Biology subjects

Beyer, D.

Publications and source records attributed to Beyer, D..

7 recordsLinked to original sources

bioRxiv2025

Development and structure-guided characterization of a novel ACE2-binding macrocyclic peptide

Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation, and the main protein receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63, developed using mRNA display, with high ACE2-binding affinity. The peptide was characterized in vitro in terms of purity, stability, hydrophilicity and ACE2 binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 [A] resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the wide catalytic cleft of the ACE2 peptidase domain, with no direct contact to the conserved zinc ion site. WJL-63 residues Q4, R7, R11 and R14 anchor the peptide deep inside the binding pocket. The opposite edges of the peptide were found to be in contact with subdomain 1 and subdomain 2 of the peptidase domain. This upright binding mode requires an open ACE2 conformation, in contrast to small molecule carboxypeptidase inhibitors, which typically bind to the closed conformation of the enzyme. As a consequence of the open conformation binding mode, the front edge of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells on which it revealed relatively strong binding with a KD value of 90 {+/-} 28 nM. WJL-63 provides a strong basis for the development of new classes of compounds for the modulation of ACE2 conformation, and for the development of imaging agents for the visualization of ACE2, for example in fluorescence or electron microscopy, or positron emission tomography (PET) imaging.

molecular biology↗

bioRxiv2025

Nanoscale PI(4,5)P2 partitioning driven by FGF2 oligomerization triggers membrane pore formation

Fibroblast Growth Factor 2 (FGF2) is a potent tumor cell survival factor. Unlike most secreted proteins, FGF2 lacks a signal peptide and is exported by an unconventional secretory pathway, bypassing the ER/Golgi system. A key event of this unusual secretory mechanism is FGF2 recruitment at the inner plasma membrane leaflet mediated by the phosphoinositide PI(4,5)P2. This interaction induces FGF2 oligomerization which, in turn triggers the formation of a transient lipidic membrane pore with a toroidal structure. FGF2 oligomers populating such membrane pores are captured and disassembled at the outer plasma membrane leaflet through interactions with heparan sulfate chains of the cell surface proteoglycan GPC1, resulting in full translocation of FGF2 across the plasma membrane. Once FGF2 appears on cell surfaces, it engages in signaling complexes initiating autocrine or paracrine signaling. Here, using a multidisciplinary and multiscale approach, we show that FGF2 self-assembles into oligomers that induce lateral PI(4,5)P2 partitioning and membrane remodeling. This process depends on PI(4,5)P2 reaching a critical local threshold concentration that, due to its non-bilayer properties destabilizes the bilayer, facilitating pore formation and FGF2 translocation across the plasma membrane. Our findings highlight a fundamental principle of how protein oligomerization can induce lateral partitioning of membrane lipids that in turn leads to a drastic membrane remodeling event: the transformation of the lipid bilayer into a toroidal membrane pore. The underlying mechanism is likely to be applicable to a wide range of membrane lipid interacting proteins that induce membrane remodeling in other cellular processes.

biochemistry↗

bioRxiv2024

Development of radiofluorinated MLN-4760 derivatives for PET imaging of the SARS-CoV-2 entry receptor ACE2

PurposeThe angiotensin converting enzyme 2 (ACE2) plays a regulatory role in the cardiovascular system and serves SARS-CoV-2 as an entry receptor. The aim of this study was to synthesize and evaluate radiofluorinated derivatives of the ACE2 inhibitor MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were demonstrated to be suitable for non-invasive imaging of ACE2, potentially enabling a better understanding of its expression dynamics. MethodsBased on computational molecular modeling, the ACE2-binding modes of F-MLN-4760 and F-Aza-MLN-4760 were similar to that of MLN-4760. Co-crystallization of the hACE2/F-MLN-4760 protein complex was performed for confirmation. Displacement experiments using [3H]MLN-4760 enabled the determination of the binding affinities of the synthesized F-MLN-4760 and F-Aza-MLN-4760 to ACE2 expressed in HEK-ACE2 cells. Aryl trimethylstannane-based and pyridine-based radiofluorination precursors were synthesized and used for the preparation of the respective radiotracers. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were evaluated with regard to the uptake in HEK-ACE2 and HEK-ACE cells and in vitro binding to tissue sections of HEK-ACE2 xenografts and normal organs of mice. Biodistribution and PET/CT imaging studies of [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were performed using HEK-ACE2 and HEK-ACE xenografted nude mice. ResultsCrystallography data revealed an equal ACE2-binding mode for F-MLN-4760 as previously found for MLN-4760 and indicated that the same would hold true for F-Aza-MLN-4760. The IC50 values were all in the high nM range, but three-fold lower for F-MLN-4760 and seven-fold lower for F-Aza-MLN-4760 than for MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were obtained in 1.4 {+/-} 0.3 GBq and 0.5 {+/-} 0.1 GBq activity with >99% radiochemical purity in a 5.3% and 1.2% radiochemical yield, respectively. Uptake in HEK-ACE2 cells was higher for [18F]F-MLN-4760 (67 {+/-} 9%) than for [18F]F-Aza-MLN-4760 (37 {+/-} 8%) after 3 h incubation while negligible uptake was seen in HEK-ACE cells (<0.3%). [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 accumulated specifically in HEK-ACE2 xenografts of mice (13 {+/-} 2% IA/g and 15 {+/-} 2% IA/g at 1 h p.i.) with almost no uptake observed in HEK-ACE xenografts (<0.3% IA/g). This was confirmed by PET/CT imaging, which also visualized unspecific accumulation in the gall bladder and intestinal tract. ConclusionBoth radiotracers showed specific and selective binding to ACE2 in vitro and in vivo. [18F]F-MLN-4760 was, however, obtained in higher yields and the ACE2-binding affinity was superior over that of [18F]F-Aza-MLN-4760. [18F]F-MLN-4760 would, thus, be the candidate of choice for further developlment to enable PET imaging of ACE2 in patients.

pharmacology and toxicology↗

bioRxiv2023

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast Growth Factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by PI(4,5)P2-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the protein-protein interaction interface through which FGF2 binds to the 1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we provide insights into a FGF2 dimerization interface that brings C95 residues in close proximity, resulting in disulfide bridged FGF2 dimers. We propose a mechanism by which they bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95 bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

biochemistry↗

bioRxiv2022

Clarifying space use concepts in ecology: range vs. occurrence distributions

Quantifying animal movements is necessary for answering a wide array of research questions in ecology and conservation biology. Consequently, ecologists have made considerable efforts to identify the best way to estimate an animals home range, and many methods of estimating home ranges have arisen over the past half century. Most of these methods fall into two distinct categories of estimators that have only recently been described in statistical detail: those that measure range distributions (methods such as Kernel Density Estimation that quantify the long-run behavior of a movement process that features restricted space use) and those that measure occurrence distributions (methods such as Brownian Bridge Movement Models and the Correlated Random Walk Library that quantify uncertainty in an animal movement path during a specific period of observation). In this paper, we use theory, simulations, and empirical analysis to demonstrate the importance of applying these two classes of space use estimators appropriately and distinctly. Conflating range and occurrence distributions can have serious consequences for ecological inference and conservation practice. For example, in most situations, home-range estimates quantified using occurrence estimators are too small, and this problem is exacerbated by ongoing improvements in tracking technology that enable more frequent and more accurate data on animal movements. We encourage researchers to use range estimators to estimate the area of home ranges and occurrence estimators to answer other questions in movement ecology, such as when and where an animal crosses a linear feature, visits a location of interest, or interacts with other animals. Open Research StatementTracking data on Aepyceros melampus, Beatragus hunteri, Bycanistes bucinator, Cerdocyon thous, Eulemur rufifrons, Glyptemys insculpta, Gyps coprotheres, Madoqua guentheri, Ovis canadensis, Propithecus verreauxi, Sus scrofa, and Ursus arctos are publicly archived in the Dryad repository (Noonan et al. 2018; https://doi.org/10.5061/dryad.v5051j2), as are data from Procapra gutturosa (Fleming et al. 2014a; https://doi.org/10.5061/dryad.45157). Data on Panthera onca were taken from (Morato et al. 2018). Additional data are publicly archived in the Movebank repository under the following identifiers: Canis latrans, 8159699; Canis lupus, 8159399; Chrysocyon brachyurus, 18156143; Felis silvestris, 40386102; Gyps africanus, 2919708; Lepus europaeus, 25727477; Martes pennanti, 2964494; Panthera leo, 220229; Papio cynocephalus, 222027; Syncerus caffer, 1764627; Tapirus terrestris, 443607536; Torgos tracheliotus, 2919708; and Ursus americanus, 8170674.

ecology↗

bioRxiv2022

IFITM3 blocks viral entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically impacts virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

molecular biology↗

bioRxiv2018

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

biochemistry↗