Biology subjects

Kotisch, H.

Publications and source records attributed to Kotisch, H..

4 recordsLinked to original sources

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↗

bioRxiv2024

PITHD1: An Endogenous Inhibitor of the 26S Proteasome During Cellular Dormancy

Cellular dormancy represents a state of regulated growth arrest essential for diverse biological processes, from reproduction to cancer progression. While mechanisms controlling protein synthesis in dormant cells have been identified, how cells regulate protein degradation during dormancy remains unclear. Using zebrafish oocytes, eggs and embryos as a model system, we discovered PITHD1 as an endogenous inhibitor of the 26S proteasome. Our high-resolution cryoEM structure reveals that PITHD1 simultaneously blocks three crucial functional sites on the 19S regulatory particle which are required for ubiquitin recognition, processing, and substrate translocation. This triple-lock mechanism effectively prevents protein degradation in dormant cells. Given PITHD1s evolutionary conservation across species, this mechanism likely represents a general strategy for reversible proteasome regulation during cellular dormancy. Our findings establish a new paradigm for controlling proteostasis in quiescent states.

biochemistry↗

bioRxiv2024

A multivalent adaptor mechanism drives the nuclear import of proteasomes

Nuclear protein homeostasis, including the turnover of transcription factors, critically depends on nuclear proteasomes. After each cell division, proteasomes need to be re-imported into the newly formed nucleus in a highly dynamic process that requires the largely unstructured protein AKIRIN2. However, how AKIRIN2 orchestrates this process and, more generally, how large protein complexes are translocated into the nucleus remains poorly understood. Here, we have used an integrated approach combining protein-wide saturation mutagenesis screens, cryoEM, and biochemical reconstitution to characterize AKIRIN2 as a scaffold protein that coordinates the stepwise assembly of an importin cluster around the proteasome. Through surveying every possible single amino acid substitution in AKIRIN2 using FACS- and microscopy-based genetic screens, we establish a comprehensive map of functionally relevant residues and binding interfaces in structured and disordered protein regions. Integrating these results with cryoEM analysis reveals a wing helix in a disordered region of AKIRIN2 that plays a crucial role in stabilizing proteasome interactions. Upon primary binding, AKIRIN2 homodimers recruit the importin IPO9, which in turn facilitates the binding of a second AKIRIN2 homodimer that recruits additional importins. Together, this multivalent molecular assembly amplifies the number of nuclear localisation signals and, thereby, triggers efficient proteasome translocation into the nucleus. Inside the nucleus, RanGTP rapidly dissociates importins, and AKIRIN2 is degraded by the proteasome in a ubiquitin-independent manner. Beyond mechanistically resolving the nuclear import of proteasomes, we propose that multivalent adaptor proteins like AKIRIN2 orchestrate the import of other macromolecular complexes and thereby dynamically control the composition of the nuclear proteome.

molecular biology↗

bioRxiv2022

Cryo-EM structure of the chain-elongating E3 ligase UBR5

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT E3 ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of the human UBR5, revealing a building block of an antiparallel dimer which can further assemble into larger oligomers. The large helical scaffold of the dimer is decorated with numerous protein-interacting motifs for substrate engagement. Using cryo-EM processing tools, we observe the dynamic nature of the domain movements of UBR5, which allows the catalytic HECT domain to reach engaged substrates. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates permits UBR5 to function in several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3s.

biochemistry↗