Biology subjects

Chen, Y. S.

Publications and source records attributed to Chen, Y. S..

7 recordsLinked to original sources

bioRxiv2025

Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates

The SARS-CoV-2 main protease (3CLpro) is essential for viral replication and a leading antiviral target. Circulating variants accumulate substitutions on this enzyme, distant from the catalytic site. Surprisingly, mutant enzymes retain full proteolytic activity, though preserved overall activity does not exclude subtler effects on substrate recognition or selectivity, arising from distal structural perturbations. In this study, we compared the steady-state kinetics of wild-type (Wuhan) 3CLpro with enzymes from the Beta (K90R), Lambda (G15S), and Omicron (P132H) variants, using two peptide substrates representing distinct viral polyprotein cleavage sites. All four proteases displayed comparable catalytic efficiencies, similar pH-rate profiles, suggesting conservation of the catalytic mechanism despite sequence variation. The crystal structure of Omicron 3CLpro bound to an Nsp8-Nsp9 peptide revealed a conserved fold and active-site geometry, with the P132H side chain adopting a substrate-dependent conformation that rebuilt its local contacts, indicating towards how a distal substitution is accommodated without perturbing catalysis. Thermal stability measurements identified the sole distinguishing effect of P132H, with Omicron showing altered stability at elevated temperature. A screen of 31 tanshinones against 3CLpro identified T06 with Ki values of 5 uM, as 3CL pro inhibitor. Thus, 3CLpro may tolerate distal substitutions through local structural adaptation, supporting its durability as an antiviral target.

biochemistry↗

bioRxiv2025

Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 {+/-} 0.1 {micro}M). A 1.9 [A] crystal structure of the hCatK-Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.

biochemistry↗

bioRxiv2024

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

For structures determined by single particle cryo-EM, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors, and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here, we combine electron energy-loss spectroscopy (EELS) with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, REEL-EM, allows 3D reconstruction of EELS data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localise abundant elements. We discuss the current limitations of the method and potential future developments.

biophysics↗

bioRxiv2024

Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1)

As the primary Ca2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca2+ release, sarcomere tension and heat production. Restoration of Ca2+ into the SR also consumes ATP, generating a further untenable metabolic load. When anesthetizing patients with known MH mutations, the non-triggering intravenous general anesthetic propofol is commonly substituted for triggering anesthetics. Evidence of direct binding of anesthetic agents to RyR1 or its binding partners is scant, and the atomic-level interactions of propofol with RyR1 are entirely unknown. Here, we show that propofol decreases RyR1 opening in heavy SR vesicles and planar lipid bilayers, and that it inhibits activator-induced Ca2+ release from SR in human skeletal muscle. In addition to confirming direct binding, photoaffinity labeling using m-azipropofol (AziPm) revealed several putative propofol binding sites on RyR1. Prediction of binding affinity by molecular dynamics simulation suggests that propofol binds at least one of these sites at clinical concentrations. These findings invite the hypothesis that in addition to propofol not triggering MH, it may also be protective against MH by inhibiting induced Ca2+ flux through RyR1.

pharmacology and toxicology↗

bioRxiv2021

Rvb1/Rvb2 proteins couple transcription and translation during glucose starvation

During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly represses translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester the mRNAs into phase-separated granules and repress their translation. Additionally, this Rvb-tethering to the mRNA drove further transcriptional upregulation of the target genes. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

molecular biology↗

bioRxiv2021

Crystal structure of a bacterial CNNM magnesium transporter

CBS-pair domain divalent metal cation transport mediators (CNNMs) are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. CNNM proteins are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determined the crystal structure of an archaeal CNNM protein with Mg2+-ATP bound. The structure reveals a novel transmembrane fold for the DUF21 domain, the largest family of domains of unknown function. The protein has a negatively charged cavity that penetrates halfway through the membrane suggesting it functions as a cation transporter. The cytosolic portion of the protein is comprised of highly charged four-helix bundle and a CBS-pair domain. HDX-MS experiments, molecular dynamics, and additional crystal structures show that the cytosolic domains undergo large conformational changes upon nucleotide binding suggesting a mechanism of regulation shared between human and bacterial orthologs. The molecular characterization of CNNM proteins has profound implications for understanding their biological functions in human diseases, including cancer, and in animals, bacteria and plants.

biophysics↗

bioRxiv2019

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. This control is particularly complicated for mitochondria, as they must coordinate gene expression from both the nuclear and mitochondrial genome. We have found that cells are able to use this dynamic morphology to post-transcriptionally coordinate protein expression with the metabolic demands of the cell through enhanced mRNA localization to the mitochondria. As yeast switch to respiratory metabolism, they increase their mitochondrial volume fraction that is, the ratio of mitochondrial volume to intracellular volume which drives the localization of nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through artificial tethering experiments, we show that this mitochondrial localization is sufficient to increase protein production, whereas sequestering mRNAs away from the mitochondrial surface decreases protein production, and those cells are deficient in growth in respiratory conditions. Furthermore, we find that this mRNA sensitivity to mitochondrial volume fraction is driven by the speed of translation downstream of the mitochondrial targeting sequence (MTS), as local ribosome stalling through a stretch of polyprolines in the nascent peptide can drive constitutive localization of mRNAs to the mitochondria. This points to a mechanism by which organelle volume fraction provides feedback to regulate organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome.

cell biology↗