Journal of Molecular Biology最新文献

{"title":"CNPI: Rapid Analyses of Human Copy Number Data","authors":"Jack Ustanik,&nbsp;Tychele N. Turner","doi":"10.1016/j.jmb.2025.169313","DOIUrl":"10.1016/j.jmb.2025.169313","url":null,"abstract":"<div><div>Tools for genotyping copy number in whole-genome sequencing (WGS) data exist. Despite their availability, there are a limited number of tools that efficiently process this data at scale with rapid compute time, low memory usage, and easy to parse outputs. This is critical as WGS continues to be housed in the “cloud.” Copy Number Private Investigator (CNPI) is a computational toolkit (<span><span>https://github.com/TNTurnerLab/CNPI</span><svg><path></path></svg></span>) that performs several tasks including genotyping of regions of interest, digital karyotype determination, assessment of chromosomal sex, plotting of copy number in regions of the genome, and generation of an Individual-level Copy Number Score (ICNS). Inputs to CNPI include copy number estimates, genome-wide, from WGS data and a corresponding reference annotation file. With these inputs, CNPI can genotype any regions of the genome rapidly. For example, all genes in the genome can be genotyped in &lt;2 s with 1 CPU and &lt;1 GB memory. Benchmarking against gold-standard NA12878 data demonstrated good results with flexibility for users to optimize copy number thresholds based on desired sensitivity and specificity. Usage of CNPI is customizable with a variety of user-tailored functions and arguments. CNPI can be deployed to cloud based environments such as AWS lambda due to its intuitive design, near-zero cost per sample, and availability of compute resources. The application of ICNS has proven to be useful by comparing unaffected individuals to individuals with autism and 9p deletion syndrome, respectively. Overall, CNPI is advantageous for its ability to quickly provide reliable and precise copy number assessments of human genomic data.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169313"},"PeriodicalIF":4.7,"publicationDate":"2025-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Condensate Formation of the Human RNA-binding Protein SMAUG1 is Controlled by its Intrinsically Disordered Regions and Interactions with 14-3-3 Proteins","authors":"Olivia Carey ,&nbsp;John Fehilly ,&nbsp;Eoghan Thomas O’Leary ,&nbsp;Stephen O’Shea ,&nbsp;Klaudia Juda ,&nbsp;Rahel Fitzel ,&nbsp;Pooja Selvaraj ,&nbsp;Katja Burk ,&nbsp;Andrew J. Lindsay ,&nbsp;Paul Young ,&nbsp;Rita Pancsa ,&nbsp;Bálint Mészáros ,&nbsp;Kellie Dean","doi":"10.1016/j.jmb.2025.169314","DOIUrl":"10.1016/j.jmb.2025.169314","url":null,"abstract":"<div><div>SMAUG1 is a human RNA-binding protein (RBP) that is dysregulated in a wide range of diseases. It is evolutionarily conserved and forms condensates containing translationally repressed RNAs. This indicates that condensation is central to SMAUG1 function. In this work, we show that a prion-like disordered region within the C-terminal half of SMAUG1 is required, but not sufficient, to drive formation of liquid-like condensates in cells. We use biochemical assays to show that SMAUG1 liquid–liquid phase separation (LLPS) appears to be independent of RNA binding and does not depend on other large, disordered regions of the protein that potentially harbor several binding sites for partner proteins. Using a combination of computational predictions, structural modeling, <em>in vitro</em> and in cell measurements, we show that SMAUG1-driven condensation is negatively regulated by direct interactions with members of the 14-3-3 protein family. These interactions are mediated by at least four distinct phospho-regulated short linear motifs within the disordered regions of SMAUG1, working synergistically. Interactions between SMAUG1 and dimeric 14-3-3 proteins drive the dissolution of condensates and are likely intertwined with other unknown regulatory mechanisms. Interestingly, a monomeric 14-3-3 variant cannot induce condensate dissolution, suggesting that the conformational constraints imposed on the SMAUG1 polypeptide chain by dimeric 14-3-3 proteins, potentially bridging distant binding sites, are important for the described phase separation-regulatory mechanism. Our results reinforce recent findings on the general regulatory role of 14-3-3 proteins in biological condensation and provide valuable novel insights into how SMAUG1 phase separation is regulated.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169314"},"PeriodicalIF":4.7,"publicationDate":"2025-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Post-Translational Modifications Orchestrate Repair of Trapped Topoisomerase-Induced DNA Breaks via TDP1 and TDP2.","authors":"Benu Brata Das, Saini Basu, Abhik Sengupta, Arpan Bhattacharyya","doi":"10.1016/j.jmb.2025.169309","DOIUrl":"10.1016/j.jmb.2025.169309","url":null,"abstract":"<p><p>DNA topoisomerases are critical for maintaining DNA topology and facilitating replication, transcription, and chromatin organization in both nuclear and mitochondrial genomes. When covalently trapped on DNA as topoisomerase cleavage complexes (Topcc's), notably Top1ccs and Top2ccs, these enzymes generate cytotoxic DNA lesions that disrupt genomic integrity and threaten cell viability. Tyrosyl-DNA phosphodiesterase (TDP1 and TDP2) has emerged as key player in the resolution of these lesions, with broader roles in the repair of diverse DNA end structures. Post-translational modifications (PTMs) dynamically regulate the DNA damage response by modulating the activity, localization, and interactions of repair factors. This review provides a comprehensive overview of the mechanisms by which PTMs modulate the activity of Top1 and Top2, and the repair of their covalently trapped complexes. We further delineate how PTMs fine-tune the functional networks of TDP1 and TDP2, enhancing their efficiency in resolving Topccs and preserving genome stability. Together, these insights highlight the multilayered regulatory mechanisms that safeguard genomic integrity and offer potential avenues for therapeutic intervention.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169309"},"PeriodicalIF":4.7,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525839","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Legionella pneumophila Dot/Icm Type IV Secretion System is Structurally and Functionally Resilient in Absence of Species-specific Proteins Dis2 and Dis3","authors":"Jacquelyn R. Roberts ,&nbsp;Arwen E. Frick-Cheng ,&nbsp;Henry J. Styron ,&nbsp;Clarissa L. Durie ,&nbsp;Louise Chang ,&nbsp;Melanie D. Ohi","doi":"10.1016/j.jmb.2025.169310","DOIUrl":"10.1016/j.jmb.2025.169310","url":null,"abstract":"<div><div><em>Legionella pneumophila</em> is a pathogenic Gram-negative bacterium that causes Legionnaires’ disease. The main virulence factor of <em>L. pneumophila</em> is the Dot/Icm Type IV Secretion System (T4SS), which translocates effector proteins into the cytoplasm of the host cell, allowing the bacterium to establish a replicative niche. The outer membrane core complex (OMCC), the T4SS machinery localized between the inner and outer membranes, is composed of at least nine proteins organized into various sub-complexes that include the dome, outer membrane cap (OMC), periplasmic ring (PR), and stalk. In this study we describe how two uncharacterized Dot/Icm T4SS components, Dis2 and Dis3, contribute to the structure of the T4SS, the ability of the T4SS to translocate effectors, and the pathogenicity of <em>L. pneumophila</em>. Using cryo-electron microscopy we show that OMCCs purified from a Δ<em>dis2</em> strain are only missing the density for Dis2, while OMCCs purified from the Δ<em>dis3</em> strain lack densities for Dis3 and DotF in the OMC. Despite missing these proteins, the OMC and PR of both mutant OMCCs remain structurally stable. Strains lacking <em>dis2</em> and or <em>dis3</em> efficiently replicate in human macrophages; however, they have subtle differences in translocation efficiency for four tested substrates. Combined these data indicate that Dis2 or Dis3 are not required for the stability or global organization of the OMCC, but each protein may contribute to the efficient translocation of specific effectors.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169310"},"PeriodicalIF":4.7,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multilayered Mechanisms for Long-range Regulatory Interactions in Eukaryotic Transcription.","authors":"Takashi Fukaya","doi":"10.1016/j.jmb.2025.169311","DOIUrl":"10.1016/j.jmb.2025.169311","url":null,"abstract":"<p><p>Transcription is a fundamental biological reaction that underlies essentially all developmental and physiological processes across species. While substantial efforts have been made to decipher the basic mechanisms of transcriptional regulation over the decades, we are still far from a comprehensive understanding of this highly intricate biological reaction including the temporal and spatial dynamics of the process. In recent years, new concepts and models have been proposed based on novel insights obtained from the use of cutting-edge technologies such as genome-editing, whole-genome assays, structural analysis, and quantitative live-imaging approaches. In this review, I summarize emerging models and concepts for the dynamic modulation of long-range regulatory interactions in the context of animal development. I suggest that the multilayered actions of enhancers and associating regulatory DNAs such as\"Facilitators\" and \"Range Extenders\" dynamically modulate clustering of transcription machineries at specific genomic loci to flexibly control the temporal and spatial dynamics of gene expression during development.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169311"},"PeriodicalIF":4.7,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144525838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human Plastins are Novel Cytoskeletal pH Sensors with a Reduced F-actin Bundling Capacity at Basic pH","authors":"Lucas A. Runyan ,&nbsp;Elena Kudryashova ,&nbsp;Richa Agrawal ,&nbsp;Mubarik Mohamed ,&nbsp;Dmitri S. Kudryashov","doi":"10.1016/j.jmb.2025.169306","DOIUrl":"10.1016/j.jmb.2025.169306","url":null,"abstract":"<div><div>Intracellular pH (pH_i) is a fundamental component of cell homeostasis. Controlled elevations in pH_i precede and accompany cell polarization, cytokinesis, and directional migration. pH dysregulation contributes to cancer, neurodegenerative diseases, diabetes, and other metabolic disorders. While cytoskeletal rearrangements are crucial for these processes, only a few cytoskeletal proteins, namely CDC42, cofilin, talin, cortactin, α-actinin, and AIP1 have been documented as pH sensors. Here, we report that actin-bundling proteins plastin 2 (PLS2, aka LCP1) and plastin 3 (PLS3) respond to physiological scale pH fluctuations by a reduced F-actin bundling at alkaline pH. The inhibition of PLS2 actin-bundling activity at elevated pH stems from the reduced affinity of the N-terminal actin-binding domain (ABD1) to actin. In fibroblast cells, elevated cytosolic pH caused the dissociation of ectopically expressed PLS2 and 3 from actin structures, whereas acidic conditions promoted their tighter association with focal adhesions and stress fibers. We identified His207 as one of the pH-sensing residues of PLS2 whose mutation to Lys and Tyr reduces pH sensitivity by enhancing and inhibiting the bundling ability, respectively. Our results suggest that weaker actin bundling by plastin isoforms at alkaline pH favors higher dynamics of the actin cytoskeleton. Therefore, like other cytoskeleton pH sensors, plastins promote disassembly and faster dynamics of cytoskeletal components during cytokinesis and cell migration. Since both plastins are implemented in cancer, their pH sensitivity may contribute to the accelerated proliferation and enhanced invasive and metastatic potentials of cancer cells at alkaline pH_i.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169306"},"PeriodicalIF":4.7,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-molecule Tracking and Kinetic Analysis in Living Cells and Multicellular Organisms.","authors":"J Christof M Gebhardt","doi":"10.1016/j.jmb.2025.169308","DOIUrl":"10.1016/j.jmb.2025.169308","url":null,"abstract":"<p><p>In a living organism, the interplay of stochastically interacting molecules brings forth structures and processes robustly organized in space and time. Single-molecule localization microscopy and tracking emerged as important techniques to visualize the super-resolved spatial distribution and real-time motion of individual fluorescently labeled molecules in a living cell or multicellular organism. Thereby, single-molecule tracking (SMT) enables quantifying kinetic mechanisms underlying vital organismal processes. This review covers the methodology of SMT in living cells and multicellular organisms, including labeling approaches and microscopy techniques, with a focus on recent developments in temporal excitation patterns facilitating extracting kinetic properties and the analysis of molecular kinetic parameters accessible by SMT. Emphasis lies on the application of this methodology to quantifying the kinetics of proteins such as transcription factors, and some recent examples are highlighted. The review concludes by envisioning future perspectives of SMT in living systems.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169308"},"PeriodicalIF":4.7,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SeqWord Motif Mapper: A Tool for Rapid Statistical Analysis and Visualization of Epigenetic Modifications in Bacterial Genomes","authors":"Christophe M.J. Lefebvre,&nbsp;Rian E. Pierneef,&nbsp;Oleg N. Reva","doi":"10.1016/j.jmb.2025.169307","DOIUrl":"10.1016/j.jmb.2025.169307","url":null,"abstract":"<div><div>Genomic methylation in bacteria plays a crucial role in gene regulation, chromosome replication, pathogenicity, and defense against phages. While single-molecule real-time (SMRT) sequencing technologies have advanced the detection of epigenetically modified bases, the statistical analysis of their distribution and the possible roles they play in bacterial cells remains challenging. To address this gap, we developed SeqWord Motif Mapper (SWMM), a computational tool designed for the statistical analysis and visualization of bacterial methylation patterns. SWMM utilizes PacBio sequencing data to identify sequence coverage, methylation motif distribution, and putative functional associations. Implemented in Python 3.9, the tool is platform-independent and requires minimal dependencies, making it accessible to a wide range of users. The SWMM command-line interface and a web-based version of the program facilitate the exploration of epigenetic modifications across bacterial genomes. Through case studies on different bacterial and archaeal taxa, we demonstrated that genome methylation in microorganisms extends beyond canonical sites and possibly influences gene expression, adaptation, and genome architecture. The tool enables detailed statistical evaluation of methylation motif distribution and provides insights into the potential regulatory roles of epigenetic modifications in bacterial genomes. SWMM is freely available at <span><span>https://begp.bi.up.ac.za</span><svg><path></path></svg></span>, with source code hosted on GitHub at <span><span>https://github.com/chrilef/BactEpiGenPro</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169307"},"PeriodicalIF":4.7,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Formation Mechanism and Antibacterial Activity of Natural Antimicrobial Lysozyme with Antibiotics Doxycycline and Tigecycline","authors":"Xiangrong Li ,&nbsp;Xianfei Liu ,&nbsp;Jingjing Zhao ,&nbsp;Yujie Sun ,&nbsp;Yuxin Liang ,&nbsp;Qiongya Hu","doi":"10.1016/j.jmb.2025.169304","DOIUrl":"10.1016/j.jmb.2025.169304","url":null,"abstract":"<div><div>The combination of lysozyme with antibiotics has emerged as a promising strategy to combat bacterial resistance; however, the lack of mechanistic understanding regarding their molecular-level interactions remains a critical knowledge gap, hindering the rational design of optimized synergistic therapies. Through integrated spectroscopic techniques (FTIR, CD, UV–vis, fluorescence), thermodynamic analyses, molecular docking, and 200-ns molecular dynamics simulation, we established that doxycycline/tigecycline bind lysozyme via moderate-affinity hydrophobic interactions (<em>K</em>_a ∼ 10⁴ L mol⁻¹) but with distinct binding patterns-tigecycline forms additional hydrogen bonds through its glycylamino side chain, yielding higher stability and lower free drug fraction (<em>f</em>_u) compare to doxycycline. Additionally, doxycycline and tigecycline have a relatively mild effect on lysozyme conformation, but doxycycline has a greater impact on lysozyme conformation than tigecycline. Key findings reveal a paradoxical efficacy relationship: while doxycycline exhibits stronger intrinsic antibacterial activity, the lysozyme-tigecycline complex demonstrates synergistic enhancement (particularly against <em>E. coli</em>) through stable binding-mediated protection and optimized release kinetics, whereas lysozyme-doxycycline’s weaker binding leads to premature release and reduced activity. These results fundamentally advance protein-drug interaction paradigms by demonstrating that optimal therapeutic outcomes require balanced binding stability, with broader implications for designing lysozyme-based delivery systems where moderately strong interactions enable synergistic effects while preserving structural integrity, offering a template for developing combination therapies against antimicrobial resistance.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169304"},"PeriodicalIF":4.7,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sequence and Structure-based Prediction of Allosteric Sites.","authors":"Juan Xie, Gaoxiang Pan, Luhua Lai","doi":"10.1016/j.jmb.2025.169305","DOIUrl":"10.1016/j.jmb.2025.169305","url":null,"abstract":"<p><p>Allosteric regulation in proteins is a critical aspect of cellular function, influencing various biological processes through conformational or dynamic changes induced by effector molecules. Allosteric drugs possess significant therapeutic value due to their unique advantages, such as high specificity and diverse regulatory types, yet their presence in clinical applications remains limited. Understanding the relationship between protein sequence, structure, and allosteric regulation can promote insights into allosteric mechanisms and facilitate allosteric drug design. In this review, we present an overview of marketed allosteric drugs, summarize recent computational methods for predicting allosteric sites based on protein sequences and structures, together with case studies of recent rational allosteric drug design. We also discuss challenges and future directions in computer-aided allosteric drug design, with emphasis on the potential of multi-modal data integration and interpretable deep learning models in improving allosteric site prediction and rational allosteric drug design.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169305"},"PeriodicalIF":4.7,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}