Biology subjects

Fottner, M.

Publications and source records attributed to Fottner, M..

6 recordsLinked to original sources

bioRxiv2025

The functional landscape of the human ubiquitinome

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. To date, hundreds of thousands of ubiquitination sites (ubi-sites) have been identified, however fewer than 1% have known functional roles. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. To pinpoint critical regulatory events requiring ubiquitination at a precise site, we mapped ubi-site conservation across proteomics data from six non-human species. Perturbation proteomics revealed that highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize site-specific ubiquitination relevant for organismal fitness, we constructed a machine learning-based positional importance score for more than 100,000 ubi-sites, which identifies sites regulating diverse protein functions and rationalizes genetic vulnerabilities. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying site-specific protein ubiquitination.

cell biology↗

bioRxiv2025

Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W

Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet access to such conjugates remains a major challenge. Existing approaches are often constrained by low yields, harsh reaction conditions, engineered recognition motifs or non-native linkage architectures. Here, we present UbyW (Ubiquitylation by UBE2W), a programmable platform for site-specific ubiquitylation that repurposes the E2 enzyme UBE2W to target genetically encoded isopeptidic neo-N-termini. UbyW enables efficient generation of near-native Ub-protein conjugates across diverse protein substrates, including endogenous ubiquitylation sites within folded domains, and can be implemented through a reconstituted intracellular cascade in Escherichia coli for streamlined high-yield production. The platform further enables installation of chemical functionalities adjacent to the isopeptidic linkage, including photocrosslinkers for capturing modification-dependent interactions. Using programmable probes targeting site-specific ubiquitylation of the small GTPase Ran, we identify USP15 as a cognate deubiquitylase and show that Ran K71 monoubiquitylation disrupts key Ran-cycle interactions.

biochemistry↗

bioRxiv2025

Structural basis for the ubiquitin chain recognition of the human 26S proteasome

Proteasomal degradation is a fundamental process for all eukaryotic life. A protein destined for degradation is first tagged with a polyubiquitin chain, which is selected by the proteasome. Different ubiquitin chain topologies serve as distinct signals, with K48-linked chains acting as the canonical degradation signal and K11/K48-branched chains providing even more potent targeting, particularly during cell cycle regulation. However, the structural basis for how the proteasome distinguishes between these different chain architectures has remained unclear. Here, we present high-resolution cryo-EM structures of the human 26S proteasome bound to both a K48-linked tetraubiquitin chain and a K11/K48-branched chain. Our structures reveal distinct binding modes for these two types of chain linkage. K48 chains wrap around the Ubiquitin interaction motif of the receptor RPN10 in an unexpected spiral conformation, while K11 branches engage the proteasome through previously uncharacterised interfaces in a cleft formed between RPN2 and RPN10. Through structure-guided mutagenesis and cellular studies, we demonstrate that these binding modes are essential for efficient substrate degradation and cell cycle progression. These findings establish how the proteasome achieves selective substrate recognition through chain topology-specific interactions.

biochemistry↗

bioRxiv2025

Hijacking a bacterial membrane transporter for efficient genetic code expansion

The site-specific encoding of non-canonical amino acids (ncAAs) provides a powerful tool for expanding the functional repertoire of proteins. Its widespread use for basic research and biotechnological applications is, however, hampered by low efficiencies of current ncAA incorporation strategies. We uncover poor cellular ncAA uptake as a main obstacle to efficient genetic code expansion and overcome this bottleneck by hijacking a bacterial membrane transporter to actively import isopeptide-linked ncAAs within easily synthesizable tripeptide-scaffolds. Using this approach, we enable efficient encoding of eleven previously inaccessible ncAAs, decorating proteins with bioorthogonal and crosslinker moieties, posttranslational modifications, and functionalities for chemoenzymatic conjugation. To enhance scalability of protein production, we evolve the membrane transporter for preferential import of isopeptide-linked tripeptides, creating a novel Escherichia coli strain that facilitates single and multi-site ncAA incorporation with wild type efficiencies. Additionally, we adapt the tripeptide-scaffolds for co-transport of two different ncAAs, enabling their efficient dual incorporation. This work underscores the importance of optimizing ncAA-uptake for high-yielding production of modified proteins and will accelerate the development of generalizable transport systems, aiding incorporation of non-canonical building blocks to broaden the chemical space of proteins without the need to design for passive membrane permeability.

synthetic biology↗

bioRxiv2024

One-Pot Dual Protein Labelling for Simultaneous Mechanical and Fluorescent Readouts in Optical Tweezers

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Forster resonance energy transfer (FRET) pairs, addresses this issue. Moreover, attaching one FRET probe to a tethered protein and the other to a protein in solution allows precise localisation of interaction sites, while probing mechanical properties. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labelling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labelled with the FRET pair maleimide-Atto550 and maleimide-Atto647N, alongside single- stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labelled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing protein conformational dynamics and protein interactions beyond the limitations of traditional optical tweezers. StatementThe developed method combines fluorescence microscopy with optical tweezers, enhancing single-molecule protein studies by overcoming the limitations of one- directional mechanical probing. Utilizing two orthogonal protein conjugation methods for one-pot dual labelling, the heat shock protein 90 was labelled with a FRET pair and single-stranded DNA oligonucleotides. Validated by comparison with published conformational changes, mechanical unfolding signatures, and FRET pair distances, this approach provides a powerful tool to explore single-molecule conformational dynamics and protein interactions.

biophysics↗

bioRxiv2022

Intestinal myofibroblasts regulate intestinal epithelial cell plasticity via YAP/TAZ

Intestinal stromal cells play a key role as the crypt niche cells during epithelial homeostasis and tumor initiation. However, the underlying cellular and molecular mechanisms remain unclear. We developed various types of three-dimensional (3D) tissue culture models to culture small intestinal myofibroblasts (SI MFs) together with enteroids. SI MFs significantly enhanced self-renewal, lumen formation and survival of enteroids, that was mediated via a paracrine mechanism in a Wnt-independent manner. Such co-cultured enteroids resembled SI organoids derived from Apc+/1638N tumors. Microarray analysis showed upregulation of genes associated with YAP signaling in enteroids co-cultured with SI MFs, which was confirmed by protein quantification by mass spectrometry and could be correlated with findings from human colorectal tumor specimens. Mass spectrometric analysis of conditioned media and inhibitor studies pointed to a role for TGF-{beta} in the SI MF-SI epithelium cross-talk. Altogether, utilizing different 3D stroma-epithelium co-culture models, we demonstrate here that SI MFs have the potential to induce a tumor-like phenotype in the intestinal crypts via a paracrine mechanism, that involves YAP and TGF-{beta}, but not canonical Wnt signaling.

cell biology↗