Biology subjects

Vasiljevic, S.

Publications and source records attributed to Vasiljevic, S..

7 recordsLinked to original sources

bioRxiv2026

Mechanism of spike opening in a mammalian gammacoronavirus

Coronavirus spike proteins mediate receptor binding and fusion and are key determinants of viral host range. To bind protein receptors, some CoV spikes undergo conformational changes termed opening, which expose the receptor-binding surface. Yet, the prerequisites and mechanism of spike opening remain poorly understood, especially outside of beta-CoVs, and many CoV spikes have been observed only in the closed conformation. The receptor specificities of gamma-CoVs, for which birds are natural hosts, await in-depth characterisation and their spikes have not been observed to open. Here, we report structures of the spike from an unusual mammalian gamma-CoV isolated from a beluga whale (BwCoV), in closed and open conformations, the latter with two S1B domains erect. BwCoV spike opening is associated with changes throughout S1 that resemble those observed in beta-CoV spikes and similarly propagate into destabilisation of the fusogenic S2 core. Furthermore, the BwCoV-S structure contains an additional density likely corresponding to a fatty acid, consistent with lipid binding stabilising the closed conformation through a pocket distinct from that identified for beta-CoVs. These structural insights reveal conserved principles regulating spike dynamics across coronavirus genera and support lipid binding as a potential modulatory mechanism of spike opening.

biochemistry↗

bioRxiv2026

The genetic architecture of milk urea concentration in dairy cattle differs across the lactation cycle

Milk urea concentration (MUC) is an indicator of dietary protein utilization and nitrogen use efficiency in dairy cows. We performed genome-wide association studies (GWAS) on MUC in early, mid, and late lactation in the Holstein (HOL) and Brown Swiss (BSW) dairy cattle breeds using imputed sequence variants. We identified 11 and 17 independent quantitative trait loci (QTL) for MUC across the three lactation stages in BSW and HOL, respectively. While many of these QTL have previously been reported for MUC and other dairy traits, our study provides evidence that some QTL exert lactation-stage specific effects. Our findings suggest that variants at the DGAT1 locus on BTA14 have pleiotropic effects on MUC and other dairy traits. This QTL showed an early lactation-specific association with MUC but impacted milk and fat yield across the entire lactation. We fine-mapped two QTL for MUC in early and mid-lactation in BSW on BTA9 (lead SNP: 9:21392941, Pcorrected = 1.1E-17) and BTA28 (lead SNP: 28:6518357; Pcorrected = 3E-11). We identified lncRNA ENSBTAG00000058688 and IBTK as positional and functional candidate genes for the BTA9 QTL, and KCNK1 as positional and functional candidate gene that harbors a highly significant missense variant for the BTA28 QTL. In conclusion, our results shed light on the genetic architecture of MUC and highlighted QTL harboring potential functional variants underpinning milk urea variation within and across breeds.

genomics↗

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗

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↗

bioRxiv2020

Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.

biochemistry↗

bioRxiv2020

Networks of HIV-1 envelope glycans maintain antibody epitopes in the face of glycan additions and deletions

Numerous broadly neutralizing antibodies (bnAbs) have been identified that target the glycans of the HIV-1 envelope spike. Neutralization breadth is notable given that glycan processing can be substantially influenced by the presence or absence of neighboring glycans. Here, using a stabilized recombinant envelope trimer, we investigate the degree to which mutations in the glycan network surrounding an epitope impact the fine glycan processing of antibody targets. Using cryo-electron microscopy and site-specific glycan analysis, we reveal the hierarchy of importance of glycans in the formation of the 2G12 bnAb epitope, and show that the epitope is only subtly impacted by variations in the glycan network. In contrast, we show that the PG9 and PG16 glycan-based epitopes at the trimer apex are dependent on the presence of the highly conserved surrounding glycans. Glycan networks underpin the conservation of bnAb epitopes and are an important parameter in immunogen design.

immunology↗

bioRxiv2019

Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP glucose:glycoprotein glucosyl-transferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecules flexibility. Principal component analysis of the MD trajectories affords a description of UGGTs overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzymes central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name "Parodi limit" the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 [A]. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a "one-size-fits-all adjustable spanner" substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzymes active site.

biochemistry↗