Biology subjects

Gasper, R.

Publications and source records attributed to Gasper, R..

4 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2025

Molecular anatomy of PLK1 master docking motifs

The Polo-box domain (PBD) localizes Polo-like kinase 1 (PLK1) near mitotic substrates required for chromosome biorientation. Recent work on mitotic kinetochores showed PLK1 docking begins hierarchically at master docking motifs on BUB1 and CENP-U. Whether master docking motifs have common molecular features remains poorly understood. Presence on CENP-U of two neighbouring motifs generated by initial CDK1 priming and subsequent PLK1 phosphorylation led us to hypothesize PBD dimerization might be involved. Using biochemical, biophysical, and modelling approaches, we gathered strong evidence that CENP-U contains a single master docking motif. The motif is very high affinity and sufficient to form extensive interactions with the PBD, engaging multiple pockets on its surface without obvious added benefits from dimerization. Comparisons with motifs in BUB1, BUBR1, and PRC1 suggests apparent commonalities of master PLK1 docking motifs. We discuss the implications of our observations for the mechanism of PLK1 activation.

biochemistry↗

bioRxiv2024

Monovalent Pseudo-Natural Product Degraders Supercharge the Native Degradation of IDO1 by KLHDC3

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degrader drugs function by directly mediating proximity between a neosubstrate and hijacked E3 ligase. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and, thus, would also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

cell biology↗

bioRxiv2019

Evolved, Selective Erasers of Distinct Lysine Acylations

Lysine acetylation, including related lysine modifications such as butyrylation and crotonylation, is a widespread post-translational modification with important roles in many important physiological processes. However, uncovering the regulatory mechanisms that govern the reverse process, deacylation, has been challenging to address, in great part because the small set of lysine deacetylases (KDACs) that remove the modifications are promiscuous in their substrate and acylation-type preference. This lack of selectivity hinders a broader understanding of how deacylation is regulated at the cellular level and how it is correlated with lysine deacylation-related diseases. To facilitate the dissection of KDACs with respect to substrate specificity and modification type, it would be beneficial to re-engineer KDACs to be selective towards a given substrate and/or modification. To dissect the differential contributions of various acylations to cell physiology, we developed a novel directed evolution approach to create selective KDAC variants that are up to 400-fold selective towards butyryl- over crotonyl-lysine substrates. Structural analyses of this non-promiscuous KDAC revealed unprecedented insights regarding the conformational changes mediating the gain in specificity. As a second case study to illustrate the power of this approach, we re-engineer the human SirT1 to increase its selectivity towards acetylated versus crotonylated substrates. These new enzymes, as well as the generic approach that we report here, will greatly facilitate the dissection of the differential roles of lysine acylation in cell physiology.\n\nSignificance StatementAcetylation of lysine residues features numerous roles in diverse physiological processes and correlates with the manifestation of metabolic diseases, cancer and ageing. The already huge diversity of the acetylome is multiplied by variations in the types of acylation. This complexity is in stark contrast to the small set of lysine deacetylases (KDACs) present in human cells, anticipating a pronounced substrate promiscuity.\n\nWe device a strategy to tackle this disarray by creating KDAC variants with increased selectivity towards particular types of lysine acylations using a novel selection system. The variants facilitate the dissection of the differential contributions of particular acylations to gene expression, development and disease. Our structural analyses shed light on the mechanism of substrate discrimination by Sirtuin-type KDACs.

biochemistry↗