Biology subjects

Davey, N. E.

Publications and source records attributed to Davey, N. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression

Investigating biological function with high reproducibility requires the generation of stable cell lines that offer precise and inducible transgene expression. However, traditional methods often suffer from the limitations of random genomic integration, including unpredictable expression levels, leaky basal activity, and disruption of endogenous loci. To address these challenges, we engineered a versatile RPE1 cell line by integrating a TetOn3G-FRT cassette into the AAVS1 safe-harbour locus using CRISPR/Cas9(D10A) nickase. This platform utilises the Flp-FRT recombination system to facilitate rapid, site-specific "plug-and-play" insertion of transgenes, combined with the Tet-On 3G system for tight, dose-dependent expression control. The resulting RPE1 AAVS1 FRT8 line enables highly consistent transgene induction across independent clones, behaving as a monoclonal population. We highlight the importance of rigorous cell line characterisation through a multi-omics approach. Whole genome sequencing identified precise heterozygous integration at the target locus and de novo genomic duplications on other chromosomes. By integrating WGS with high-resolution proteomics and cell cycle analysis, we confirmed that these alterations did not significantly perturb cellular homeostasis or phenotypic fitness. This work establishes a robust and reliable platform for the efficient generation of inducible AAVS1 FRT stable cell lines, facilitating reproducible research in modern molecular biology.

bioengineering↗

bioRxiv2026

ZATT/ZNF451 promotes release of stalled TOP2 cleavage complexes

Topoisomerase II (TOP2) resolves DNA topological constraints through a tightly regulated cycle of DNA double-strand cleavage and religation. Nearby DNA damage or chemotherapeutic agents such as etoposide block the DNA religation step, stabilizing TOP2-DNA cleavage complexes (TOP2ccs) at DNA double-strand breaks (DSBs). The SUMO E3 ligase ZATT (ZNF451) has recently emerged as a key effector of TOP2cc repair, but its mechanism of action remains poorly understood. Here, we show that ZATT is sufficient to resolve TOP2ccs independently of TDP2, TOP2 proteolysis, and canonical DSB repair pathways. Using Xenopus egg extracts and biochemical reconstitution, we find that ZATT salvages trapped TOP2 by promoting TOP2 release from its stalled cleavage complex. Structural modeling and targeted mutagenesis in Xenopus egg extracts and human cells identify a highly conserved hydrophobic pocket in the tower domain of TOP2 where the ZATT coiled-coil "hooks on" to promote TOP2cc resolution. Our findings reveal a new strategy to resolve TOP2ccs that bypasses the exposure of dangerous DNA breaks.

molecular biology↗

bioRxiv2026

An Atlas of Short Linear Motif-Mediated Human Protein-Protein Interactions

Short linear motifs (SLiMs) within intrinsically disordered protein regions mediate transient interactions crucial for cell physiology1. However, the global interaction landscape of human SLiMs remains largely uncharted. Here we present the Atlas of SLiM-mediated Human protein-protein Interactions (ASHI), which maps more than 20,000 interactions by screening over 800 human protein domains against a library of one million peptides tiling the human disordered proteome. ASHI expands the SLiM interactome, uncovers novel binding modes for known peptide-binding domains, and reveals unexpected peptide-binding activities in enzymes, chaperones, RNA-binding proteins, and modification-reader domains. Furthermore, intrinsically disordered regions emerge as densely encoded interaction platforms where interaction specificity is governed by diverse mechanisms, including key motif determinants, flanking residues, competition, and multivalency. These data provide an unprecedented foundation for modeling dynamic interaction networks, interpreting disease-associated variants, and decoding the dark proteome.

biochemistry↗

bioRxiv2026

Uncovering cancer dependencies in peptide-interacting protein pockets

Cancer cells often become dependent on specific molecular functions. As many proteins perform multiple functions mediated by different pockets and interfaces, we hypothesized that we could identify distinct cancer dependencies and therapeutic vulnerabilities by disrupting peptide-binding pockets. To test this hypothesis, we screened a proteome-wide library of 7152 genetically encoded peptides across nine cancer cell lines. We identify common and selective dependencies on peptide-binding pockets and find that gene knockout and peptide-mediated inhibition of pockets often drive divergent phenotypes. For the common-essential gene HCF1, we identify a therapeutic window by using inhibitory peptides with varying affinity. Moreover, peptides targeting TLE1-4 reveal a dependency hidden in genetic screens by homolog redundancy. We also uncover that peptides inhibiting cyclin D drive specific suppression of leukemia cell proliferation and demonstrate that these peptides improve the potency of CDK4/6 inhibitors. Overall, our screening platform facilitates data-driven prioritization of molecular pockets for subsequent therapeutic translation.

cancer biology↗

bioRxiv2025

Systematic Discovery of Motif-based Interactions of the Auxiliary Domains of USP Family Deubiquitinases

The ubiquitin-specific proteases (USPs) family is the largest family of human deubiquitinating enzymes (DUBs). While most USPs are agnostic to polyubiquitin linkage-type, their substrate specificity is thought to be mediated by the recognition of the ubiquitylated protein itself. In addition to their catalytic domain, USPs have one or more auxiliary domains (ADs) with key functions in regulating DUB activity and subcellular localization. We hypothesize that some ADs bind short linear motifs (SLiMs) typically found in intrinsically disordered regions of proteins to achieve targeting to substrates and multiprotein complexes. To test this hypothesis, we systematically assessed the potential of 29 USP-ADs and two full-length USPs for SLiM binding using a combination of proteomic-peptide phage display, peptide arrays and affinity measurements. We discovered SLiM-based interactions for 14 ADs from 9 USP-DUBs, including CYLD, USP11, USP19, USP20, USP22 and USP33, and define the consensus motif and properties of the SLiM-AD binding. Interestingly, we established that the zf-UBP and DUSP2 domains of USP20 and USP33 are SLiM binding ADs with similar binding profiles, explaining the functional redundancy between the two DUBs. Our work reveals unique motifs recognized by the auxiliary domains CAP-Gly, UBL, zf-UBP and DUSP, with potential functional implications for substrate recognition and complex assemblies.

biochemistry↗

bioRxiv2025

A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

cell biology↗

bioRxiv2024

High-throughput discovery and deep characterization of cyclin-CDK docking motifs

Cyclin-CDKs are master regulators of cell division. In addition to directly activating the CDK, the cyclin subunit regulates CDK specificity by binding short peptide "docking" motifs in CDK substrates. Here, we measure the relative binding strength of [~]100,000 peptides to 11 human cyclins from five cyclin families (D, E, A, B and F). Using a quantitative intracellular binding assay and large-scale tiled peptide screening, we identified a range of non-canonical binders that unveil a broader than anticipated repertoire of cyclin docking motif types. Structural and saturation mutagenesis studies revealed distinct binding modes and sequence features that govern motif recognition, binding strength, and cyclin preference. Docking motifs vary from highly selective to pan-cyclin, thereby fine-tuning the timing of CDK phosphorylation during cell cycle progression. Overall, these findings provide an unprecedented depth of understanding about the rules encoding specificity and affinity within a group of related but distinct protein domains.

molecular biology↗

bioRxiv2024

A quantitative intracellular peptide binding assay reveals recognition determinants and context dependence of short linear motifs

Transient protein-protein interactions play key roles in controlling dynamic cellular responses. Many examples involve globular protein domains that bind to peptide sequences known as Short Linear Motifs (SLiMs), which are enriched in intrinsically disordered regions of proteins. Here we describe a novel functional assay for measuring SLiM binding, called Systematic Intracellular Motif Binding Analysis (SIMBA). In this method, binding of a foreign globular domain to its cognate SLiM peptide allows yeast cells to proliferate by blocking a growth arrest signal. A high-throughput application of the SIMBA method involving competitive growth and deep sequencing provides rapid quantification of the relative binding strength for thousands of SLiM sequence variants, and a comprehensive interrogation of SLiM sequence features that control their recognition and potency. We show that multiple distinct classes of SLiM-binding domains can be analyzed by this method, and that the relative binding strength of peptides in vivo correlates with their biochemical affinities measured in vitro. Deep mutational scanning provides high-resolution definitions of motif recognition determinants and reveals how sequence variations at non-core positions can modulate binding strength. Furthermore, mutational scanning of multiple parent peptides that bind human tankyrase ARC or YAP WW domains identifies distinct binding modes and uncovers context effects in which the preferred residues at one position depend on residues elsewhere. The findings establish SIMBA as a fast and incisive approach for interrogating SLiM recognition via massively parallel quantification of protein-peptide binding strength in vivo.

biochemistry↗

bioRxiv2024

A proteome-wide dependency map of protein interaction motifs

Short linear motifs (SLiMs) are the most ubiquitous protein interaction modules within unstructured regions of the human proteome, yet their contribution to cellular homeostasis remains poorly understood. To systematically assess SLiM function, we applied base editing to mutate all reported and a set of computationally predicted SLiMs defined by SLiM-like evolutionary patterns. By screening 7,293 SLiM-containing regions with 80,473 mutations in HAP1 cells, we define a SLiM dependency map identifying 450 known and 264 predicted SLiMs required for normal cell proliferation. Mutational consequences were highly reproducible in RPE1 cells, with differences attributed to cell line-specific gene essentiality. We show that most essential predicted SLiMs belong to novel classes and identify binding partners for several of these, providing mechanistic insight into a disease-associated ANKRD17 mutation. Our study provides a proteome-wide resource on SLiM essentiality and uncovers numerous uncharacterized yet essential SLiMs.

cell biology↗

bioRxiv2024

Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning

Deep mutational scanning (DMS) has emerged as a powerful approach for evaluating the effects of mutations on binding or function. Here, we developed a multiplexed DMS by phage display protocol to define the binding determinants of short linear motifs (SLiMs) binding to peptide binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well-known ligands for eleven different peptide binding domains, including the talin-1 PTB domain. Systematic benchmarking against a gold-standard set of motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage analysis correctly identifies known motif binding determinants. The DMS analysis further defined a non-canonical PTB binding motif, with a putative extended conformation. A second DMS library was designed aiming to provide information on the binding determinants for 19 SLiM-based interactions between human and SARS-CoV-2 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins, and refined the consensus motifs in human peptides binding to five domains from SARS-CoV-2 proteins, including the non-structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity improved cell-permeable NSP9-binding peptide was found to exert stronger antiviral effects as compared to the initial wild-type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants, and shows how DMS by phage display can guide peptide-engineering efforts.

biochemistry↗

bioRxiv2024

High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells

The cell cycle governs a precise series of molecular events, regulated by coordinated changes in protein and phosphorylation abundance, that culminates in the generation of two daughter cells. Here, we present a proteomic and phosphoproteomic analysis of the human cell cycle in hTERT-RPE-1 cells using deep quantitative mass spectrometry by isobaric labelling. Through analysing non-transformed cells, and improving the temporal resolution and coverage of key cell cycle regulators, we present a dataset of cell cycle-dependent protein and phosphorylation site oscillation that offers a foundational reference for investigating cell cycle regulation. These data reveal uncharacterised regulatory intricacies including proteins and phosphorylation sites exhibiting previously unreported cell cycle-dependent oscillation, and novel proteins targeted for degradation during mitotic exit. Integrated with complementary resources, our data link cycle-dependent abundance dynamics to functional changes and are accessible through the Cell Cycle database (CCdb), an interactive web-based resource for the cell cycle community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/599917v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@bb2a69org.highwire.dtl.DTLVardef@1dd55ecorg.highwire.dtl.DTLVardef@34073eorg.highwire.dtl.DTLVardef@1c7a8c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Benchmarking computational tools for de novo motif discovery

BackgroundOver the past twenty years, numerous motif discovery bioinformatic tools have been developed for discovering short linear motifs (SLiMs) from high-throughput experimental data on domain-peptide interactions. However, these tools are generally evaluated individually and mostly using synthetic data that do not accurately capture the motif context observed within proteomic data. Consequently, it is unclear how these tools perform in real-world use cases and how they perform compared to each other. ResultsHere, we benchmarked five motif discovery tools and seven general sequence alignment tools on their capacity to find SLiMs. For this purpose we have built MEP-Bench, a benchmarking dataset of peptides of varying complexity from curated SLiM instances from the Eukaryotic Linear Motif database. MEP-Bench allows tools to be tested for the effect of dataset size, peptide length, background noise level and motif complexity on motif discovery. The main metric used to compare all tools was the percentage of correctly aligned SLiM containing peptides. Two motif discovery tools (DEME and SLiMFinder) and a sequence alignment tool (Opal) outperformed the rest of the tools when benchmarked with this metric, averaging over 70% correctly aligned motif-containing peptides. The performance of the motif discovery tools and Opal were not affected by the sizes of the datasets. However, increasing peptide lengths and noise levels decreased all tools performances. While all tools performed well for N-/C-terminal motifs, for low-complexity motifs only DEME and SLiMFinder returned correctly aligned motifs for 50% or more of the datasets. ConclusionsThis study highlights DEME, SLiMFinder and Opal as the best performing tools for finding motifs in short peptides, and it indicates experimental parameters that should be considered given the limitations of the available tools. However, there is room for improvement, as no tool was able to identify all motif types. We propose that MEP-Bench can serve as a valuable resource for the SLiM community to compare new motif discovery methods with those benchmarked here.

bioinformatics↗

bioRxiv2023

Catalytic and non-catalytic functions of DNA polymerase kappa in translesion DNA synthesis

Translesion DNA synthesis (TLS) is an essential process that allows cells to bypass lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (Pol() has the unique ability to bypass minor groove DNA adducts in vitro. However, Pol(is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Pol(during lesion bypass. Strikingly, we show that Pol(has two main functions during TLS, which are differentially regulated via Rev1 binding. On the one hand, Pol(is essential to replicate across minor groove DNA lesions in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, via its cooperative interaction with Rev1 and ubiquitylated PCNA, Pol(stabilizes the Rev1-Pol(extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of Pol(catalytic activity. Together, our work identifies catalytic and non-catalytic functions of Pol(in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases.

molecular biology↗

bioRxiv2023

Global substrate identification and high throughput in vitro dephosphorylation reactions uncover PP1 and PP2A-B55 specificity principles

Phosphoprotein phosphatases (PPPs) dephosphorylate Serine (Ser)/Threonine (Thr) residues to regulate major signaling pathways and cellular transitions. Despite the central role of PPPs the substrates in most cellular processes and the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A- B55, which reveals common and unique preferences for the two phosphatases. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE:Dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit, resulting in defective cytokinesis thus underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and demostrate how binding of phosphatases to substrates shapes dephosphorylation specificity. Collectively, we develop novel approaches to advance our ability to investigate protein phosphatases and use these to provide a framework for understanding mitotic exit regulation by dephosphorylation.

cell biology↗

bioRxiv2022

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.

molecular biology↗

bioRxiv2022

MIADE metadata guidelines: Minimum Information About a Disorder Experiment

An unambiguous description of an experimental setup and analysis, and the subsequent biological observation is vital for accurate data interpretation and reproducible results. Consequently, experimental analyses should be described in a concise, unequivocal, and digestible manner. The aim of minimum information guidelines is to define the fundamental complement of data that can support an unambiguous conclusion on experimental observations. In this document, we present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the minimal fundamental parameters required for non-experts to understand the key findings of an experiment studying intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. We give examples of the application of these guidelines in common use cases and describe the implementation of an update to the DisProt IDP database to allow MIADE-compliant annotation. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardise the dissemination of the key metadata on an IDP experiment by IDP data sources.

scientific communication and education↗

bioRxiv2022

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1,712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

biochemistry↗

bioRxiv2022

Large-scale identification of phospho-modulated motif-based protein-protein interactions

Phosphorylation is an extensively studied post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Deciphering which of the hundreds of thousands of phosphosites in the proteome that regulate interactions remains challenging. We generated a proteomic peptide-phage display (ProP-PD) library to screen for phosphosites that regulate short linear motif-based interactions. The phage peptidome covers 13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome, each phosphosite being represented as a wildtype and a phosphomimetic variant. We screened 73 modular domains and identified 252 putative phospho-modulated interactions. Affinity measurements confirmed the phosphomodulation of 16 out of 21 tested interactions. We discovered a novel phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP). We validated the phospho-dependent clathrin interaction in a cellular context and found it to be essential for the mitotic function of HURP. Structural characterisation elucidated the molecular basis for the phospho-dependency of the clathrin-HURP interaction. Collectively, our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated SLiM-based interactions required for cellular function.

biochemistry↗