2025 Hyper Recent •CC0 1.0 Universal

This work is dedicated to the public domain. No rights reserved.

Access Preprint From Server
April 1st, 2025
Version: 1
University Freiburg
molecular biology
biorxiv

A NanoBRET-based assay monitoring interactions in the USP18 signaling hub identifies the first cell-penetrant small molecule compromising USP18/ISG15 binding

Hess, S.Open in Google Scholar•Campos-Alonso, M.Open in Google Scholar•Brand, M.Open in Google Scholar•Lauw, S.Open in Google Scholar•Lindenmann, U.Open in Google Scholar•Goebel, K.Open in Google Scholar•Geurink, P. P.Open in Google Scholar•Fritz, G.Open in Google Scholar•Riedl, R.Open in Google Scholar•Knobeloch, K.-P.Open in Google Scholar

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico, this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro. To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.

Similar Papers

biorxiv
Wed Apr 02 2025
Genome reannotation and effector candidate identification in Meloidogyne chitwoodi through gland-specific transcriptome analysis
The root-knot nematode (RKN) Meloidogyne chitwoodi is a threat for potato production in the western United States, negatively impacting potato yield and product value. Meloidogyne chitwoodi produce proteins, called effectors, in their esophageal glands that are secreted during parasitism and play integral roles in plant-nematode interactions. Because the esophageal glands are the main effector sec...
Teixeira, M.
•
Ko, I.
•
Bali, S.
•
Vieira, P.
...•
Gleason, C.
biorxiv
Wed Apr 02 2025
Autoregulation of RPL7B by inhibition of a structural splicing enhancer
Yeast ribosomal protein gene RPL7B is autoregulated by inhibition of splicing. The first intron has a "zipper stem" that brings the 5' splice near the branch point and serves as an enhancer of splicing that is required for efficient splicing because it has non-consensus branch point sequence of UGCUAAC. The intron also contains an alternative, and mutually exclusive structure, that is conserved ac...
Stormo, G. D.
•
Granas, D.
•
Hewa, I. G.
•
White, M. A.
biorxiv
Wed Apr 02 2025
AquIRE reveals multiple mechanisms of clinically induced RNA damage and the conservation and dynamics of glycoRNAs
RNA is subject to many modifications, from small chemical changes such as methylation through to conjugation of biomolecules such as glycans. As well as these endogenously written modifications, RNA is also exposed to damage induced by its environment. Certain clinical compounds are known to drive covalent modifications of RNA with a growing appreciation for how these affect function. To understan...
Zhang, Z.
•
Kotopanova, Z. V.
•
Dang, K.
•
Kong, X.
...•
Knight, J. R.
biorxiv
Tue Apr 01 2025
Pseudogymnoascus destructans transcriptional response to chronic copper stress
Copper (Cu) is an essential metal micronutrient, and a fungal pathogens\' ability to thrive in diverse niches across a broad range of bioavailable copper levels is vital for host-colonization and fungal-propagation. Recent transcriptomic studies have implemented that trace metal acquisition is important for the propagation of the white nose syndrome (WNS) causing fungus, Pseudogymnoascus destructa...
Anne, S.
•
McDonald, M. R.
•
Lu, Y.
•
Peterson, R. L.
biorxiv
Tue Apr 01 2025
Surveillance of Japanese Encephalitis Virus in Piggery Effluent and Environmental Samples: A Complementary Tool for Outbreak Detection
Japanese encephalitis virus (JEV) is an emerging public health and biosecurity concern in Australia, with recent human cases and detections in mosquitoes and pigs across multiple states highlights the risk to susceptible human and animal populations. While traditional surveillance relies on mosquito trapping, sentinel chicken surveillance and direct testing of samples collected from pigs, monitori...
Ahmed, W.
•
Gebrewold, M.
•
Williams, D. T.
•
Wang, J.
...•
Simpson, S.
biorxiv
Tue Apr 01 2025
Inherent Specificity and Mutational Sensitivity as Quantitative Metrics for RBP Binding
Interactions between RNAs and RNA binding proteins (RBPs) regulate gene expression in eukaryotic cells. RNA-RBP affinities measured in vitro reveal diverse binding specificities, yet approaches to directly compare specificities across RBPs are lacking. Here, we introduce two quantitative metrics: inherent specificity, which measures how selectively an RBP distinguishes its strongest binding motif ...
Yi, S.
•
Singh, S. S.
•
Ye, X.
•
Krishna, R.
...•
Luna, J. M.
biorxiv
Tue Apr 01 2025
Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA
Single-stranded DNA-binding proteins (SSBs) are essential for genome stability, facilitating replication, repair, and recombination by binding ssDNA, recruiting other proteins, and dynamically relocating in response to cellular demands. Using single-molecule fluorescence resonance energy transfer (smFRET) assays, we elucidated the mechanisms underlying direct strand transfer from one locale to ano...
Paul, T.
•
Lee, I.-R.
•
Pangeni, S.
•
Rashid, F.
...•
Ha, T.
biorxiv
Tue Apr 01 2025
Negative regulation of miRNAs sorting in EVs: the RNA-binding protein PCBP2 impairs SYNCRIP-mediated miRNAs EVs loading
While it is accepted that Extracellular Vesicles (EVs)-mediated transfer of microRNAs contributes to intercellular communication, the knowledge about molecular mechanisms controlling the selective and dynamic miRNA-loading in EVs is still limited to few specific RNA-binding proteins interacting with sequence determinants. Moreover, although mutagenesis analysis demonstrated the presence/function o...
Marocco, F.
•
Garbo, S.
•
Montaldo, C.
•
Colantoni, A.
...•
Tripodi, M.
biorxiv
Tue Apr 01 2025
The break in the cycle: Inositol pyrophosphate fluxomics disentangled via mathematical modelling
This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line {Delta}SPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthe...
Hermes, J.
•
Kim, G.-D.
•
Liu, G.
•
De Leo, M. G.
...•
Timmer, J.