Biology subjects

Fernandez-Leiro, R.

Publications and source records attributed to Fernandez-Leiro, R..

11 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↗

bioRxiv2026

The novel viper FETUA-3 protein evolved a unique mode of inhibiting snake venom metalloproteinases

Molecular innovation and coevolution play major roles in the evolution of biological diversity, yet we have relatively few instances in which we understand the biological and molecular bases of coevolution. Rattlesnakes and other vipers have evolved resistance to their own novel venom metalloproteinase (MP) toxins through the invention and coevolution of serum-borne inhibitors (FETUAs) derived from the ancestral, non-inhibitory, serum glycoprotein Fetuin-A. However, how FETUAs acquired their function and their mechanism of action are unknown. Here, we use a combination of structure prediction, site-directed mutagenesis, functional analyses, and molecular dynamics to elucidate how the rattlesnake FETUA-3 protein evolved to inhibit three classes of venom MP toxins. We show that FETUA-3 uses a combination of structural motifs to bind multiple MPs with high-affinity and acts as a potent noncompetitive inhibitor by inserting its N-terminus into MP active sites and directly disrupting substrate cleavage. We also find strong selective constraints on key N-terminal residues that maintain FETUA-3 specificity for multiple MP targets. These results reveal that FETUA-3 evolved a unique mechanism of action distinct from other vertebrate metalloproteinase inhibitors and how a small number of FETUA proteins controls the activities of a large family of venom toxins.

evolutionary biology↗

bioRxiv2026

A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks

Mitochondrial DNA (mtDNA) maintenance is essential for cellular homeostasis, and defects in mtDNA replication are linked to a broad spectrum of mitochondrial diseases. During replication, DNA polymerase {gamma} (Pol{gamma}) must traverse duplex junctions and stable secondary structures, yet how the human enzyme overcomes these barriers remains incompletely understood. Here, cryo-electron microscopy structures of Pol{gamma} bound to forked DNA, G-quadruplex (G4)-containing DNA and DNA bound to mitochondrial single-stranded DNA-binding protein (mtSSB) reveal a common template-entry path along the catcher domain across distinct substrate and active-site configurations. Within this domain, an arginine-rich helix containing R1026, R1030 and R1034 constitutes a Template Stabilising Motif (TSM). Biochemical reconstitution and DNA-binding experiments show that disrupting the TSM selectively impairs strand displacement, RNA-DNA hybrid displacement and synthesis through G4-forming sequences, while largely preserving synthesis on unstructured templates. Single-molecule optical-tweezers experiments further show that mechanical destabilisation of the fork partially restores mutant strand-displacement activity, whereas force or mtSSB restores primer-extension kinetics on ssDNA templates. Together, these findings establish the role of the TSM in maintaining productive template engagement and identify template stabilisation as a common mechanism enabling Pol{gamma} to traverse structurally diverse barriers in mtDNA.

molecular biology↗

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↗

bioRxiv2025

The tyrosine kinase inhibitor GNF-7 targets senescent cells through allosteric activation of GCN2.

The one-two-punch approach refers to the sequential administration of two different chemotherapies, the second of which targets cancer cells that resisted the initial treatment. To find such a second punch, we performed a chemical screen to find drugs that are preferentially toxic for cells with an activated DNA damage response (DDR). This screen identified the tyrosine kinase inhibitor GNF-7 as a top hit. Subsequent work revealed that GNF-7 is a potent senolytic, even when senescence is triggered by therapies that do not activate the DDR. Consistently, GNF-7 is highly efficacious to kill cancer cells previously treated with CDK4/6 inhibitors, including in patient-derived organoids and mouse xenografts. Surprisingly, the senolytic effect of GNF-7 is not mediated by the inhibition of a tyrosine kinase (TK), but rather by the activation of GCN2, an effect previously reported for other TK inhibitors. Together, our study reports the discovery of a novel senolytic agent that strongly synergizes with CDK4/6 inhibitors when applied sequentially and expands our understanding of the mechanisms behind the anticancer effects of TK inhibitors.

cancer biology↗

bioRxiv2024

Auto-regulation of the real-time kinetics of the human mitochondrial replicative helicase

The human mitochondrial replicative helicase, Twinkle, is essential for the replication and integrity of mitochondrial DNA (mtDNA). Therefore, investigating the real-time kinetics of Twinkles activities and their regulation is crucial for understanding mtDNA maintenance. Here, we combine biochemical and single-molecule manipulation and visualization techniques to investigate the loading of Twinkle onto the DNA fork, its real-time DNA unwinding and rewinding kinetics, and the regulation of these processes by the amino- (N) and carboxyl- (C) terminal ends of the helicase and the mitochondrial SSB protein (mtSSB). We observed that Twinkle rapidly diffuses along dsDNA scanning for the DNA fork, where it establishes specific interactions that halt further diffusion. Our results show that during DNA unwinding, the real-time kinetics of the helicase are downregulated by interactions of its N-terminal Zinc-binding domain (ZBD) with DNA and the control of the ATPase activity by the C-terminal tail. We found that binding of mtSSB to DNA likely outcompetes the ZBD-DNA interactions, alleviating the down regulatory effects of this domain. Furthermore, we show that ZBD-DNA interactions, together with the ATP binding, also control the real-time kinetics of the DNA rewinding events that follow helicase stalling. Our findings reveal that ZBD and C-terminal tail play a major role in regulation of Twinkles real-time kinetics. Their interplay constitutes an auto-regulatory mechanism that may be crucial for coordination of the mtDNA maintenance activities of the helicase.

biophysics↗

bioRxiv2024

A comprehensive genetic catalog of human double-strand break repair

The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. By employing parallel in-pool profiling of Cas9-induced indels within a genome-wide knockout library, we present a comprehensive catalog detailing how virtually every human gene influences the DSB repair process. This REPAIRome resource is validated through the identification of novel mechanisms, pathways and factors involved in DSB repair, including unexpected opposing roles for XLF and PAXX in DNA end processing, a molecular explanation for Cas9-induced multi-nucleotide insertions, the identification of HLTF as a DSB-repair factor, the involvement of the SAGA complex in microhomology-mediated end joining, and importantly, an indel mutational signature linked to VHL loss, renal carcinoma and hypoxia. Collectively, these results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.

molecular biology↗

bioRxiv2024

Disruption of CAD oligomerization by pathogenic variants

CAD is a multi-enzymatic protein essential for initiating the de novo biosynthesis of pyrimidine nucleotides, forming large hexamers whose structure and function are not fully understood. Defects in CAD result in a severe neurometabolic disorder that is challenging to diagnose. We developed a cellular functional assay to identify defective CAD variants, and in this study, we characterized five pathogenic missense mutations in CADs dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains. All mutations impaired enzymatic activities, with two notably disrupting the formation of DHO dimers and ATC trimers. Combining crystal structures and AlphaFold predictions, we modeled the hexameric CAD complex, highlighting the central role of the DHO and ATC domains in its assembly. Our findings provide insight into CADs stability, function, and organization, revealing that correct oligomerization of CAD into a supramolecular complex is required for its function in nucleotide synthesis and that mutations affecting this assembly are potentially pathogenic.

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↗

bioRxiv2019

The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. Crystal structures, solved at 3.18[A] resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the pre-catalytic towards the active conformation, a molecular mechanism that was supported by site directed mutagenesis and targeted MD simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations. Significance StatementThe present crystal structure of phenylalanine hydroxylase (PAH) provides the first and long-awaited 3D-structure of the full-length human PAH, both unbound and complexed with the tetrahydrobiopterin (BH4) cofactor. The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (Kuvan(R)) is the only drug-based therapy for a subset of phenylketonuria patients. We found two tetramer conformations in the same crystal, depending on the active site occupation by BH4, which aid to understand the stabilization by BH4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human-compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human. Classification: Biological Science (major), biochemistry (minor) The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6HYC and 6HPO). The EM maps have been deposited in the EMDB with accession s EMD-4605 This article contains supporting information.

biochemistry↗

bioRxiv2018

DNA and RNA binding mediate the toxicity of arginine-rich peptides encoded by C9ORF72 GGGGCC repeats

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

neuroscience↗