https://hdl.handle.net/10481/14661 2026-04-27T18:48:42Z https://hdl.handle.net/10481/113013 Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike’s S2 subunit Polo-Megías, Daniel; Cano Muñoz, Mario; Trolese, Philipp; Lestani, Sara; La Rocchia, Ilaria; Pierangelini, Andrea; Fongaro, Benedetta; Polverino de Laureto, Patrizia; Morales-Yánez, Francisco J.; Vaneyck, Jonathan; Vanderplasschen, Alain; Decoville, Thomas; Laumond, Géraldine; Salinas-Garcia, M. Carmen; Cámara-Artigas, Ana; Gavira Gallardo, José Antonio; Moog, Christiane; Dumoulin, Mireille; Conejero Lara, Francisco The formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1 region and block its interaction with HR2, inhibiting viral fusion. Moreover, they are recognized by plasma antibodies from COVID-19 convalescent patients. In this work, we generated camelid heavy-chain-only antibody fragments (VHHs), also named nanobodies (NBs), against a CoVS-HR1 variant mimicking the full HR1 region. A first generation of selected NBs bound HR1 with high affinity and competed with HR2. Notably, this set of NBs exclusively recognized the C-terminal half of HR1, and two of them showed mild neutralizing activity in cell infection assays. Using a truncated CoVS-HR1 variant (N2C), we selected a second generation of NBs targeting specifically the N- terminal half of HR1. However, these NBs did not demonstrate neutralizing activity, possibly due to their low binding affinities. Several NB epitopes were delineated by hydrogen‑deuterium exchange and mass spectrometry analysis, and the crystal structure of a ternary complex between an HR1-mimetic protein and two NBs was determined, confirming competition with HR2. Intriguingly, we found cooperative binding effects between NBs targeting each half of HR1, but these did not result in detectable inhibitory synergy. These findings demonstrate he existence of neutralizing epitopes in the S2 HR1 region and provide a foundation for future development of enhanced neutralizing NBs focused on specific epitopes using HR1-mimetic proteins. This research was funded by grant PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033). Additional support was provided by ANRS (Agence Nationale de Recherches sur le SIDA et les h´epatites virales), the Investissements d'Avenir program administered by the ANR (grant ANR-10-LABX-77), and EHVA (Grant No. 681032, Horizon 2020), with co-funding from the ERDF/ESF under the initiatives “A way to make Europe” and “Investing in your future.” We are also grateful to the Andalusian Regional Government for the predoctoral fellowship awarded to Daniel Polo-Megías. Mario Cano-Munoz ˜ was supported by a Postdoctoral Research Program from the Spanish Research Agency: Juan de la Cierva (JDC2022- 049681-I). Francisco Morales-Y´ anez ˜ was supported by a COS-R funding from the University of Li`ege. Jonathan Vaneyck was supported by CIP funding. Mireille Dumoulin is a research associate from the FRS-FNRS. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain, and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of beamtime and staff assistance at XALOC (ALBA, BAG number 2023087670) and ID30B and ID23-2 (ESRF, BAG number MX2650) beamlines during diffraction data collection. We also acknowledge the Robotein® platform of the BE Instruct-ERIC Centre for providing access to the EasyPick Microlab STARlet Hamilton workstation (https://www.robotein.uliege.be/cms/ c_14301428/en/robotein). Funding for open access charge: Universidad de Granada/CBUA. https://hdl.handle.net/10481/112968 Impact of Ser81 phosphorylation on alanine: glyoxylate aminotransferase associated with Primary hyperoxaluria type I Milosevic, Sara; Salido, Eduardo; Mesa-Torres, Noel; Pey, Angel L.; Cano-Muñoz, Mario Phosphorylation is a fundamental post‑translational modification that contributes to the dynamic control of protein function and stability. More than 300,000 site‑specific phosphorylation sites have been detected across > 20,000 human proteins, yet only a small fraction (~ 5%) have been experimentally characterized, and their roles in health and disease remain largely unexplored. In particular, the functional consequences of most phosphorylation events in metabolic enzymes remain unknown. Here, we investigated the functional impact of modifying Ser81 in alanine:glyoxylate aminotransferase (AGT), the key enzyme responsible for glyoxylate detoxification and whose loss‑of‑function causes Primary Hyperoxaluria Type I (PH1). We examined phosphomimetic substitutions at Ser81 in the WT (wild‑type) enzyme, the common polymorphic minor allele (LM, containing two variations, p.P11L and p. I340M), and the two most frequent PH1‑associated variants (LM‑p.G170R and LM‑p.I244T). Using biochemical, biophysical and cell‑based approaches, we found that introducing a negative charge at Ser81 (through the S81D substitution) strongly perturbs PLP (pyridoxal 5´‑phosphate)/PMP (pyridoxamine 5´‑phosphate) binding pose and disrupts catalytic activity, while preserving secondary and tertiary structure as well as peroxisomal localization. In contrast, the non‑charged S81A substitution produced milder effects. These results indicate that Ser81 contributes to stabilizing the cofactor interaction network at the AGT active site. Our findings therefore identify Ser81 as a previ ously uncharacterized regulatory position that can critically influence AGT activity. Although further work is required to determine the physiological frequency and regulatory context of this modification in vivo, our results suggest that phosphorylation at this position could represent an additional modulatory layer influencing AGT function and genotype–phenotype relationships in PH1, with potential implications for understanding regulatory mechanisms affecting AGT activity in disease. https://hdl.handle.net/10481/109761 Identification of polyketide inhibitors targeting 3-dehydroquinate dehydratase in the shikimate pathway of Enterococcus faecalis Cheung, Vivian W.N.; Xue, Bo; Hernandez-Valladares, Maria; Go, Maybelle K.; Tung, Alvin; Aguda, Adeleke H.; Robinson, Robert C.; Yew, Wen S. Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort". Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis. This research was supported by grants from the Ministry of Education, the National Medical Research Council and the National Research Foundation of Singapore to W.S.Y, and grants from the Biomedical Research Council of A*STAR to R.C.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://hdl.handle.net/10481/109760 The role of palmitoylation in regulating Ras localization and function Eisenberg, Sharon; Laude, Alex J.; Beckett, Alison J.; Mageean, Craig J.; Aran, Verónica; Hernández Valladares, María del Carmen; Henis, Yoav I.; Prior, Ian A. Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras. We gratefully acknowledge funding from the Royal Society, North West Cancer Research Fund, the Wellcome Trust (to I.A.P.) and from the DGF-DIP [grant numbers KL 1948/1-1 and GA 309/10-1] and the Ministry of Science & Technology, Israel (to Y.I.H.). Y.I.H. is an incumbent of the Zalman Weinberg Chair in Cell Biology. https://hdl.handle.net/10481/109759 Actin Polymerization Dynamics - Insights from In vitro TIRF Microscopy Kannan, Balakrishnan; Larsson, Marten; Lee, Wei Lin; Hernandez-Valladares, Maria; Robinson, Robert C Actin elongation is a bi-molecular reaction between monomeric actin (G-actin) and filamentous actin (F-actin), in the first approximation. It can be controlled by changing the ability of either G-actin or F-actin to participate in the reaction. Either of the two mechanisms alone is not sufficient to maintain a large pool of G-actin ready to polymerize in a signal-controlled fashion [1]. Mammalian cells have hundreds of actin-binding proteins (ABP) which bind either or both the forms of actin. Profilin sequesters G-actin and makes them pre-disposed towards F-actin barbed-end addition, cofilin severs F-actin and deploymerizes it into G-actin. On the other hand, capping protein (CP) caps the barbed-end and stops further elongation of F-actin [2]. Gelsolin-family of proteins [3] sever F-actin as well as cap filaments. In vitro TIRF microscopy [4] has been used to monitor real-time actin dynamics in the presence of ABPs [5], [6]. Representative results on some ABPs which alter actin assembly will be presented.