Biology subjects

Caputo, A. T.

Publications and source records attributed to Caputo, A. T..

3 recordsLinked to original sources

bioRxiv2026

Knockdown of fibrillarin reveals cell- and virus- dependent restriction of henipavirus replication and enhanced antiviral immune signalling

The henipavirus genus includes several highly pathogenic zoonotic viruses that lack medical countermeasures. The 2-O-methyltransferase fibrillarin (FBL) has been identified by genome-wide functional genomics studies as a henipavirus pro-viral factor. While FBL is a potential target for host-directed antiviral therapeutics, its impact on henipavirus replication in cell types relevant to henipavirus disease has not been explored. Here we demonstrate that depleting cellular FBL using short interfering RNAs (siRNAs) has varying impacts on henipavirus replication, depending on cell type and virus. FBL depletion in small airway epithelial cells (HSAEC1-KT) significantly impaired viral gene transcription for Hendra virus (HeV) genotypes 1 and 2 (HeV-g1, HeV-g2), and Nipah virus (NiV) Bangladesh strain (NiV-B), but not NiV Malaysia (NiV-M) strain or Cedar virus (CedV). Conversely, reduced FBL expression in bronchial epithelial (NuLi-1) cells, aortic endothelial (Telo HAEC) cells or neuroblastoma (BE(2)-M17) cells did not inhibit HeV-g1 replication. Transcriptomic analyses revealed that impaired virus replication in FBL knockdown cells was associated with enhanced activation of innate immune pathways and robust upregulation of interferon stimulated genes (ISGs). Together, these findings demonstrate a virus- and cell-type dependent impact of FBL on henipavirus replication and support a role for FBL in modulation of the antiviral innate immune response.

microbiology↗

bioRxiv2026

Selective Hydrolytic Defluorination of Branched Perfluorooctanoic Acid Isomers by a Haloacid Dehalogenase

Per- and polyfluoroalkyl substances (PFAS) are highly resistant to enzymatic C-F bond cleavage, and hydrolytic defluorination of long-chain PFAS has rarely been demonstrated. Here, we report selective hydrolytic defluorination of branched perfluorooctanoic acid (PFOA) isomers by a haloacid dehalogenase (4A) from Delftia acidovorans strain D4B. A fluoride-specific riboswitch biosensor was used for initial substrate screening, followed by scaled-up assays in which fluoride release was quantified using a fluoride ion-selective electrode. Defluorination products were subsequently identified by liquid chromatography-mass spectrometry (LC-MS). Although purified 4A (10 M) readily catalyzed hydrolytic defluorination of fluoroacetic acid, incubation of PFOA (0.5 mM) with purified 4A resulted in a statistically significant increase in fluoride release at elevated enzyme loading (500 M). High-resolution LC-MS/MS analysis revealed that defluorination products originated from minor branched PFOA isomers rather than linear PFOA. Molecular docking analyses supported catalytically plausible binding geometries for branched PFOA isomers, positioning the substrate -carbon within [~]4 [A] of the catalytic aspartate residue. These findings demonstrate previously unrecognized hydrolytic reactivity of a haloacid dehalogenase toward branched PFAS isomers and expand the known catalytic scope of the haloacid dehalogenase family. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/719434v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d56316org.highwire.dtl.DTLVardef@15c7961org.highwire.dtl.DTLVardef@24de56org.highwire.dtl.DTLVardef@80f19d_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISEnzymatic defluorination of PFAS is rarely observed in environmental systems. This study identifies hydrolytic defluorination of branched PFOA isomers, improving understanding of PFAS defluorination at the enzyme level.

synthetic biology↗

bioRxiv2022

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 WY conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 {micro}M binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 {micro}M in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan. Significance StatementWhen a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell - availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.

biochemistry↗