Biology subjects

Llorca, O.

Publications and source records attributed to Llorca, O..

9 recordsLinked to original sources

bioRxiv2026

Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Phosphatidylinositol 3-kinase-related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

molecular biology↗

bioRxiv2025

OT-Curtains: an approach for studying protein interactions with DNA-ends using optical tweezers and confocal fluorescence microscopy

DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected and resolved to preserve genome integrity. We present OT-Curtains, a single-molecule method inspired by DNA Curtains that uses a custom branched DNA substrate containing multiple accessible ends for simultaneous observation on dual-trap optical tweezers coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering force-free experiments, OT-Curtains overcomes common limitations of flow-stretch-based methods. OT-Curtains allows real-time visualization and quantification of end recognition, protection, resection and cleavage at several DNA ends in parallel. We demonstrate compatibility with well-studied DNA binding systems by monitoring Ku-mediated DNA break recognition, AddAB-mediated DNA break resection, ParB-mediated DNA condensation and KpnI-mediated DNA cleavage. We show that kinetic and mechanistic parameters can be extracted from the data under defined forces and solution conditions. OTCurtains offers an accessible and multiplexed route to interrogate DNA-end transactions central to double-stranded DNA break repair pathways and telomere biology, as well as a general framework for benchmarking proteins acting at DNA ends.

biophysics↗

bioRxiv2025

Automated and modular protein binder design with BinderFlow

Deep learning has revolutionised de novo protein design, with new models achieving unprecedented success in creating novel proteins with specific functions, including artificial protein binders. However, current methods remain computationally demanding and challenging to operate without specialised infrastructure and expertise. To overcome these limitations, we developed BinderFlow, a structured and parallelised pipeline for protein binder design. Its batch-basednature enables live monitoring of design campaigns, seamless coexistence with other GPU-intensive processes, and reduces human intervention. Furthermore, BinderFlows modular structure enables straightforward modifications to the design pipeline to incorporate new models and tools or to implement alternative design strategies. Complementing this, we developed BFmonitor, a web-based dashboard that simplifies campaign monitoring, design evaluation, and hit selection. Together, these tools lower the entry barrier for non-specialised users and streamline expert workflows, making generative protein design more accessible, scalable and practical for both exploratory and production-level research.

bioinformatics↗

bioRxiv2024

SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53-deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, confirming that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibitors correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

cancer biology↗

bioRxiv2024

Conserved Lysine in transmembrane helix 5 is key for the inner gating of the LAT transporter BasC

L-amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these transporters is related to cancer. BasC is a bacterial LAT transporter with an APC fold. In this study, to monitor the cytoplasmic motion of BasC, we developed a smFRET assay that can characterize the conformational states of the intracellular gate in solution at room temperature. Based on combined biochemical and biophysical data and molecular dynamics simulations, we propose a model in which the conserved lysine residue in TM5 supports TM1a to explore both open and closed states within the cytoplasmic gate under apo conditions. This equilibrium can be altered by substrates, mutation of conserved lysine 154 in TM5, or transport-blocking nanobodies. Overall, these findings provide insights into the transport mechanism of BasC and highlight the significance of the lysine residue in TM5 in the cytoplasmic gating of LATs.

biochemistry↗

bioRxiv2024

The structure of the R2T complex reveals a different architecture of the related HSP90 co-chaperones R2T and R2TP

Heat shock protein 90 (HSP90) is a molecular chaperone that contributes to the maturation and activation of substrates in multiple cellular pathways. Its activity is supported by various co-chaperones. One of these is R2TP, a complex of RuvBL1-RuvBL2-RPAP3-PIH1D1 in humans, which is involved in the assembly of various multiprotein complexes, including mTORC1 and Box C/D and Box H/ACA snoRNPs. Structural analyses have shown that the complex is organized around a heterohexameric ring of the ATPases RuvBL1-RuvBL2 in both yeast and humans. In addition, several R2TP-like co-chaperones have been identified in humans, such as R2T, which lacks PIH1D1, but these are less well characterized. In seed plants, there are no PIH1D1 orthologs. Here, we have identified the R2T complex of Arabidopsis and determined its cryoEM structure. R2T associates with the prefoldin-like complex in vivo and is located in the cytosolic and nuclear compartments. R2T is organized as a dodecamer of AtRuvBL1-AtRuvBL2a that forms two rings, with one AtRPAP3 anchored to each ring. AtRPAP3 has no effect on the ATPase activity of AtRuvBL1-AtRuvBL2a and binds with a different stoichiometry than that described for human R2TP. We show the interaction of AtRPAP3 with AtRuvBL2a and AtHSP90 in vivo and describe the residues involved. Taken together, our results show that AtRPAP3 recruits AtRuvBL1-AtRuvBL2a and AtHSP90 via a mechanism that is also conserved in other eukaryotes, but that R2T and R2TP co-chaperone complexes have distinct structures that also suggest differences in their functions and mechanisms.

biochemistry↗

bioRxiv2021

CDK1 and PLK1 cooperate to regulate BICD2, dynein activation and recruitment to the nucleus as well as centrosome separation in G2/M

The activity of dynein is regulated by a number of adaptors that mediate its interaction with dynactin, effectively activating the motor complex while also connecting it to different cargos. The regulation of adaptors is consequently central to dynein physiology, but remains largely unexplored. We now describe that one of the best-known dynein adaptors, BICD2, is effectively activated through phosphorylation. In G2 phosphorylation of BICD2 by CDK1 promotes its interaction with PLK1. In turn, PLK1 phosphorylation of a single residue in the N-terminus of BICD2 results in a conformational change that facilitates interaction with dynein and dynactin, allowing the formation of active motor complexes. BICD2 phosphorylation is central for dynein recruitment to the nuclear envelope, centrosome tethering to the nucleus and centrosome separation in G2/M. This work reveals adaptor activation through phosphorylation as crucial for the spatiotemporal regulation of dynein activity.

cell biology↗

bioRxiv2020

Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

biochemistry↗

bioRxiv2020

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

biophysics↗