Biology subjects

Bolhuis, D.

Publications and source records attributed to Bolhuis, D..

3 recordsLinked to original sources

bioRxiv2024

USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S-phase chromatin and promotes normal cell cycle progression. Proteomics and enzyme assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, thus antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S-phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

cell biology↗

bioRxiv2024

Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41

The ubiquitin-like protein ISG15 (interferon-stimulated gene 15) regulates the host response to bacterial and viral infections through its conjugation to proteins (ISGylation) following interferon production. ISGylation is antagonized by the highly specific cysteine protease USP18, which is the major deISGylating enzyme. However, mechanisms underlying USP18s extraordinary specificity towards ISG15 remains elusive. Here, we show that USP18 interacts with its paralog USP41, whose catalytic domain shares 97% identity with USP18. However, USP41 does not act as a deISGylase, which led us to perform a comparative analysis to decipher the basis for this difference, revealing molecular determinants of USP18s specificity towards ISG15. We found that USP18 C-terminus, as well as a conserved Leucine at position 198, are essential for its enzymatic activity and likely act as functional surfaces based on AlphaFold predictions. Finally, we propose that USP41 antagonizes conjugation of the understudied ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) from substrates in a catalytic-independent manner. Altogether, our results offer new insights into USP18s specificity towards ISG15, while identifying USP41 as a negative regulator of FAT10 conjugation.

biochemistry↗

bioRxiv2020

In silico APC/C substrate discovery reveals cell cycle degradation of chromatin regulators including UHRF1

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates and we show that several chromatin proteins bind APC/C, oscillate during the cell cycle and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

cell biology↗