Biology subjects

Andreeva, A.

Publications and source records attributed to Andreeva, A..

9 recordsLinked to original sources

bioRxiv2026

Stereociliary localization and function of the tip-link protein PCDH15-CD2 are regulated by the PDZ-scaffold NHERF-1

Sensory hair cells in the inner ear convert mechanical sound deflections into electrical signals via the stereociliary hair bundle on their apical surface. Proper bundle function requires that structural, adhesion, and motor proteins be delivered to specific stereocilia sub-compartments and maintained at precise levels throughout life, yet the underlying mechanisms remain poorly understood. Here we investigated how spatiotemporal localization of Protocadherin-15 (PCDH15), a component of kinociliary links, transient lateral links, and tip links, is regulated in mouse auditory hair cells. Using biochemical assays in vitro, we identified the PDZ-domain scaffolds NHERF-1/2 as direct binding partners of CD2, a PCDH15 isoform essential for hearing. We mapped the interaction to NHERF-1's PDZ1 domain and CD2's PDZ-binding motif (PBM), and showed that this binding is regulated by phosphorylation of the CD2 PBM. Using Nherf1 knockout and Pcdh15-CD2 knock-in mice defective in this interaction, we found CD2-NHERF-1 binding is required for proper CD2 levels and localization in the developing hair bundle. Loss of this interaction largely spared early bundle morphogenesis and MET function but caused high-frequency hearing loss and degeneration of the shortest stereociliary row in adult mice. Together, these findings define an important regulatory mechanism with therapeutic relevance for hair bundle repair.

cell biology↗

bioRxiv2026

Large-scale structure prediction of DUF-containing protein-protein interactions

Motivation: Continuing advances in genome and metagenome sequencing expand the number of identified conserved protein families that remain functionally uncharacterized and contain domains of unknown function (DUFs). Functional-association resources such as STRING provide biological context, but mostly do not distinguish indirect association from physical interaction. We assessed whether AlphaFold 3 complex prediction, combined with STRING evidence and domain-level analysis of interfaces and interaction partners, can help identify and characterize DUF-containing proteins. Results: We generated four structural-prediction cohorts from STRING associations involving DUF-containing proteins and evaluated the predicted complexes using interface ipSAE, average pLDDT and buried surface area. An L2-regularized logistic regression model was trained on an initial cohort of predictions from high-confidence STRING associations to prioritize DUF-containing candidates likely to produce structurally confident AlphaFold 3 complexes. The model was then applied across all 12,535 organisms represented in STRING v12.0, followed by grouping into DUF-family and partner-architecture modules, covering 2,076 unique DUF families. The final L2-model screen contained 12,298 successfully modelled protein pairs, including 1,208 (9.82%) complexes meeting a strict-confidence criterion and 2,433 (19.78%) meeting a more liberal confidence criterion. Two examples suggest roles for DUF4130 in nucleic-acid-associated radical-SAM biology and DUF5819 in a bacterial system related to vitamin-K-dependent carboxylation. Availability and implementation: Predicted structures and associated metadata are available through Zenodo at https://doi.org/10.5281/zenodo.21875362. The model implementation and code used to generate the analyses and figures are available at https://github.com/linoriep/Proteome-scale-structure-prediction-of-DUF-containing-protein-protein-interactions.

bioinformatics↗

bioRxiv2026

Structural basis of biofilm formation mediated by the Pseudomonas aeruginosa fibrillar adhesin CdrA

Many bacteria, including the important human pathogen Pseudomonas aeruginosa, are naturally found in antibiotic-tolerant, multicellular biofilms. Cell-cell interactions within P. aeruginosa biofilms are mediated by a large fibrillar adhesin called CdrA in an extracellular polysaccharide-dependent manner. Here, we report an electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus, which combined with electron cryotomography of focused-ion beam milled specimens, allows us to derive a complete in situ model of the native adhesin. Our structure reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the P. aeruginosa pangenome, with structural similarity to previously reported sugar-binding domains in multiple bacterial species. Inhibitory nanobodies targeting CdrA that reduce biofilm formation bind to epitopes in, or close to, the ADEPT on bacterial cells. Furthermore, structure-guided mutagenesis of residues within the ADEPT abolishes bacterial aggregation, and genomic deletion of the whole ADEPT leads to strong attenuation of biofilm formation. Our data forms a rational basis for future targeted inhibition of pathogenic P. aeruginosa biofilms and elucidates the mechanism of biofilm formation mediated by fibrillar adhesins that are widespread in bacteria.

microbiology↗

bioRxiv2025

A genomic atlas of the human gut virome elucidates genetic factors shaping host interactions

Viruses are key modulators of human gut microbiome composition and function. While metagenomic sequencing has enabled culture-independent discovery of gut bacteriophage diversity, existing genomic catalogues suffer from limited geographic representation, sparse taxonomic classification, and insufficient functional annotation, hindering detailed investigation into phage biology. Here, we present the Unified Human Gastrointestinal Virome (UHGV), a collection of 873,994 viral genomes from globally diverse populations that addresses these limitations. UHGV provides high-quality virome references with extensive host predictions, comprehensive functional annotations, protein structures, a classification framework for comparative analysis, and a web portal to facilitate data access. Using UHGV to profile worldwide metagenomes, we found that host range breadth is strongly associated with phage prevalence. Additionally, we identified diversity-generating retroelements and DNA methyltransferases as key factors enabling phage populations to access diverse hosts, revealing how specific genomic features contribute to global phage distribution patterns. UHGV is available at http://uhgv.jgi.doe.gov.

genomics↗

bioRxiv2025

A global survey of intramolecular isopeptide bonds

Many protein domains harbour covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large-scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template-based software Isopeptor. Our findings reveal an extensive phyletic distribution in surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA-like or CnaB-like, from a relatively small set of related Pfam families, including ten novel families that we predict to contain intramolecular isopeptide bonds. One CnaA-like domain of unknown function, DUF11 (renamed here to "CLIPPER") is broadly distributed in cell-surface proteins from Gram-positive bacteria, Gram-negative bacteria, and archaea, and is structurally and biophysically characterised in this work. Using X-ray crystallography, we resolve a CLIPPER domain from a Gram-negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond-containing protein domains in nature, and structurally resolve the previously cryptic CLIPPER domain.

bioinformatics↗

bioRxiv2024

Isopeptor: a tool for detecting intramolecular isopeptide bonds in protein structures

MotivationIntramolecular isopeptide bonds contribute to the structural stability of proteins, and have primarily been identified in domains of bacterial fibrillar adhesins and pili. At present, there is no systematic method available to detect them in newly determined molecular structures. This can result in mis-annotations and incorrect modelling. ResultsHere, we present Isopeptor, a computational tool designed to predict the presence of intramolecular isopeptide bonds in experimentally determined structures. Isopeptor utilizes structure-guided template matching via the Jess software, combined with a logistic regression classifier that incorporates Root Mean Square Deviation (RMSD) and relative solvent accessible area (rASA) as key features. The tool demonstrates a recall of 1.0 and a precision of 0.95 when tested on a Protein Data Bank (PDB) subset of domains known to contain intramolecular isopeptide bonds that have been deposited with incorrectly modelled geometries. Isopeptors python-based implementation supports integration into bioinformatics workflows, enabling early detection and prediction of isopeptide bonds during protein structure modelling. Availability and implementationIsopeptor is implemented in python and can be accessed via the command line, through a python API or via a Google Colaboratory implementation (https://colab.research.google.com/github/FranceCosta/Isopeptor_development/blob/main/notebooks/Isopeptide_finder.ipynb). Source code is hosted on GitHub (https://github.com/FranceCosta/isopeptor) and can be installed via the python package installation manager PIP.

bioinformatics↗

bioRxiv2024

A defining member of the new cysteine-cradle family is a transient aECM protein involved in signalling skin damage

Apical extracellular matrices (aECMs) act as crucial barriers, and communicate with the epidermis to trigger protective responses following injury or infection. In Caenorhabditis elegans, the skin aECM, the cuticle, is produced by the epidermis and is decorated with periodic circumferential furrows. We previously showed that mutants lacking cuticle furrows exhibit persistent immune activation (PIA). In a genetic suppressor screen, we identified spia-1 as a key gene downstream of furrow collagens and upstream of immune signalling. spia-1 expression oscillates during larval development, peaking between each moult together with patterning cuticular components. It encodes a secreted protein that localises to furrows. SPIA- 1 shares a novel cysteine-cradle domain with other aECM proteins. SPIA-1 mediates immune activation in response to furrow loss and is proposed to act as a sensor of cuticle damage. This research provides a molecular insight into intricate interplay between cuticle integrity and epidermal immune activation in C. elegans. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/589058v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@152abadorg.highwire.dtl.DTLVardef@85169eorg.highwire.dtl.DTLVardef@18ff2d3org.highwire.dtl.DTLVardef@18f31d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

What is hidden in the darkness? Deep-learning assisted large-scale protein family curation uncovers novel protein families and folds

Driven by the development and upscaling of fast genome sequencing and assembly pipelines, the number of protein-coding sequences deposited in public protein sequence databases is increasing exponentially. Recently, the dramatic success of deep learning-based approaches applied to protein structure prediction has done the same for protein structures. We are now entering a new era in protein sequence and structure annotation, with hundreds of millions of predicted protein structures made available through the AlphaFold database. These models cover most of the catalogued natural proteins, including those difficult to annotate for function or putative biological role based on standard, homology-based approaches. In this work, we quantified how much of such "dark matter" of the natural protein universe was structurally illuminated by AlphaFold2 and modelled this diversity as an interactive sequence similarity network that can be navigated at https://uniprot3d.org/atlas/AFDB90v4. In the process, we discovered multiple novel protein families by searching for novelties from sequence, structure, and semantic perspectives. We added a number of them to Pfam, and experimentally demonstrate that one of these belongs to a novel superfamily of translation-targeting toxin-antitoxin systems, TumE-TumA. This work highlights the role of large-scale, evolution-driven protein comparison efforts in combination with structural similarities, genomic context conservation, and deep-learning based function prediction tools for the identification of novel protein families, aiding not only annotation and classification efforts but also the curation and prioritisation of target proteins for experimental characterisation.

bioinformatics↗

bioRxiv2021

Substrate recognition determinants of human eIF2α phosphatases

Phosphorylation of the translation initiation factor eIF2 is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2 phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.

cell biology↗