and ticks were also higher in invasion sites. Top soil temperature and relative humility were similar across the three habitats, but invasion sites harbored more Rattus losea rat, on which infested chiggers and ticks were more well fed than those from the most commonly trapped species (Suncus murinus shrew), implicating that abundance of superior hosts instead of microclimate, might determine the abundance of both vectors.\n\nConclusions/SignificanceThis study highlights an important but largely neglected issue that socioeconomic change can have unexpected consequence for human health mediated particularly through invasive plants, which could become a hotspot for emerging infectious diseases but usually are very hard to be eradicated. In the future, a more holistic perspective that integrates socioeconomy, land use, exotic species, and human health should be considered to fully understand potential emergence of vector-borne diseases.\n\nAuthor summaryUnderstanding how environmental factors, such as land use change, affect risks to vector-borne diseases helps control and prevent human diseases, but ecological preference of vectors of most neglected diseases remain little investigated. In this study, we found that vectors of scrub typhus (chigger mites) and spotted fever (hard ticks), two emerging neglected diseases, were much more abundant in sites invaded by exotic plants than the other major land cover types in a small island of Taiwan; moreover, populations of chigger mite in invasion sites were more stable across seasons, suggesting that plant invasion sites could be a refuge for disease vectors under unfavorable climate. Higher abundance of chigger mites and ticks was related to higher abundance of a superior rodent host instead of a difference in soil micro-climate. More significantly, these invasive plants are facilitated by extensive abandonment of farmlands driven by industrialization and rural to urban migration, thus demonstrating an important but largely neglected issue that socioeconomic change, when mediated through a change in land cover, can have unexpected downstream effect on emerging neglected tropical diseases.

ecology↗

bioRxiv2018

Low-Density Lipoprotein Receptor-Mediated Lipidome-Transcriptome Reprogramming Impulses to Cisplatin Insensitivity

Platinum-based therapy remains the cornerstone for cancer patient management; however, its efficacy varies. Theis study demonstrated the differential expressions of low-density lipoprotein receptor (LDLR) in subtypes of epithelial ovarian carcinoma (EOC) determines cisplatin sensitivity. Its sensitive in serous EOCs (low LDLR), where insensitive in endometrioid and clear cell EOCs (high LDLR). Meanwhile, knocked-down or overexpressed LDLR in EOC could reversed the chemosensitivity pattern both in vitro and in vivo. Mechanistic dissection with transcriptome vs. lipidome trans-omics analyses elucidated the LDLR[->]LPC (Lyso-PhosphotidylCholine)[->]FAM83B (phospholipase-related)[->]FGFRs (cisplatin sensitivity and phospholipase-related) regulatory axis in cisplatin insensitivity. Implementing LPC-liposome encapsulated cisplatin could facilitate DNA-adduct formation via lipid droplets (LDs) delivery. Furthermore, Bioinformatics analyses found that the LDL/R[->]LD homeostasis alteration is critical for therapeutic prognosis. Lastly, using LPC-liposome-cisplatin improved cisplatin sensitivities in gastric cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, and pancreatic adenocarcinoma cells. In conclusion, this report discovered a LDL/R-reprogrammed transcriptome-lipidome network, by which impulses platinum insensitivity and disease outcome. The drug specific lipidome for liposome manufacture might be an efficienct pharmaceutics strategy for chemoagents.\n\nSignificanceLDLR reprograms cellular lipidome and transcriptome profiles to determines chemotherapy therapeutic efficacy. The LDLR-reduced LPC abundance disturbs phospholipids homeostasis of Lands cycle in LD, by which attenuates intracellular platinum transportation for DNA-adduct formation. Targeting LDLR-LD-lipidome with LPC-liposome-platinum could boost therapeutic efficacy for insensitivity.

cancer biology↗