Biology subjects

Mailand, N.

Publications and source records attributed to Mailand, N..

9 recordsLinked to original sources

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

mnDINO: Accurate and robust segmentation of micronuclei with vision transformer networks

Recent advances in cell segmentation successfully produce models that generalize across various cell-lines and imaging types. However, these methods still fail to recognize subcellular structures such as micronuclei (MN), which are rare and tiny DNA-containing structures found outside of the main nucleus and observable under the microscope. While they can be hard to recognize in images, studying MN formation is of great interest because of their relationship to chromosome instability, genotoxicity, and cancer progression. Here we present a segmentation model, mnDINO, to segment micronuclei in DNA stained images under diverse experimental conditions with very high efficiency and accuracy. To train this model, we collected a heterogeneous set of images with more than five thousand annotated micronuclei. Trained with this diverse resource, the mnDINO model improves the accuracy of MN segmentation, and exhibits strong generalization across microscopes and cell lines. The dataset, code, and pre-trained model are made publicly available to facilitate future research in MN biology.

bioinformatics↗

bioRxiv2024

Targeted disruption of linkage-specific ubiquitylation reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin chains dictate the functional outcome of numerous critical ubiquitin-dependent signaling processes. However, the functions and targets of several poly-ubiquitin topologies remain poorly defined due to a paucity of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin chain types in human cells to profile system-wide impacts of disabling formation of individual chain types. Focusing on K29-linked ubiquitylation, we reveal a strong association of this linkage type with chromatin-associated proteins and show that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Preventing K29-linked ubiquitylation-mediated control of SUV39H1 stability deregulates the H3K9me3 landscape, but not other histone marks. Collectively, our ubiquitin replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.

cell biology↗

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↗

bioRxiv2023

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Protein SUMOylation provides a principal driving force for cellular stress responses including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL) for DPCs, combining ubiquitin ligase activity through its RING domain with poly-SUMO chain binding via a cluster of SUMO-interacting motifs, analogous to the STUbL RNF4. Surprisingly, the STUbL activities of TOPORS and RNF4 are both required for SUMO-dependent DPC repair, PML degradation and other stress responses, making overlapping and distinct contributions to ubiquitin chain formation on SUMOylated targets to enable p97/VCP unfoldase recruitment. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, leading to defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Together, our findings establish TOPORS as a novel STUbL whose concerted action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk. HighlightsO_LIThe RING E3 ligase TOPORS is required for SUMO-dependent DPC repair C_LIO_LITOPORS is a novel SUMO-targeted ubiquitin ligase (STUbL) C_LIO_LITOPORS promotes multiple STUbL-driven processes in conjunction with RNF4 C_LIO_LICombined TOPORS and RNF4 loss is synthetic lethal in human cells C_LI

molecular biology↗

bioRxiv2023

VCF1 is an unconventional p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1 (VCP/p97 Cofactor FAM104 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel -helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

molecular biology↗

bioRxiv2023

Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organising protein Dbn1 as a novel major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCF{beta}-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized {beta}-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA-damage dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAXs ability to identify novel targets of stimulus-regulated ubiquitylation and reveal an SCF{beta}-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

molecular biology↗

bioRxiv2021

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

PP2A is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

cell biology↗

bioRxiv2020

Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Dominant missense mutations in the human serine protease FAM111A underlie perinatally lethal gracile bone dysplasia and Kenny-Caffey syndrome 1-3, yet how FAM111A mutations lead to disease is not known. We show that FAM111A proteolytic activity suppresses DNA replication and transcription by displacing key effectors of these processes from chromatin, triggering rapid programmed cell death by Caspase-dependent apoptosis to potently undermine cell viability. Patient-associated point mutations in FAM111A exacerbate these phenotypes by hyperactivating its intrinsic protease activity. Moreover, FAM111A forms a complex with the uncharacterized homologous serine protease FAM111B, point mutations in which cause a hereditary fibrosing poikiloderma syndrome 4, and we demonstrate that disease-associated FAM111B mutants display amplified proteolytic activity and phenocopy the cellular impact of deregulated FAM111A catalytic activity. Thus, patient-associated FAM111A and FAM111B mutations may drive multisystem disorders via a common gain-of-function mechanism that relieves inhibitory constraints on their protease activities to powerfully undermine cellular fitness.

cell biology↗