Biology subjects

Peters, N.

Publications and source records attributed to Peters, N..

4 recordsLinked to original sources

bioRxiv2025

An optimised system for rapid auxin-inducible protein degradation in budding yeast

The auxin system for inducible protein degradation is a powerful tool to investigate protein function. It consists of a degron fused to a target protein, an auxin-related ligand that binds to the degron, and a receptor that recognises the auxin-bound degron and mediates proteasomal degradation of the target protein. Variants of all system components are available, and we here test three degrons, three auxins and three degron receptors to identify optimal combinations of these variants in budding yeast. We show that the degrons mIAA7 or AID* together with adamantyl-auxin and the degron receptor OsTIR1(F74G) allow particularly rapid and extensive degradation. Basal degradation in the absence of auxin is generally low and can be prevented entirely by inducible expression of OsTIR1(F74G). Finally, we demonstrate that the remarkable efficiency of this system makes it competitive with established chemical inhibitors, such as tunicamycin and MG132, and with temperature-sensitive mutant alleles. These findings will aid the effective application of the auxin system.

cell biology↗

bioRxiv2025

Pathogenic DVL frameshifting variants in Robinow syndrome disrupt WNT signaling and cellular dynamics

Robinow syndrome (RS) is a genetically heterogeneous rare disorder involving six genes in the WNT/planar cell polarity (PCP) signaling pathway. Frameshifting variants in DVL genes that introduce a novel basic C-terminus are a common cause of autosomal dominant RS, accounting for [~]33% of individuals without ROR2 variants. Here, we review ClinVar and literature variants affecting DVL paralogs resulting in RS and investigate the cellular effects of pathogenic DVL frameshift variants with mutant tails replacing at least 82 amino acids. In silico analysis of the new C-termini revealed altered intrinsically disordered regions (IDRs), charge distribution, and predicted protein structures. To explore potential altered biological mechanisms caused by novel C-termini, we generated wild-type (WT), frameshift, and truncated constructs of DVL1-3, and analyzed their behavior in a transfection-based in vitro systems. DVL proteins normally polymerize into cytoplasmic puncta that redistribute upon WNT stimulation. Immunocytochemistry showed that mutant DVL proteins failed to change their localization in response to WNT ligands, in contrast to WT alleles--a consistent observation across all three DVLs. In line with this, TOPFlash reporter assays demonstrated that mutant DVL1 and DVL3 failed to activate canonical WNT signaling, while WT proteins induced strong activation. Additionally, the mutant C-terminal tail interfered with CSNK1E-induced phosphorylation, offering a potential mechanism underlying the impaired WNT response. Our results provide further understanding of the cellular consequences of pathogenic DVL frameshifting variants and offers insights into the effects of such alleles on WNT signaling, and the perturbations thereof, that may lead to developmental phenotypes observed in RS.

genetics↗

bioRxiv2024

Reprograming of the ubiquitin ligase Ubr1 by intrinsically disordered Roq1 through cooperating multifunctional motifs

One way cells control the speed and specificity of protein degradation is by regulating the activity of ubiquitin ligases. Upon proteotoxic stress in yeast, the intrinsically disordered protein Roq1 binds the ubiquitin ligase Ubr1 as a pseudosubstrate, thereby modulating the degradation of substrates of the N-degron pathway and promoting the elimination of misfolded proteins. The mechanism underlying this reprograming of Ubr1 is unknown. Here, we show that Roq1 controls Ubr1 by means of two cooperating multifunctional motifs. The N-terminal arginine and a short hydrophobic motif of Roq1 interact with Ubr1 as part of a heterobivalent binding mechanism. Via its N-terminal arginine, Roq1 regulates the ubiquitination of various N-degron substrates and folded proteins. Via its hydrophobic motif, Roq1 accelerates the ubiquitination of misfolded proteins. These findings reveal how a small, intrinsically disordered protein with a simple architecture engages parallel channels of communication to reprogram a functionally complex ubiquitin ligase.

biochemistry↗

bioRxiv2023

Pre-existing Subclones Determine Radioresistance in Rectal Cancer Organoids

More than half of all cancer patients receive radiation therapy, but resistance is commonly observed. Currently, it is unknown whether resistance to radiation therapy is acquired or inherently present. Here, we employed organoids derived from rectal cancer and single-cell whole genome sequencing to investigate the long-term evolution of subclones in response to radiation. Comparing single-cell whole genome karyotypes between unirradiated and irradiated organoids revealed three patterns of subclonal evolution: (i) subclonal persistence, (ii) subclonal extinction, and (iii) subclonal expansion. Only organoids in which subclonal shifts occurred (i.e., expansion or extinction) became more resistant to radiation. Although radioresistant subclones did not share recurrent copy number alterations that could explain their radioresistance, resistance was associated with reduced chromosomal instability; an association that was also observed in 529 human cancer cell lines. These data suggest resistance to radiation is inherently present and associated with reduced chromosomal instability.

cancer biology↗