Biology subjects

Hoogendoorn, S.

Publications and source records attributed to Hoogendoorn, S..

5 recordsLinked to original sources

bioRxiv2026

Chemical Interrogation and Reprogramming of ATAT1-Mediated Tubulin Acetylation

Acetylation of -tubulin K40 by -tubulin acetyltransferase 1 (ATAT1) using acetyl-coenzyme A (Ac-CoA) marks stable microtubule populations, yet chemical tools to directly measure ATAT1 ligand engagement, inhibit its activity, or visualize ATAT1-mediated modification on intact microtubules remain limited. Through the development of a quantitative binding assay, we uncovered that ATAT1 can bind unnatural cofactors but fails to efficiently use them in acyl-transfer reactions. Structure-guided mutation subsequently yielded ATAT1-L163A, which successfully installed clickable handles at the native -tubulin K40 site of synthetic tubulin peptides, -tubulin, and intact microtubules. Cu(I)-catalyzed azide-alkyne cycloaddition enabled visualization of modified microtubules by in-gel fluorescence and microscopy. Moreover, we report a p11-CoA bisubstrate inhibitor that suppressed both native acetylation and engineered acylation. Together, these tools provide chemically controlled access to ATAT1 activity and a site-verified, clickable K40 modification on intact microtubules.

biochemistry↗

bioRxiv2026

A biochemical probe for microtubule lattice integrity uncovers motor-caused lattice damage

Microtubules experience mechanical and enzymatic stresses that can compromise lattice integrity, yet where and how lattice damage forms remains poorly understood due to the lack of tools that directly visualize damage as it occurs. Existing approaches infer damage indirectly through repair events or rely on static ultrastructural snapshots, precluding dynamic analysis. Here, we introduce MT-DS (Microtubule Damage Sensor), a damage-selective fluorescent probe that directly labels microtubule lattice openings. MT-DS combines taxane-based microtubule binding with a multivalent protein scaffold to restrict intraluminal diffusion and selectively retain the probe at sites of lattice openings. Using MT-DS, we visualize intrinsic lattice defects in stabilized microtubules, uncover a strong enrichment of damage at annealing sites, and demonstrate that kinesin-1{Delta}6 actively generates de novo lattice damage during motility. By enabling direct, time-resolved detection of microtubule damage, MT-DS establishes lattice integrity as an experimentally accessible parameter and provides a chemical tool to investigate how mechanical stress reshapes the microtubule cytoskeleton.

biochemistry↗

bioRxiv2026

Tipping the balance: Synthesis and evaluation of centrinone-based degraders of polo-like kinase 4

Polo-like kinase 4 (PLK4) is a serine/threonine-protein kinase that plays a pivotal role in centriole biogenesis and, as such, represents a master regulator of centriole duplication. Due to its importance in cancer development and progression, PLK4 represents an attractive target for the development of novel therapeutics. Herein, we present a series of molecular degraders of PLK4, based on the highly selective PLK4 inhibitor centrinone, with the aim of targeting PLK4 for degradation via the ubiquitin-proteasome system. While all synthesized degraders retained low nanomolar binding affinities to the kinase domain of PLK4, large differences were found with respect to their ability to change cellular PLK4 levels. We uncover a complex pharmacological profile of the most potent degraders, D6 and D10, consisting of concomitant lowering of PLK4 levels through degradation, and enhancing PLK4 levels through inhibition of its autoregulation - dependent on its localization at the centrioles.

cell biology↗

bioRxiv2022

Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog Pathway Inhibitor-1

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Illustrative of these are the many screens that have been conducted to find inhibitors for the Hedgehog (Hh) signaling pathway - a major developmental pathway with many implications in health and disease - with many hits but very few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We developed a PROTAC based on the downstream Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using our Hedgehog Pathway PROTAC (HPP) we identified and validated BET bromodomains to be the cellular targets of HPI-1. Furthermore, we found that HPP-9 has a unique mechanism of action as a long-acting Hh pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that has answered the longstanding question of the cellular target of HPI-1 and yielded the first PROTAC that acts on the Hh pathway.

cell biology↗

bioRxiv2017

A comprehensive portrait of cilia and ciliopathies from a CRISPR-based screen for Hedgehog signaling

The primary cilium organizes Hedgehog signaling, shapes embryonic development and is the unifying cause of the ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures including a protein complex containing {varepsilon}- and {delta}- tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many forms of congenital heart defects are ciliopathies. Collectively, this screen enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

genetics↗