Journal of Molecular Biology最新文献

{"title":"Optogenetic Control of Condensates: Principles and Applications","authors":"Zikang Dennis Huang ,&nbsp;Lukasz J. Bugaj","doi":"10.1016/j.jmb.2024.168835","DOIUrl":"10.1016/j.jmb.2024.168835","url":null,"abstract":"<div><div>Biomolecular condensates appear throughout cell physiology and pathology, but the specific role of condensation or its dynamics is often difficult to determine. Optogenetics offers an expanding toolset to address these challenges, providing tools to directly control condensation of arbitrary proteins with precision over their formation, dissolution, and patterning in space and time. In this review, we describe the current state of the field for optogenetic control of condensation. We survey the proteins and their derivatives that form the foundation of this toolset, and we discuss the factors that distinguish them to enable appropriate selection for a given application. We also describe recent examples of the ways in which optogenetic condensation has been used in both basic and applied studies. Finally, we discuss important design considerations when engineering new proteins for optogenetic condensation, and we preview future innovations that will further empower this toolset in the coming years.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168835"},"PeriodicalIF":4.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142492282","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":"Cryo-EM Structure of raiA ncRNA From Clostridium Reveals a New RNA 3D Fold","authors":"Nagendar Goud Badepally,&nbsp;Tales Rocha de Moura,&nbsp;Elżbieta Purta,&nbsp;Eugene F. Baulin,&nbsp;Janusz M. Bujnicki","doi":"10.1016/j.jmb.2024.168833","DOIUrl":"10.1016/j.jmb.2024.168833","url":null,"abstract":"<div><div>Advancements in genome-wide sequence analysis have led to the discovery of numerous novel bacterial non-coding RNAs (ncRNAs). These ncRNAs have been categorized into various RNA families and classes based on their size, structure, function, and evolutionary relationships. One such ncRNA family, <em>raiA,</em> is notably abundant in the bacterial phyla Firmicutes and Actinobacteria and is remarkably well-conserved across many Gram-positive bacteria. In this study, we integrated cryo-electron microscopy single-particle analysis with computational modeling and biochemical techniques to elucidate the structural characteristics of <em>raiA</em> from <em>Clostridium</em> sp. CAG 138. Our findings reveal the globular 3D fold of <em>raiA</em>, providing valuable structural insights. This analysis paves the way for future investigations into the functional properties of <em>raiA</em>, potentially uncovering new regulatory mechanisms in bacterial ncRNAs.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168833"},"PeriodicalIF":4.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142492279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling the Membrane Topology of TMEM151A: A Step Towards Understanding its Cellular Role","authors":"Lisastella Morinelli ,&nbsp;Beatrice Corradi ,&nbsp;Pietro Arnaldi ,&nbsp;Katia Cortese ,&nbsp;Martina Muià ,&nbsp;Federico Zara ,&nbsp;Luca Maragliano ,&nbsp;Bruno Sterlini ,&nbsp;Anna Corradi","doi":"10.1016/j.jmb.2024.168834","DOIUrl":"10.1016/j.jmb.2024.168834","url":null,"abstract":"<div><div>Transmembrane protein 151A (TMEM151A) has been identified as a causative gene for paroxysmal kinesigenic dyskinesia, though its molecular function remains almost completely unknown. Understanding the membrane topology of transmembrane proteins is crucial for elucidating their functions and possible interacting partners. In this study, we utilized molecular dynamics simulations, immunocytochemistry, and electron microscopy to define the topology of TMEM151A. Our results validate a starting AlphaFold model of TMEM151A and reveal that it comprises a transmembrane domain with two membrane-spanning alpha helices connected by a short extracellular loop and an intramembrane helix-hinge-helix structure. Notably, most of the protein is oriented towards the intracellular side of the membranes with a large cytosolic domain featuring a combination of alpha-helix and beta-sheet structures, as well as the protein N- and C-termini. These insights into TMEM151A’s topology and orientation of its domains with respect of the cell membranes provide essential information for future functional studies and represent a first fundamental step for understanding its role in the pathogenesis of paroxysmal kinesigenic dyskinesia.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168834"},"PeriodicalIF":4.7,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142492283","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":"Disruption of CAD Oligomerization by Pathogenic Variants","authors":"Francisco Del Caño-Ochoa ,&nbsp;Lobna Ramadane-Morchadi ,&nbsp;Lluís Eixerés ,&nbsp;María Moreno-Morcillo ,&nbsp;Rafael Fernández-Leiro ,&nbsp;Santiago Ramón-Maiques","doi":"10.1016/j.jmb.2024.168832","DOIUrl":"10.1016/j.jmb.2024.168832","url":null,"abstract":"<div><div>CAD, the multi-enzymatic protein essential for initiating the <em>de novo</em> biosynthesis of pyrimidine nucleotides, forms large hexamers whose structure and function are not fully understood. Defects in CAD cause 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 CAD’s dihydroorotase (DHO) and aspartate transcarbamoylase (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 CAD’s 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.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168832"},"PeriodicalIF":4.7,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142492280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Phenotypes Segregate Missense Mutations in SLC13A5 Epilepsy","authors":"Valeria Jaramillo-Martinez,&nbsp;Souad R. Sennoune,&nbsp;Elena B. Tikhonova,&nbsp;Andrey L. Karamyshev,&nbsp;Vadivel Ganapathy,&nbsp;Ina L. Urbatsch","doi":"10.1016/j.jmb.2024.168820","DOIUrl":"10.1016/j.jmb.2024.168820","url":null,"abstract":"<div><div>The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na⁺ gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons and astrocytes. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. Here we mechanistically classify six frequent SLC13A5 mutations by phenotyping their protein cell surface expression and citrate transport functions. Mutants C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. In contrast, mutations G219R, S427L, and L488P show low total protein expression levels, absence of mature, glycosylated proteins at the cell surface, retention of the proteins in the endoplasmic reticulum, and diminished transport activity. This mechanistic classification divides SLC13A5 mutants into two groups, Class I (C50R, T142M, and T227M) and Class II (G219R, S427L, and L488P). Importantly, mutants’ mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. Together, these experiments provide a comprehensive understanding of the disease-causing mutation’s defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different approaches for treatment to either restore transport function of the mutant protein that is capable of reaching the cell surface (Class I), or therapies that enable the correction of protein folding defects to enable escape to the cell surface where it may restore transport function (Class II).</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168820"},"PeriodicalIF":4.7,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142492281","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":"Engineered mRNAs With Stable Structures Minimize Double-stranded RNA Formation and Increase Protein Expression","authors":"Qianshan Qin ,&nbsp;Huayuan Yan ,&nbsp;Weixiang Gao ,&nbsp;Ruyin Cao ,&nbsp;Guopeng Liu ,&nbsp;Xiaojing Zhang ,&nbsp;Niangang Wang ,&nbsp;Wenjie Zuo ,&nbsp;Lei Yuan ,&nbsp;Peng Gao ,&nbsp;Qi Liu","doi":"10.1016/j.jmb.2024.168822","DOIUrl":"10.1016/j.jmb.2024.168822","url":null,"abstract":"<div><div>The therapeutic use of synthetic message RNA (mRNA) has been validated in COVID-19 vaccines and shows enormous potential in developing infectious and oncological vaccines. However, double-stranded RNA (dsRNA) byproducts generated during the <em>in vitro</em> transcription (IVT) process can diminish the efficacy of mRNA-based therapeutics and provoke innate immune responses. Existing methods to eliminate dsRNA byproducts are often cumbersome and labor-intensive. In this study, we revealed that a loose mRNA secondary structure and more unpaired U bases in the sequence generally lead to the formation of more dsRNA byproducts during the IVT process. We further developed a predictive model for dsRNA byproducts formation based on sequence characteristics to guide the optimization of mRNA sequences, helping to minimize unwanted immune response and improve the protein expression of mRNA products. Collectively, our study provides novel clues and methodologies for developing effective mRNA therapeutics with minimized dsRNA byproducts and increased protein expression.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168822"},"PeriodicalIF":4.7,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454992","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":"A Hydrophobic Core Stabilizes the Residual Structure in the RRM2 Intermediate State of the ALS-linked Protein TDP-43","authors":"Brian C. Mackness ,&nbsp;Brittany R. Morgan ,&nbsp;Laura M. Deveau ,&nbsp;Sagar V. Kathuria ,&nbsp;Jill A. Zitzewitz ,&nbsp;Francesca Massi","doi":"10.1016/j.jmb.2024.168823","DOIUrl":"10.1016/j.jmb.2024.168823","url":null,"abstract":"<div><div>Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43 (TAR DNA-binding protein-43), using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168823"},"PeriodicalIF":4.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454991","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":"Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces","authors":"Stefania Pilati ,&nbsp;Klemens Wild ,&nbsp;Andrea Gumiero ,&nbsp;Iris Holdermann ,&nbsp;Yvonne Hackmann ,&nbsp;Mauro Dalla Serra ,&nbsp;Graziano Guella ,&nbsp;Claudio Moser ,&nbsp;Irmgard Sinning","doi":"10.1016/j.jmb.2024.168821","DOIUrl":"10.1016/j.jmb.2024.168821","url":null,"abstract":"<div><div>Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. <em>Vitis vinifera</em> LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplast membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interface. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168821"},"PeriodicalIF":4.7,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sifting through the noise: A survey of diffusion probabilistic models and their applications to biomolecules.","authors":"Trevor Norton, Debswapna Bhattacharya","doi":"10.1016/j.jmb.2024.168818","DOIUrl":"10.1016/j.jmb.2024.168818","url":null,"abstract":"<p><p>Diffusion probabilistic models have made their way into a number of high-profile applications since their inception. In particular, there has been a wave of research into using diffusion models in the prediction and design of biomolecular structures and sequences. Their growing ubiquity makes it imperative for researchers in these fields to understand them. This paper serves as a general overview for the theory behind these models and the current state of research. We first introduce diffusion models and discuss common motifs used when applying them to biomolecules. We then present the significant outcomes achieved through the application of these models in generative and predictive tasks. This survey aims to provide readers with a comprehensive understanding of the increasingly critical role of diffusion models.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168818"},"PeriodicalIF":4.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398992","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":"HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells","authors":"Javier Martínez del Río ,&nbsp;Estrella Frutos-Beltrán ,&nbsp;Alba Sebastián-Martín ,&nbsp;Fátima Lasala ,&nbsp;Kiyoshi Yasukawa ,&nbsp;Rafael Delgado ,&nbsp;Luis Menéndez-Arias","doi":"10.1016/j.jmb.2024.168815","DOIUrl":"10.1016/j.jmb.2024.168815","url":null,"abstract":"<div><div>Nucleotide incorporation and <em>lacZ</em>-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg²⁺) using an <em>in vitro</em>-transcribed RNA were around 7.3 × 10⁻⁵. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg²⁺ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10⁻⁵). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg²⁺ (7.4 × 10⁻⁵) were similar to those determined with the <em>in vitro</em>-transcribed RNA, and were reduced to 1.8 × 10⁻⁵ in the presence of 0.5 mM Mg²⁺. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168815"},"PeriodicalIF":4.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}