Biology subjects

Roumeliotis, T. I.

Publications and source records attributed to Roumeliotis, T. I..

At least 19 recordsLinked to original sources

bioRxiv2026

BCOR mutations establish a persistent culture-adaptive state in human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are widely used for disease modelling and regenerative medicine, yet their utility depends on maintaining molecular integrity during long-term culture. BCOR (BCL6 Co-Repressor) mutations are among the most recurrent culture-acquired alterations in hiPSCs, but their functional consequences remain poorly understood. Here, we show that hiPSC BCOR mutations are predominantly truncating indels enriched in exon 7, defining a mutational landscape distinct from that observed in cancer. Multi-omics profiling reveals that BCOR loss drives widespread chromatin, transcriptomic and proteomic remodelling, with coordinated activation of developmental, pluripotency-associated and mitochondrial metabolism programmes. To facilitate routine surveillance, we develop a cost-effective TaqMan qPCR assay that accurately identifies BCOR-mutant hiPSCs across independent cell lines. Finally, we demonstrate that correction of BCOR mutation by CRISPR-Cas9 only partially restores the wildtype molecular state, highlighting the importance of early detection and monitoring of adaptive mutations in hiPSC cultures.

cell biology↗

bioRxiv2026

Structural basis of polyspecific drug recognition by MRP2

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a hepatobiliary ATP-binding cassette transporter that mediates the efflux of endogenous conjugates and many therapeutic agents, yet the basis of its broad ligand specificity remains poorly understood. Here we combine cryo-electron microscopy, ATPase measurements and cell-based transport inhibition assays to define how MRP2 recognizes clinically relevant compounds from distinct chemical classes. We find that chemically diverse ligands occupy overlapping regions of a shared transmembrane cavity but adopt different poses, contact networks and stoichiometries. Ligand recognition is mediated predominantly by a permissive environment complemented by ligand-specific polar interactions, rather than by a single conserved pharmacophore. These findings establish a structural framework for MRP2 polyspecificity and provide insight into hepatobiliary drug disposition and transporter-mediated drug-drug interactions.

biochemistry↗

bioRxiv2026

Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

The cyclin-dependent kinase CDK11 functions in transcription, mitotic progression, and mRNA splicing. Specifically, spliceosome activation during the B to Bact transition depends on phosphorylation of the U2 snRNP component SF3B1 by the CDK11-cyclin L-SAP30BP complex. Here, we present the structure of this spliceosome-activating CDK-cyclin complex, determined by cryogenic electron microscopy at 2.3 [A] resolution. Our structure and biochemical experiments show that SAP30BP forms extensive interactions with cyclin L2, thereby stabilising it, and forms critical interactions with the C-terminal kinase lobe of CDK11 that promote complex assembly. Destabilisation of cyclin L2 in the absence of SAP30BP suggests that these principles are applicable to all CDK11-cyclin L complexes. Furthermore, we identify a pseudo-substrate sequence near the CDK11 C-terminus and provide evidence for a role of this segment in CDK11 auto-regulation. Finally, the structure of the CDK11-cyclin L2-SAP30BP complex bound to the clinical high-affinity CDK11 inhibitor OTS964 and a comparison to OTS964-bound off-target complexes provide insight into the mechanism of OTS964 selectivity and specificity.

molecular biology↗

bioRxiv2025

CCDC103-mediated assembly of the R2C complex links RUVBL1-RUVBL2 to Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetic disorder caused by defective cilia motility, powered by axonemal dynein motors. Assembly of these motors is facilitated by the molecular chaperone HSP90, its co-chaperone RUVBL1-RUVBL2 and adaptor proteins such as CCDC103 (aka DNAAF19). Mutations in CCDC103 identified in PCD patients impair dynein assembly, contributing to the disease pathology. Here, we present the cryo-electron microscopy structure of the human RUVBL1-RUVBL2-CCDC103 complex at 3.2[A] resolution, a chaperone assembly we refer to as R2C. It comprises a hetero-hexameric RUVBL1-RUVBL2 ring bound to three CCDC103 molecules via their RUVBL2-binding domains (RBDs), which have additional functions. Unlike RPAP3 of R2TP, a previously defined co-chaperone of HSP90, CCDC103 lacks a PIH1D1-binding motif and TPR domains, but its flexible N-terminal region regulates RUVBL1-RUVBL2 oligomerisation. Our characterisation of the R2C complex presented here enhances understanding of the intricate protein network involved in Hsp90-mediated assembly of dynein motors and how adaptor mutations contribute to PCD.

biochemistry↗

bioRxiv2025

MAGELLAN: Automated Generation of Interpretable Computational Models for Biological Reasoning

Computational models have become essential tools for understanding signalling networks and their non-linear dynamics. However, these models are typically constructed manually using prior knowledge and can be over-reliant on study bias. These limitations hinder their ability to make accurate predictions and incorporate new evidence. Scaling up the construction of models to take advantage of increasingly abundant omics data can bridge these gaps by providing a comprehensive view of signalling events and how they influence cellular phenotypes. In this study, we present MAGELLAN, a method leveraging message passing graph neural networks to build computational models directly from pathway data and discrete rules representing experimental results. We used this to construct a computational model of breast cancer signalling and re-parameterize a previously published non-small cell lung cancer (NSCLC) model, showing that MAGELLAN can predict genetic dependencies and achieve comparable model quality to expert-curated and manually trained models. Our approach enables the integration of prior knowledge networks and experimental data to build predictive models that are mechanistically interpretable. This approach simplifies model creation, making it more accessible and practical for experimentalists, and supports broader applications in drug discovery and biological research.

systems biology↗

bioRxiv2025

Selection of pre-leukemic hematopoietic stem cells driven by distinct extracellular matrix molecules

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

cell biology↗

bioRxiv2024

Chromosome end protection by RAP1-mediated inhibition of DNA-PK

During classical non-homologous end joining (cNHEJ), DNA-dependent protein kinase (DNA-PK) encapsulates free DNA ends, forming a recruitment platform for downstream end-joining factors including Ligase 4 (LIG4)1. DNA-PK can also bind telomeres and regulate their resection2-4, but does not initiate cNHEJ at this position. How the end joining process is regulated in this context-specific manner is currently unclear. Here we show that the shelterin components TRF2 and RAP1 form a complex with DNA-PK that directly represses its end joining function at telomeres. Biochemical experiments and cryo-electron microscopy reveal that when bound to TRF2, RAP1 establishes a network of interactions with KU and DNA that prevents DNA-PK from recruiting LIG4. In mouse and human cells, RAP1 is redundant with the Apollo nuclease in repressing cNHEJ at chromosome ends, demonstrating that the inhibition of DNA-PK prevents telomere fusions in parallel with overhang-dependent mechanisms. Our experiments show that the end joining function of DNA-PK is directly and specifically repressed at telomeres, establishing a molecular mechanism for how individual linear chromosomes are maintained in mammalian cells.

biochemistry↗

bioRxiv2024

Cell cycle-dependent S-sulfenyl proteomics uncover a redox switch in p21-CDK feedback governing the proliferation-senescence decision

Reactive oxygen species (ROS) influence cell proliferation and fate decisions by oxidizing cysteine residues (S-sulfenylation) of proteins, but specific targets and underlying regulatory mechanisms remain poorly defined. Here, we employ redox proteomics to identify cell cycle-coordinated S-sulfenylation events and investigate their functional role in proliferation control. Although ROS levels rise during cell cycle progression, overall oxidation of the proteome remains constant with dynamic S-sulfenylation restricted to a subset of cysteines. Among these, we identify a critical redox-sensitive cysteine residue (C41) in the cyclin-dependent kinase (CDK) inhibitor p21. C41 oxidation regulates the interaction of p21 with CDK2 and CDK4, controlling a double-negative feedback loop that determines p21 stability. When C41 remains reduced, p21s half-life increases in G2 phase resulting in more p21 inheritance to daughter cells suppressing proliferation and promoting senescence after irradiation. Notably, we identify dynamic S-sulfenylation on further cell cycle regulators implying coordination of cell cycle and redox control.

cell biology↗

bioRxiv2024

Integrated multiomic profiling reveals SWI/SNF subunit-specific pathway alterations and targetable vulnerabilities

Mutations in subunits of the SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex occur in {approx}20% of cancers and represent a highly unmet medical need. To identify novel therapeutic approaches, we systematically characterized transcriptomic and proteomic changes caused by the loss of SWI/SNF subunits or other epigenetic enzymes in isogenic cell lines, which we subsequently integrated with high-throughput drug screening and independent genetic screens of the DepMap project. Using an optimized bioinformatics pipeline for pathway enrichment, we identified Metabolism of proteins as the most frequently dysregulated Reactome pathway category in SWI/SNF-defective cell lines. Drug screening and multiomic integration revealed multiple chemicals selectively cytotoxic for SWI/SNF-defective models, including CBP/EP300 or mitochondrial respiration inhibitors. A novel algorithm for the analysis of DepMap CRISPR screens independently identified synthetic lethality between SWI/SNF defects and EP300 or mitochondrial respiration genes, which we further revalidated in disease-relevant models. These results unravel novel genetic dependencies for SWI/SNF-defective cancers.

cancer biology↗

bioRxiv2024

PBRM1 directs PBAF to pericentromeres and protects centromere integrity

The specialised structure of the centromere is critical for effective chromosome segregation, but its repetitive nature makes it vulnerable to rearrangements. Centromere fragility can drive tumorigenesis, but protective mechanisms preventing fragility are still not fully understood. The PBAF chromatin remodelling complex is frequently misregulated in cancer, but its role in cancer is still not fully characterized. Here, we identify PBAF as a protector of centromere and pericentromere structure with profound consequences for genome stability. A conserved feature of isogenic cell lines lacking PBRM1, a subunit of PBAF, is compromised centromere and pericentromere integrity. PBAF is present at these regions, and the binding pattern changes when PBRM1 is absent. PBRM1 loss creates a dependence on the spindle assembly checkpoint, which represents a therapeutic vulnerability. Importantly, we find that even in the absence of any perturbations, PBRM1 loss leads to centromere fragility, thus identifying a new player in centromere protection.

molecular biology↗

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↗

bioRxiv2023

Spatial control of the APC/C ensures the rapid degradation of Cyclin B1

The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is under the control of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The Checkpoint prevents the APC/C from recognizing Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live cell imaging, in vitro reconstitution, biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We further show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an arginine-anchor in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the nucleosome binding motif on Cyclin B1 reduces its interaction with APC/C and delays its degradation, and cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes constitute a platform to promote the efficient interaction between Cyclin B1 and APC/C and ensure the timely degradation of Cyclin B1 and genomic stability.

cell biology↗

bioRxiv2023

m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate expression of the gene programs shaping neuronal morphology are poorly understood. To address this, we analysed how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the differentiation of human neurons. We performed a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we showed that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

molecular biology↗

bioRxiv2023

Mechanism of assembly, activation and lysine selection by the SIN3B histone deacetylase complex

Histone deacetylase complexes remove histone lysine acetylation, a key post-translational modification that activates transcription at each gene. Although these complexes are drug targets and crucial regulators of organismal physiology, their structure and mechanisms of action are largely unclear. Here, we present the first structure of a complete human SIN3B histone deacetylase holo-complex with and without a substrate mimic. Remarkably, SIN3B encircles the deacetylase and contacts its allosteric basic patch thereby stimulating catalysis. A SIN3B loop inserts into the catalytic tunnel, rearranges to accommodate the acetyl-lysine moiety and stabilises the substrate for specific deacetylation, which is guided by a substrate receptor subunit. Our findings provide a model of specificity for a main transcriptional regulator conserved from yeast to human and a resource of protein-protein interactions for future drug designs.

molecular biology↗

bioRxiv2022

Characterization of proteome-size scaling by integrative omics reveals mechanisms of proliferation control in cancer

Almost all living cells maintain size uniformity through successive divisions. Proteins that sub- or super-scale with size act as rheostats which regulate cell progression. A comprehensive atlas of these proteins is lacking; particularly in cancer cells where both mitogen and growth signalling are dysregulated. Utilising a multi-omic strategy, that integrates quantitative single cell imaging, phosphoproteomic and transcriptomic datasets, we leverage the inherent size heterogeneity of melanoma cells to investigate how peptides, post-translational modifications, and mRNAs scale with cell size to regulate proliferation. We find melanoma cells have different mean sizes, but all retain uniformity. Across the proteome, we identify proteins and phosphorylation events that sub and super scale with cell size. In particular, G2/M, biosynthetic, and cytoskeletal regulators sub- and super-scale with size. In small cells growth and proliferation processes are tightly coupled by translation which promotes CCND1 accumulation and anabolic increases in mass. Counter intuitively, anabolic growth pathways and translational process are low in large cells, which throttles the expression of factors such as CCND1 and thereby coupling proliferation from anabolic growth. Strikingly, these cells exhibit increased growth and comparable proliferation rates. Mathematical modelling suggests that decoupling growth and proliferative signalling fosters proliferation under mitogenic inhibition. As factors which promote adhesion and actin reorganization super-scale with size or are enriched in large cells, we suggest that growth/proliferation in these cells may be decoupled by cell spreading and mechanics. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology determines the chemistry of the cell.

systems biology↗

bioRxiv2022

HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5, in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity, reveal how PP5 is activated by recruitment to HSP90 complexes to comprehensively dephosphorylate client proteins.

biochemistry↗

bioRxiv2021

Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the comprehensive RNA interactome of Leishmania mexicana

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labelling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of twenty Leishmania deubiquitinases including the recently characterised L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterise the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 145 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterise the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCEAdvances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and sub-tropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labelling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterise for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.

microbiology↗

bioRxiv2020

DOCK5 couples CDC42-GSK3beta activation with focal adhesion morphogenesis to reinforce the establishment of polarity and promote YAP/TAZ activation during migration.

YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells driving proliferation, invasion, and drug resistance. Through multiplexed quantitative genetic screens for YAP/TAZ localisation and cell shape, we found that the RhoGEF DOCK5 is essential for YAP/TAZ activation in metastatic cells and is required for the maintenance of polarity during migration. DOCK5 regulates cell shape and thus YAP/TAZ through different genetic interactions with CDC42, RAC, and RHOA GTPases. DOCK5 regulates focal adhesion (FA) morphogenesis in RAC-dependent fashions that promote RHOA mediated actomyosin engagement of FA. Using unbiased systems-level quantification of protein levels by mass spectrometry we show that DOCK5 maintains polarity by stabilising protein levels of the CDC42 effector GSK3{beta}. We conclude DOCK5 acts as a coincidence detector to promote leading edge persistence in subcellular locations where there is both RAC and RHOA dependent FA morphogenesis and active CDC42 mediated cell polarisation.

cell biology↗