Pub Date : 2024-10-26DOI: 10.1016/j.jmb.2024.168840
Claire Abéza , Philipp Busse , Ana C.F. Paiva , Marie-Eve Chagot , Justine Schneider , Marie-Cécile Robert , Franck Vandermoere , Christine Schaeffer , Bruno Charpentier , Pedro M.F. Sousa , Tiago M. Bandeiras , Xavier Manival , Sarah Cianferani , Edouard Bertrand , Céline Verheggen
The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.
{"title":"The HSP90/R2TP Quaternary Chaperone Scaffolds Assembly of the TSC Complex","authors":"Claire Abéza , Philipp Busse , Ana C.F. Paiva , Marie-Eve Chagot , Justine Schneider , Marie-Cécile Robert , Franck Vandermoere , Christine Schaeffer , Bruno Charpentier , Pedro M.F. Sousa , Tiago M. Bandeiras , Xavier Manival , Sarah Cianferani , Edouard Bertrand , Céline Verheggen","doi":"10.1016/j.jmb.2024.168840","DOIUrl":"10.1016/j.jmb.2024.168840","url":null,"abstract":"<div><div>The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168840"},"PeriodicalIF":4.7,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142566756","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}
Pub Date : 2024-10-26DOI: 10.1016/j.jmb.2024.168841
Ziwei Sun, Kai Song
Microbiome research has increasingly underscored the profound link between microbial compositions and human health, with numerous studies establishing a strong correlation between microbiome characteristics and various diseases. However, the analysis of microbiome data is frequently compromised by inherent sparsity issues, characterized by a substantial presence of observed zeros. These zeros not only skew the abundance distribution of microbial species but also undermine the reliability of scientific conclusions drawn from such data. Addressing this challenge, we introduce GEMimp, an innovative imputation method designed to infuse robustness into microbiome data analysis. GEMimp leverages the node2vec algorithm, which incorporates both Breadth-First Search (BFS) and Depth-First Search (DFS) strategies in its random walks sampling process. This approach enables GEMimp to learn nuanced, low-dimensional representations of each taxonomic unit, facilitating the reconstruction of their similarity networks with unprecedented accuracy.
Our comparative analysis pits GEMimp against state-of-the-art imputation methods including SAVER, MAGIC and mbImpute. The results unequivocally demonstrate that GEMimp outperforms its counterparts by achieving the highest Pearson correlation coefficient when compared to the original raw dataset. Furthermore, GEMimp shows notable proficiency in identifying significant taxa, enhancing the detection of disease-related taxa and effectively mitigating the impact of sparsity on both simulated and real-world datasets, such as those pertaining to Type 2 Diabetes (T2D) and Colorectal Cancer (CRC). These findings collectively highlight the strong effectiveness of GEMimp, allowing for better analysis on microbial data. With alleviation of sparsity issues, it could be greatly facilitated in downstream analyses and even in the field of microbiology.
{"title":"GEMimp: An Accurate and Robust Imputation Method for Microbiome Data Using Graph Embedding Neural Network","authors":"Ziwei Sun, Kai Song","doi":"10.1016/j.jmb.2024.168841","DOIUrl":"10.1016/j.jmb.2024.168841","url":null,"abstract":"<div><div>Microbiome research has increasingly underscored the profound link between microbial compositions and human health, with numerous studies establishing a strong correlation between microbiome characteristics and various diseases. However, the analysis of microbiome data is frequently compromised by inherent sparsity issues, characterized by a substantial presence of observed zeros. These zeros not only skew the abundance distribution of microbial species but also undermine the reliability of scientific conclusions drawn from such data. Addressing this challenge, we introduce GEMimp, an innovative imputation method designed to infuse robustness into microbiome data analysis. GEMimp leverages the node2vec algorithm, which incorporates both Breadth-First Search (BFS) and Depth-First Search (DFS) strategies in its random walks sampling process. This approach enables GEMimp to learn nuanced, low-dimensional representations of each taxonomic unit, facilitating the reconstruction of their similarity networks with unprecedented accuracy.</div><div>Our comparative analysis pits GEMimp against state-of-the-art imputation methods including SAVER, MAGIC and mbImpute. The results unequivocally demonstrate that GEMimp outperforms its counterparts by achieving the highest Pearson correlation coefficient when compared to the original raw dataset. Furthermore, GEMimp shows notable proficiency in identifying significant taxa, enhancing the detection of disease-related taxa and effectively mitigating the impact of sparsity on both simulated and real-world datasets, such as those pertaining to Type 2 Diabetes (T2D) and Colorectal Cancer (CRC). These findings collectively highlight the strong effectiveness of GEMimp, allowing for better analysis on microbial data. With alleviation of sparsity issues, it could be greatly facilitated in downstream analyses and even in the field of microbiology.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168841"},"PeriodicalIF":4.7,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142566750","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}
Pub Date : 2024-10-24DOI: 10.1016/j.jmb.2024.168835
Zikang Dennis Huang , Lukasz J. Bugaj
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.
{"title":"Optogenetic Control of Condensates: Principles and Applications","authors":"Zikang Dennis Huang , 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}
Pub Date : 2024-10-24DOI: 10.1016/j.jmb.2024.168833
Nagendar Goud Badepally, Tales Rocha de Moura, Elżbieta Purta, Eugene F. Baulin, Janusz M. Bujnicki
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, raiA, 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 raiA from Clostridium sp. CAG 138. Our findings reveal the globular 3D fold of raiA, providing valuable structural insights. This analysis paves the way for future investigations into the functional properties of raiA, potentially uncovering new regulatory mechanisms in bacterial ncRNAs.
{"title":"Cryo-EM Structure of raiA ncRNA From Clostridium Reveals a New RNA 3D Fold","authors":"Nagendar Goud Badepally, Tales Rocha de Moura, Elżbieta Purta, Eugene F. Baulin, 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}
Pub Date : 2024-10-23DOI: 10.1016/j.jmb.2024.168834
Lisastella Morinelli , Beatrice Corradi , Pietro Arnaldi , Katia Cortese , Martina Muià , Federico Zara , Luca Maragliano , Bruno Sterlini , Anna Corradi
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.
跨膜蛋白 151A（TMEM151A）已被确定为阵发性运动障碍的致病基因，但其分子功能几乎完全未知。了解跨膜蛋白的膜拓扑结构对于阐明其功能和可能的相互作用伙伴至关重要。在本研究中，我们利用分子动力学模拟、免疫细胞化学和电子显微镜确定了 TMEM151A 的拓扑结构。我们的研究结果验证了 TMEM151A 的起始 AlphaFold 模型，并揭示了它由一个跨膜结构域和两个跨膜 alpha 螺旋组成，两个跨膜 alpha 螺旋由一个短的胞外环和一个膜内螺旋-铰链-螺旋结构连接。值得注意的是，该蛋白质的大部分都面向膜的细胞内侧，其中一个大的细胞膜结构域具有α-螺旋和β-片状结构的组合，以及蛋白质的 N 端和 C 端。对 TMEM151A 的拓扑结构及其结构域在细胞膜上的取向的深入研究为今后的功能研究提供了重要信息，也为了解其在阵发性运动障碍发病机制中的作用迈出了基础性的第一步。
{"title":"Unraveling the Membrane Topology of TMEM151A: A Step Towards Understanding its Cellular Role","authors":"Lisastella Morinelli , Beatrice Corradi , Pietro Arnaldi , Katia Cortese , Martina Muià , Federico Zara , Luca Maragliano , Bruno Sterlini , 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}
Pub Date : 2024-10-22DOI: 10.1016/j.jmb.2024.168832
Francisco Del Caño-Ochoa , Lobna Ramadane-Morchadi , Lluís Eixerés , María Moreno-Morcillo , Rafael Fernández-Leiro , Santiago Ramón-Maiques
CAD, the multi-enzymatic protein essential for initiating the de novo 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.
{"title":"Disruption of CAD Oligomerization by Pathogenic Variants","authors":"Francisco Del Caño-Ochoa , Lobna Ramadane-Morchadi , Lluís Eixerés , María Moreno-Morcillo , Rafael Fernández-Leiro , 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}
Pub Date : 2024-10-22DOI: 10.1016/j.jmb.2024.168820
Valeria Jaramillo-Martinez, Souad R. Sennoune, Elena B. Tikhonova, Andrey L. Karamyshev, Vadivel Ganapathy, Ina L. Urbatsch
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).
{"title":"Molecular Phenotypes Segregate Missense Mutations in SLC13A5 Epilepsy","authors":"Valeria Jaramillo-Martinez, Souad R. Sennoune, Elena B. Tikhonova, Andrey L. Karamyshev, Vadivel Ganapathy, 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<sup>+</sup> 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}
Pub Date : 2024-10-19DOI: 10.1016/j.jmb.2024.168822
Qianshan Qin , Huayuan Yan , Weixiang Gao , Ruyin Cao , Guopeng Liu , Xiaojing Zhang , Niangang Wang , Wenjie Zuo , Lei Yuan , Peng Gao , Qi Liu
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 in vitro 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.
{"title":"Engineered mRNAs With Stable Structures Minimize Double-stranded RNA Formation and Increase Protein Expression","authors":"Qianshan Qin , Huayuan Yan , Weixiang Gao , Ruyin Cao , Guopeng Liu , Xiaojing Zhang , Niangang Wang , Wenjie Zuo , Lei Yuan , Peng Gao , 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}
Pub Date : 2024-10-18DOI: 10.1016/j.jmb.2024.168823
Brian C. Mackness , Brittany R. Morgan , Laura M. Deveau , Sagar V. Kathuria , Jill A. Zitzewitz , Francesca Massi
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.
折叠中间体既介导蛋白质折叠，也介导包括肌萎缩性脊髓侧索硬化症（ALS）在内的人类疾病中观察到的错误折叠和聚集，是治疗干预的主要目标。在这项研究中，我们采用实验和计算相结合的方法，在与 ALS 相关的 RNA 结合蛋白 TDP-43 的第二个 RNA 识别基序（RRM2）中确定了折叠中间体的核心结构核。尿素平衡展开研究发现，RRM2 中间状态由坍塌的残余二级结构组成，这些二级结构位于 RRM2 的 N 端半部分，而 C 端大部分处于无序状态。这些研究结果表明，RRM2 中间体在正常的 TDP-43 功能中发挥作用，并通过低稳定性和非原生二级结构成为错误折叠和聚集的模板。
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Pub Date : 2024-10-18DOI: 10.1016/j.jmb.2024.168819
Eliot Courtney, Amitava Datta, David H Mathews, Max Ward
Determining RNA secondary structure is a core problem in computational biology. Fast algorithms for predicting secondary structure are fundamental to this task.Department of Biochemistry & Biophysics, University of Rochester Medical Center, Rochester, NY, USA We describe a modified formulation of the Zuker-Stiegler algorithm with coaxial stacking, a stabilising interaction in which the ends of helices in multi-loops are stacked. In particular, optimal coaxial stacking is computed as part of the dynamic programming state, rather than in an inner loop. We introduce a new notion of sparsity, which we call replaceability. Replaceability is a more general condition and applicable in more places than the triangle inequality that is used by previous sparse folding methods. We also introduce non-monotonic candidate lists as an additional sparsification tool. Existing usages of the triangle inequality for sparsification can be thought of as an application of both replaceability and monotonicity together. The modified recurrences along with replaceability allows sparsification to be applied to coaxial stacking as well, which increases the speed of the algorithm. We implemented this algorithm in software we call memerna, which we show to have the fastest exact (non-heuristic) implementation of RNA folding under the complete Turner 2004 model with coaxial stacking, out of several popular RNA folding tools supporting coaxial stacking. We also introduce a new notation for secondary structure which includes coaxial stacking, terminal mismatches, and dangles (CTDs) information. The memerna package 0.1 release is available at https://github.com/Edgeworth/memerna/tree/release/0.1.
{"title":"memerna: Sparse RNA Folding Including Coaxial Stacking.","authors":"Eliot Courtney, Amitava Datta, David H Mathews, Max Ward","doi":"10.1016/j.jmb.2024.168819","DOIUrl":"https://doi.org/10.1016/j.jmb.2024.168819","url":null,"abstract":"<p><p>Determining RNA secondary structure is a core problem in computational biology. Fast algorithms for predicting secondary structure are fundamental to this task.Department of Biochemistry & Biophysics, University of Rochester Medical Center, Rochester, NY, USA We describe a modified formulation of the Zuker-Stiegler algorithm with coaxial stacking, a stabilising interaction in which the ends of helices in multi-loops are stacked. In particular, optimal coaxial stacking is computed as part of the dynamic programming state, rather than in an inner loop. We introduce a new notion of sparsity, which we call replaceability. Replaceability is a more general condition and applicable in more places than the triangle inequality that is used by previous sparse folding methods. We also introduce non-monotonic candidate lists as an additional sparsification tool. Existing usages of the triangle inequality for sparsification can be thought of as an application of both replaceability and monotonicity together. The modified recurrences along with replaceability allows sparsification to be applied to coaxial stacking as well, which increases the speed of the algorithm. We implemented this algorithm in software we call memerna, which we show to have the fastest exact (non-heuristic) implementation of RNA folding under the complete Turner 2004 model with coaxial stacking, out of several popular RNA folding tools supporting coaxial stacking. We also introduce a new notation for secondary structure which includes coaxial stacking, terminal mismatches, and dangles (CTDs) information. The memerna package 0.1 release is available at https://github.com/Edgeworth/memerna/tree/release/0.1.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168819"},"PeriodicalIF":4.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454993","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}
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