Biology subjects

Sunderlikova, V.

Publications and source records attributed to Sunderlikova, V..

4 recordsLinked to original sources

bioRxiv2026

Direct observation of ATP-driven ubiquitin chain handling by Cdc48

Cdc48 (p97 or VCP in metazoans) targets polyubiquitin to selectively disassemble and degrade proteins. Cdc48 is believed to move along the ubiquitin chain towards linked proteins, but this has not been directly observed. By following single molecules in time, we find that the polypeptide branch points of ubiquitin chains are repeatedly inserted and rejected from the Cdc48 pore in ATP-driven manner, in bursts lasting up to seconds. This non-processive mode either ends by terminal substrate rejection, or advances to processive action, which drives ubiquitin unfolding in two steps, branch point translocation, and the extrusion of ubiquitin and linked protein as polypeptide loops in trans. Final retrograde movement can bring polypeptide segments back to cis. Our results establish the dynamics of ubiquitin chain handling by Cdc48, reveal key hallmarks of kinetic proofreading. We speculate that Cdc48 translocation may play a role in ubiquitin chain selection.

biophysics↗

bioRxiv2025

Translation-driven temporal control for intertwined protein assembly

Protein complexes are essential to cells. However, how structurally intertwined protein subunits can assemble faithfully is poorly understood. Here, we reveal a "temporal control" mechanism driven by coupled ribosomes to form intertwined dimers. Using Disome Selective Profiling and optical tweezers, we show that the BTB domains of KEAP1, KLHL12, and PATZ1 form stable closed states as monomers, thus impeding proposed domain-swapping assembly routes. By contrast, the timed emergence of nascent chain segments during translation enables alternative folding-assembly pathways that bypass the closed monomeric state. Analysis indicates that this mechanism works in concert with dimerization quality control by the E3 ligase SCF-FBXL17, and is relevant across the BTB domain family. This study shows that ribosome cooperation expands the range of possible protein architectures.

molecular biology↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2020

Polypeptide collapse modulation and folding stimulation by GroEL-ES

Unfolded proteins ubiquitously collapse into a compact yet dynamic state1,2. While this compaction is pivotal to protein folding3, aggregation4,5, intrinsic disorder6, and phase separation7, its role in protein quality control mechanisms remains obscure8. Collapse has been characterized mainly for polypeptides that are free in solution, in terms of kinetics, chain expansion, and effect on folding9,10. Yet, theory suggests that the solvent-mediated forces driving collapse can be altered near hydrophobic and charged surfaces, which are observed for many proteins including GroEL-ES11,12. Notably, while GroEL-ES is the archetypal protein-folding chaperone, its folding mechanism remains unresolved13,14. GroEL-ES is proposed to sterically confine polypeptides within its closed chamber15, unfold misfolded states16,17, or promote folding indirectly by suppressing aggregation18,19. Here, using integrated protein manipulation and imaging, we show that GroEL-ES can strengthen the collapse of polypeptide substrates, and hence stimulate folding directly. Strikingly, attractive forces pull substrate chains into the open GroEL cavity -unclosed by GroES-, and hence trigger a gradual compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL, and is aided by GroES binding to the cavity rim, and by the amphiphilic C-terminal tails at the cavity bottom. Peptides corresponding to these C-termini alone are sufficient to strengthen the collapse. The results show a mechanism that allows folding to be stimulated: by strengthening the collapse, residues are brought together that must contact to fold. The notion that one protein can modulate the collapse of another may be generally important in protein conformation and coacervation control, for systems ranging from the GroEL-ES homologue TRiC/CCT20, to the oncogenic c-Myc/Max complex21, and the nuclear pore transporter transportin22.

biophysics↗