Biology subjects

Mayans, O.

Publications and source records attributed to Mayans, O..

9 recordsLinked to original sources

bioRxiv2026

A p53-dependent FBXO44-RAD18 axis limits mutagenesis by terminating translesion DNA synthesis

DNA lesions continually challenge genome replication and threaten genome integrity. DNA damage tolerance pathways, including translesion DNA synthesis (TLS), allow cells to bypass lesions and prevent stalled forks from collapsing into double-strand breaks. Because TLS polymerases are intrinsically error-prone, however, this pathway must be tightly restrained; persistent or deregulated TLS can increase mutagenesis, create therapeutic vulnerabilities, and promote aggressive cancer phenotypes. Through integrated transcriptional profiling, genome-wide CRISPR/Cas9 screening for replication-stress sensitivity, and complementary proteomic analyses, we identify F-box protein 44 (FBXO44) as a late p53-responsive regulator of the TLS mediator RAD18. FBXO44 promotes RAD18 ubiquitination during recovery from replication stress and facilitates shutdown of RAD18-dependent PCNA monoubiquitination. Consistently, FBXO44 loss delays resolution of replication stress and TLS signaling, increases mutation frequency, and is associated with elevated mutational burden and therapy resistance in experimental models and patient datasets. These findings define a p53-FBXO44-RAD18 regulatory axis that limits mutagenic TLS and helps safeguard genome integrity after replication stress.

cancer biology↗

bioRxiv2026

Twitchin kinase, a mechanoreceptor in the muscle sarcomere, is a catalytically-primed moonlighting kinase

To explore conserved mechanisms and functions across mechanosensory kinases associated with the skeletal architectures of the cell, we investigated in vitro and in vivo the substrate targeting of twitchin kinase (TwcK), a mechanoreceptor from the muscle sarcomere. Specifically, we elucidated the crystal structure of TwcK in complex with substrates, used real-time 31P-NMR spectroscopy and luminescence-based assays to identify the phosphorylation site on a model peptide substrate, mined the C. elegans proteome to reveal the myosin regulatory protein MLC-4 as a substrate candidate and used CRISPR/Cas9 genome-edited and transgenic C. elegans strains to query the relation of twitchin and MLC-4 in muscle. Contrary to expectations, we find that TwcK undergoes activating conformational changes that are regulated by an N-terminal tail sequence that blocks hinge dynamics in the kinase fold. This distinct mechanism is conserved across sarcomeric, but not cytoskeletal, kinases. Functionally, cytoskeletal and sarcomeric kinases share an evolutionarily conserved phosphorylation targeting of myosin light chain (MLC) proteins. Yet, we find TwcK and its MLC4 substrate to segregate in vivo and not to constitute a functional kinase/substrate pair. Thus, canonical substrate targeting cannot be delivered by TwcK in its cellular context, where it has adopted a moonlighting role. We deduce this result to apply to other intrasarcomeric kinases. Our findings highlight how the cell context confers functional individuality to non-diffusible, otherwise conserved skeletal kinases.

biophysics↗

bioRxiv2025

The Lipid-binding PX Domain of RRC-1 (ARHGAP32/33) is Required for Optimal Assembly and Function of Integrin Adhesion Complexes at the Muscle Cell Boundary in C. elegans

Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article SummaryIntegrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

cell biology↗

bioRxiv2025

Defining the molecular determinants of titin kinase by analysing distantly evolved fish orthologues

Titin kinase (TK) is a pseudokinase specific to the striated muscle of vertebrates. Embedded within the contractile sarcomere and flanked by extensible regulatory tails, TK is thought to sense mechanical signals arising from muscle function. Studies on TK to date have focused narrowly on the human representative. To investigate if a pseudokinase character is a hallmark of TK, we have studied sequences of distantly evolved fish representatives and rationalized conservation patterns by resolving the crystal structure of TK from medaka isoform b. We find that sequence alterations in catalytic motifs involved in ATP and magnesium binding, respectively {theta}xK ({theta}: bulky hydrophobic residue) and EFG, are evolutionarily conserved in TK. Beyond the kinase domain, N- and C-terminal flanking tails show remarkable structural similarity across human and medaka orthologues, even though sequence conservation is limited to individual residues and short motifs: a YD-motif in the N-terminal tail; a [R/K]H[R/K]RYY sequence, a R-7x-R motif and position -2 of the latter in the C-terminal tail. We observe that motifs in the C-terminal tail consistently covary with the divergent functional motifs of TK, being part of its pseudokinase signature. In contrast, the proposed inhibition of the catalytic aspartate by a tyrosine residue from the P+1 loop is not significantly conserved outside mammals. Finally, based on these features and sequence clustering analysis, we propose a classification of titin gene duplicates from fish into a and b isoforms (ttna and ttnb) that can assist future comparative studies. A curated genomic annotation is provided here.

bioinformatics↗

bioRxiv2024

Indel-driven evolution of the canavanine tRNA-editing deacetylase enzyme CtdA

AbstractProteins are heteropolymers composed of twenty standard amino acids. However, over 500 non-proteogenic amino acids exist in nature that can get misincorporated into proteins. Canavanine is an antimetabolite of L-arginine, with which it shares high chemical similarity. It can be utilized by bacteria such as Pseudomonas canavaninivorans in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine is also incorporated in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNAArg with both canavanine and arginine. The recently discovered canavanyl-tRNAArg deacetylase (CtdA) removes canavanine from misloaded tRNAArg and thereby prevents its incorporation in proteins. CtdA is the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated its crystal structure to 1.5 [A] resolution and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme that presents a central, deep cavity that predictably constitutes the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3 tRNA attachment sequence. The stand-alone, trans-editing CtdA is distantly related to the B3/B4 cis-editing domains of the large multi-subunit enzyme Phenylalanine tRNA synthetase (PheRS). Our comparative study reveals that CdtA and B3/B4 domains from bacterial and archeal/eukaryotic origin are three subclasses of a same conserved 3D-fold that differ in type-specific indels, which distinctly shape the substrate binding cleft of these proteins. We propose a unifying nomenclature of secondary structure elements for this 3D-fold. In CtdA, residues E191, Y104, N105 and E118 prove to be relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. No other shared residues of catalytic relevance could be identified across enzymes of this class, so that a shared mechanism of catalysis appears unlikely in these editing enzymes.

biochemistry↗

bioRxiv2024

Sequence clustering with PaSiMap in Jalview

Pairwise similarity mapping, implemented in the software PaSiMap, can be used as an alternative to principal component analysis (PCA) to analyse protein-sequence relationships. It provides the advantage of distinguishing between systematic and random differences in the dataset. Here, we present a protocol to use PaSiMap inside Jalview. You will be guided through the installation and use of the required software. Furthermore, we present an R script to prepare publication-ready graphs of the obtained data and aid in the subsequent data analysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/621149v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1daf3d2org.highwire.dtl.DTLVardef@1bd82cborg.highwire.dtl.DTLVardef@d60c7aorg.highwire.dtl.DTLVardef@cd5a89_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Liver Receptor Homolog-1 (LRH-1/NR5A2) orchestrates hepatic inflammation and TNF-induced cell death

Liver Receptor Homolog-1 (LRH-1/NR5A2) is a nuclear receptor that has been shown to promote apoptosis resistance in various tissues and disease contexts, however, its role in liver cell death remains unexplored. Deletion of LRH-1 in hepatocytes developed into a mild steatosis and inflammation already under steady-state conditions. Unexpectedly, hepatocyte-specific deletion of LRH-1 also resulted in a profound protection of mice from TNF-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes showed elevated NF-B activity, while LRH-1 overexpression inhibited NF-B activity. This inhibition was based on direct physical interaction of the ligand-binding domain of LRH-1 and the Rel homology domain of NF-B subunit RelA. Mechanistically, we found that increased transcription of anti-apoptotic NF-B target genes, together with proteasomal degradation of pro-apoptotic BIM via regeneration-driven EGF receptor signaling, prevented mitochondrial apoptosis, ultimately protecting mice from TNF-induced liver damage. Collectively, our study demonstrates that LRH-1 is a critical modulator of cell death and inflammation in the healthy and diseased liver. HighlightsO_LIHepatic LRH-1 deletion causes mild liver steatosis, fibrosis, and inflammation. C_LIO_LIFemale LRH-1-deficient mice are protected from TNF-induced liver damage. C_LIO_LILRH-1 interacts with NF-B and inhibits its activity. C_LIO_LILRH-1 deletion-provoked inflammation causes degradation of pro-apoptotic protein BIM. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/542039v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@13cd609org.highwire.dtl.DTLVardef@1080012org.highwire.dtl.DTLVardef@3cd9c4org.highwire.dtl.DTLVardef@fd0438_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Pairwise sequence similarity mapping with PaSiMap: reclassification of immunoglobulin domains from titin as case study

Sequence comparison is critical for the functional assignment of newly identified protein genes. As uncharacterised protein sequences accumulate, there is an increasing need for sensitive tools for their classification. Here, we present a novel multidimensional scaling pipeline, PaSiMap, which creates a map of pairwise sequence similarities. Uniquely, PaSiMap distinguishes between unique and shared features, allowing for a distinct view of protein-sequence relationships. We demonstrate PaSiMaps efficiency in detecting sequence groups and outliers using titins 169 immunoglobulin (Ig) domains. We show that Ig domain similarity is hierarchical, being firstly determined by chain location, then by the loop features of the Ig fold and, finally, by super-repeat position. The existence of a previously unidentified domain repeat in the distal, constitutive I-band is revealed. Prototypic Igs, plus notable outliers, are identified and thereby domain classification improved. This re-classification can now guide future molecular research. In summary, we demonstrate that PaSiMap is a sensitive tool for the classification of protein sequences, which adds a new perspective in the understanding of inter-protein relationships. PaSiMap is applicable to any biological system defined by a linear sequence, including nucleotides.

bioinformatics↗

bioRxiv2021

Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic C. elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signalling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g zebrafish).

biophysics↗