Biology subjects

Michels, A.

Publications and source records attributed to Michels, A..

4 recordsLinked to original sources

bioRxiv2026

NanoPro - a Framework for Probiotically-Guided Nanovector Therapy

The 'antigen problem', i.e. the disconnect between the broad spectrum of therapeutic modes enabled by the range of viral and synthetic nanoparticles for which workable receptor-targeting architectures exist and the relative dearth of addressable target receptors on most tumors, is a long-standing challenge in targeted tumor nanotherapy. Here we provide in vitro-proof-of-principle for NanoPro, a platform for guiding nanoscale therapeutics to otherwise antigenically indistinct tumors via probiotically-released tumor cell-binding tags, ProTags. These consist of an orthogonal antigen (GFP or ALFA) fused to the heparin-binding domain of placenta growth factor-2 and serve as attachment receptors for ProTag-targeted nanovectors. ProTag release from Escherichia coli Nissle 1917 was facilitated by deletion of the membrane-tethering lpp gene, which increased ProTag supernatant concentrations 16.5-fold compared to the parental strain without impacting viability. HBD-dependent decoration with ProTag in bacterial supernatant followed by treatment with receptor-targeted aGFP- or aALFA-lentiviral vectors resulted in majority transduction of MC-38 and MDA-MB-468 tumor cell lines with antigen specificities of 96.5% and 98.5%, respectively, depending on the choice of nanobody. This ProTag-mediated interaction was notably robust, with selective transduction still observed at 625-fold supernatant dilution, warranting further exploration of the utility of the NanoPro system for tumor-restricted delivery of nanotherapeutics.

synthetic biology↗

bioRxiv2023

The N-coil and the globular N-terminal domain of plant ARGONAUTE1 are interaction hubs for regulatory factors

The effector complex of RNA interference (RNAi) contains at its core an ARGONAUTE (AGO) protein bound to a small guide RNA. AGO proteins adopt a two-lobed structure in which the N-terminal (N) and Piwi-Argonaute-Zwille (PAZ) domains make up one lobe, while the middle (MID) and Piwi domains make up the other. Specific biochemical functions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N-terminal domain remain less clear. Here, we use yeast two-hybrid screening with the N-terminal domain of the founding member of the AGO protein family, arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated proteolysis. Interaction with a large group of proteins, including the autophagy cargo receptors ATI1 and ATI2, requires residues in a short, linear region, the N-coil, that joins the MID-Piwi lobe in the three-dimensional structure of AGO. In contrast, the F-box protein AUF1 interacts with AGO1 independently of the N-coil and requires distinct residues in the globular N domain itself. Mutation of AGO1 residues necessary for interaction with protein degradation factors in yeast stabilizes reporters fused to the AGO1 N-terminal domain in plants, supporting their in vivo relevance. Our results define distinct regions of the N domain implicated in protein-protein interaction, and point to a particular importance of the AGO1 N-coil as a site of interaction with regulatory factors.

molecular biology↗

bioRxiv2022

Distinction between small RNA-bound and free ARGONAUTE via an N-terminal protein-protein interaction site

ARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA Induced Silencing Complexes (RISCs). In the RNA-bound state, AGO proteins are stable while RNA-free AGOs turn over rapidly. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we show that a confined, linear region in Arabidopsis AGO1, the N-coil, is accessible to antibodies preferentially in the RNA-free state of AGO1. Reanalysis of hydrogen-deuterium exchange data on human Ago2 indicates similar structural flexibility of the N-coil depending on small RNA binding. Unloaded Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 via direct contact to specific amino acid residues in the N-coil, and mutation of residues required for ATI1 interaction reduces the degradation rate of unloaded AGO1 in vivo. These results provide insight into the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins.

molecular biology↗

bioRxiv2020

Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split {beta}-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

microbiology↗