Biology subjects

Hofer, F.

Publications and source records attributed to Hofer, F..

4 recordsLinked to original sources

bioRxiv2024

Probing the role of Nogo receptor homolog NgR2 in setting up cochlear connectivity

Sound encoding depends on the precise and reliable neurotransmission at the afferent synapses between the sensory inner hair cells (IHCs) and spiral ganglion neurons (SGNs). The molecular mechanisms contributing to the formation, as well as interplay between the pre- and postsynaptic components remain largely unclear. Here, we tested the role of the synaptic adhesion molecule and Nogo/RTN4 receptor homolog RTN4RL2 (also referred to as NgR2) in the development and function of afferent IHC-SGN synapses. Upon deletion of RTN4RL2 in mice (RTN4RL2-/-), presynaptic IHC active zones showed enlarged synaptic ribbons and a depolarized shift in the activation of CaV1.3 Ca2+ channels. The postsynaptic densities (PSDs) of SGNs were smaller and deficient of GluA2-4 AMPA receptor subunits despite maintained Gria2 mRNA expression in SGNs. Next to synaptically engaged PSDs we observed "orphan" PSDs located away from IHCs. They likely belong to a subset of SGN peripheral neurites that do not contact the IHCs in RTN4RL2-/- cochleae as found by volume electron microscopy reconstruction of SGN neurites. Auditory brainstem responses of RTN4RL2-/- mice showed increased sound thresholds indicating impaired hearing. Together, these findings suggest that RTN4RL2 contributes to the proper formation and function of auditory afferent synapses and is critical for normal hearing.

neuroscience↗

bioRxiv2022

ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning

Tight regulation of Arp2/3 complex is required to allow productive nucleation of force-generating, branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into Arp2/3 complex. Specifically, both isoforms of the ArpC5 subunit, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. Elevated levels of ArpC5 have also been linked to increased cancer progression and metastasis. Here, we have combined genetic engineering of cells and cellular structural biology to describe how ArpC5 and ArpC5L differentially regulate cell migration. They do so by defining the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, protein dynamics, and actin network ultrastructure. ArpC5 isoforms also have an impact on the positioning of actin assembly factors from the Ena/VASP family, which act downstream of Arp2/3 complex-mediated nucleation. This suggests that ArpC5 and Ena/VASP proteins, both predictors for poor outcome in cancer, are part of a signaling pathway enhancing cell migration and, by inference, metastasis.

cell biology↗

bioRxiv2020

Structural basis for variable IgE reactivities of Cor a 1 hazelnut allergens

A major proportion of allergic reactions to hazelnuts (Corylus avellana) are caused by immunologic cross-reactivity of IgE antibodies to pathogenesis-related class 10 (PR-10) proteins. Intriguingly, the four known isoforms of the hazelnut PR-10 allergen Cor a 1, denoted as Cor a 1.0401-Cor a 1.0404, share sequence identities exceeding 97 % but possess different immunologic properties. In this work we describe the NMR solution structures of these proteins and provide an in-depth study of their biophysical properties. Despite sharing highly similar three-dimensional structures, the four isoforms exhibit remarkable differences regarding structural flexibility, hydrogen bonding and thermal stability. Our experimental data reveal an inverse correlation between structural flexibility and IgE-binding, with the most flexible isoform having the lowest IgE-binding potential, while the isoform with the most rigid backbone scaffold displays the highest immunologic reactivity. These results point towards a significant entropic contribution to the process of antibody binding.

molecular biology↗

bioRxiv2020

In silico design of Phl p 6 variants with altered folding stability significantly impacts antigen processing, immunogenicity and immune polarization

AbstactO_ST_ABSIntroductionC_ST_ABSProtein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. MethodsUsing the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. ResultsFour stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 {degrees}C. Destabilization led to faster proteolytic processing in vitro, whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo, stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines. ConclusionMAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

immunology↗