Biology subjects

Sicheri, F.

Publications and source records attributed to Sicheri, F..

10 recordsLinked to original sources

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2025

Mitotic single-stranded DNA suppression by DDIAS

Incomplete DNA replication and chromosome breakage during mitosis pose major threats to chromosome segregation. The CIP2A-TOPBP1 complex acts to mitigate this peril, but its exact role is not yet understood. Here, we report that DDIAS acts as a DNA-binding effector of the CIP2A-TOPBP1 complex. DDIAS directly interacts with TOPBP1 and the disruption of this interaction or inactivation of its single-stranded DNA (ssDNA)-binding ability impairs genome integrity and causes synthetic lethality with BRCA1 and BRCA2 deficiency. DDIAS does not promote the putative end-tethering function of CIP2A-TOPBP1 but rather acts to suppress ssDNA during mitosis. This role is emphasized by a pronounced genetic interaction between the genes coding for DDIAS and DNA polymerase (. We conclude that DDIAS defines a mitotic DNA damage response that mitigates the threat of mitotic ssDNA arising from DNA replication stress, which we infer is a liability to accurate chromosome segregation. (144 words).

molecular biology↗

bioRxiv2025

Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

The evolutionarily conserved, intrinsically disordered protein Bora is critical for initiating the activation of mitotic kinases. Once phosphorylated at Ser112 by Cyclin A-Cdk1 kinase, phospho-(p)Bora activates unphosphorylated Aurora A kinase (AURKA), directing it towards Polo-like kinase 1 (Plk1), thus promoting Cyclin B-Cdk1 activation and mitotic entry. Here, by combining structural modelling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKAs T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the Alpha C helix of the Plk1 kinase domain through a conserved motif, guiding AURKA activity towards the Plk1 T-loop, which is otherwise impervious to phosphorylation by AURKA. We highlight the importance of this motif for Bora function in vitro and during mitotic entry in Xenopus egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors. HighlightsO_LIMITOKINAC: a versatile approach that reconstitutes Bora and AURKA-dependent Plk1 T-loop phosphorylation in E. coli. C_LIO_LIAlphaFold 3 structural modeling of the phospho-Bora - AURKA complex tested by MITOKINAC using 39 Bora variants. C_LIO_LIPhospho-Bora wraps around the N-terminal lobe of AURKA to position its essential phospho-Serine 112 in the activation loop of AURKA. C_LIO_LIpBora delivers the AURKA activity to the Plk1 T-loop, which is otherwise impervious to AURKA phosphorylation. C_LI

biochemistry↗

bioRxiv2025

ProteoSync, a program for ortholog selection, automated sequence alignment and conservation projection onto protein atomic coordinates

The projection of conservation onto the surface of a proteins 3D structure is a powerful way of inferring functionally important regions. At present, the workflow for doing so can be involved and tedious. For this reason, we created ProteoSync, a Python program that semi-automates the process. The program creates an annotated sequence alignment of orthologs from a diverse set of selectable species, and enables the fast projection of amino acid conservation onto a predicted or known 3D model in PyMOL[1].

bioinformatics↗

bioRxiv2025

WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Mechanistically, WEE1i treatment triggers a translational shut-down, which is lethal in combination with the reduced translation of EIF2AKO cells. A genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinases GCN1/2 rescued WEE1i-mediated cytotoxicity. WEE1i induced GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. Loss of the collided ribosome sensor ZNF598 conversely increased sensitivity to WEE1i. Notably, the ISR was not required for WEE1i to induce DNA damage, premature mitotic entry or sensitization to DNA-damaging chemotherapeutics. ISR activation was independent of CDK1/2 activation. Importantly, WEE1i-mediated ISR activation was independent of WEE1 presence, pointing at off-target effects, which are shared by multiple chemically distinct WEE1i. This response was also observed in peripheral blood mononuclear cells. Importantly, low-dose WEE1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i triggers toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.

cancer biology↗

bioRxiv2024

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

biochemistry↗

bioRxiv2023

Engineered Antigen-Binding Fragments for Enhanced Crystallisation of Antibody:Antigen Complexes

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallisation, making the endeavour a significant effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallisation by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallise. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in a striking improvement in Fab and Fab:antigen complex crystallisability, revealing a synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallisation screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallisation and how they complement each other in the process.

biochemistry↗

bioRxiv2022

SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination

FBXW7, which encodes a substrate specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

cancer biology↗

bioRxiv2019

Competitive inhibitors of Ras effector binding

The small GTPases H, K and NRas are molecular switches that are indispensable for the correct regulation of cellular proliferation and growth. Mutations in Ras are associated with cancer and result in unwanted activation of signaling processes caused by aberrant recruitment of downstream effector proteins. In this study, we have engineered variants of the Ras-binding domain (RBD) of CRAF kinase that bind with highly improved affinity the effector binding site of Ras. Structural characterization demonstrates how the engineered RBD variants outcompete effector binding and inhibit Ras signaling in cells leading to apoptosis, growth arrest and senescence. The optimized RBD variants provide new insights in Ras biology and enabled the functional stratification of Ras dependency in patient-derived colorectal cancer organoids. One sentence summaryInhibition of effector kinase binding to Ras induces senescence in non-tumorigenic cells and reveals Ras dependency in patient-derived cancer organoids.

cancer biology↗

bioRxiv2018

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay

The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labelled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2 and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. As a sensitive, quantitative, flexible and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

biochemistry↗