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The HSP90/R2TP quaternary chaperone scaffolds assembly of the TSC complex. HSP90/R2TP四级伴侣支架了TSC复合体的组装。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-26 DOI: 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.

R2TP 合子由 RUVBL1/RUVBL2 AAA+ ATP 酶和两个适配蛋白 RPAP3 和 PIH1D1 组成。它与 HSP90 一起在大分子复合物的组装过程中发挥作用,这些复合物通常参与细胞增殖。在这里,利用分离的 PIH 结构域进行的蛋白质组学实验揭示了 R2TP 的其他合作伙伴,包括结节性硬化症复合体(TSC)和许多转录复合体。TSC 是 mTORC1 的关键调节因子,由 TSC1、TSC2 和 TBC1D7 组成。我们展示了 TSC1 与 PIH1D1 的 PIH 磷酸化结合域的直接相互作用,令人惊讶的是,这种相互作用与磷酸化无关。通过使用突变体和 KO 细胞系,我们观察到 TSC2 与 HSP90 和 RPAP3 的 TPR 结构域有独立的相互作用。此外,PIH1D1 或 RUVBL1/2 ATPase 活性的失活抑制了 TSC1 与 TSC2 的结合。综上所述,这些数据提出了一个模型,在该模型中,R2TP 通过 PIH1D1 招募 TSC1,通过 RPAP3 和 HSP90 招募 TSC2,并利用 RUVBL1/2 的伴侣样活性来刺激它们的组装。
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引用次数: 0
Cryo-EM Structures of the Plasmodium falciparum Apicoplast DNA Polymerase. 恶性疟原虫 apicoplast DNA 聚合酶的冷冻电镜结构。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-26 DOI: 10.1016/j.jmb.2024.168842
Chen-Yu Lo, Adron R Ung, Tirthankar Koley, Scott W Nelson, Yang Gao

The apicoplast DNA polymerase (apPol) from Plasmodium falciparum is essential for the parasite's survival, making it a prime target for antimalarial therapies. Here, we present cryo-electron microscopy structures of the apPol in complex with DNA and incoming nucleotide, offering insights into its molecular mechanisms. Our structural analysis reveals that apPol contains critical residues for high-fidelity DNA synthesis, but lacks certain structural elements to confer processive DNA synthesis during replication, suggesting the presence of additional accessory factors. The enzyme exhibits large-scale conformational changes upon DNA and nucleotide binding, particularly within the fingers and thumb subdomains. These movements reveal potential allosteric sites that could serve as targets for drug design. Our findings provide a foundation for advancing the understanding of apPol's unique functional mechanisms and potentially offering new avenues for the development of novel inhibitors and therapeutic interventions against malaria.

恶性疟原虫的 apicoplast DNA 聚合酶(apPol)对寄生虫的生存至关重要,因此成为抗疟疗法的主要靶标。在这里,我们展示了apPol与DNA和传入核苷酸复合物的冷冻电子显微镜结构,为了解其分子机制提供了线索。我们的结构分析表明,apPol含有高保真DNA合成的关键残基,但缺乏某些结构元素,无法在复制过程中进行DNA合成,这表明还存在其他辅助因素。该酶在与 DNA 和核苷酸结合时,尤其是在手指和拇指亚域内,表现出大规模的构象变化。这些变化揭示了潜在的异构位点,可作为药物设计的靶点。我们的发现为进一步了解 apPol 的独特功能机制奠定了基础,并有可能为开发新型抑制剂和疟疾治疗干预措施提供新的途径。
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引用次数: 0
GEMimp: An Accurate and Robust Imputation Method for Microbiome Data Using Graph Embedding Neural Network. GEMimp:利用图嵌入神经网络对微生物组数据进行准确而稳健的估算方法。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-26 DOI: 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.

微生物组研究日益凸显微生物组成与人类健康之间的深刻联系,大量研究证实微生物组特征与各种疾病之间存在密切联系。然而,微生物组数据的分析经常受到固有稀疏性问题的影响,其特点是存在大量观测到的零。这些零不仅扭曲了微生物物种的丰度分布,还破坏了从这些数据中得出的科学结论的可靠性。为了应对这一挑战,我们引入了 GEMimp,这是一种创新的估算方法,旨在为微生物组数据分析注入稳健性。GEMimp 利用了 node2vec 算法,该算法在随机游走采样过程中同时采用了广度优先搜索(BFS)和深度优先搜索(DFS)策略。这种方法使 GEMimp 能够学习每个分类单元的细微、低维表征,从而以前所未有的准确性重建它们的相似性网络。我们将 GEMimp 与最先进的估算方法(包括 SAVER、MAGIC 和 mbImpute)进行了比较分析。结果清楚地表明,与原始数据集相比,GEMimp 取得了最高的皮尔逊相关系数,表现优于同类方法。此外,GEMimp 在识别重要类群、增强疾病相关类群的检测以及有效减轻稀疏性对模拟数据集和真实数据集(如与 2 型糖尿病(T2D)和结直肠癌(CRC)相关的数据集)的影响方面表现出了显著的能力。这些发现共同凸显了 GEMimp 的强大功效,使其能够更好地分析微生物数据。随着稀疏性问题的缓解,它可以极大地促进下游分析,甚至是微生物学领域的分析。
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引用次数: 0
Identification of a non-canonical function of prefoldin subunit 5 in proteasome assembly. 鉴定前折叠素亚基 5 在蛋白酶体组装中的非规范功能。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-25 DOI: 10.1016/j.jmb.2024.168838
Somayeh Shahmoradi Ghahe, Krzysztof Drabikowski, Monika Stasiak, Ulrike Topf

The prefoldin complex is a heterohexameric, evolutionarily conserved co-chaperone that assists in folding of polypeptides downstream of the protein translation machinery. Loss of prefoldin function leads to impaired solubility of cellular proteins. The degradation of proteins by the proteasome is an integral part of protein homeostasis. Failure of regulated protein degradation can lead to the accumulation of misfolded and defective proteins. We show that prefoldin subunit 5 is required for proteasome activity by contributing to the assembly of the 26S proteasome. In particular, we found that absence of the prefoldin subunit 5 impairs formation of the Rpt ring subcomplex of the proteasome. Concomitant deletion of PFD5 and HSM3, a chaperone for assembly of the ATPase subunits comprising the Rpt ring, exacerbates this effect, suggesting a synergistic relationship between the two factors in proteasome assembly. Thus, our findings reveal a regulatory mechanism wherein prefoldin subunit 5 plays a crucial role in maintaining proteasome integrity, thereby influencing the degradation of proteins.

预折叠素复合物是一种异构六聚体,在进化过程中得到了保守的共伴侣蛋白,可协助蛋白质翻译机制下游的多肽折叠。预折叠素功能的缺失会导致细胞蛋白质的可溶性受损。蛋白酶体对蛋白质的降解是蛋白质平衡不可或缺的一部分。蛋白质降解失调会导致错误折叠和缺陷蛋白质的积累。我们的研究表明,前折叠素亚基 5 是蛋白酶体活性所必需的,它有助于 26S 蛋白酶体的组装。特别是,我们发现预折叠素亚基 5 的缺失会影响蛋白酶体 Rpt 环亚复合物的形成。同时缺失 PFD5 和 HSM3(组成 Rpt 环的 ATPase 亚基的组装伴侣)会加剧这种影响,这表明蛋白酶体组装过程中这两个因子之间存在协同作用关系。因此,我们的研究结果揭示了一种调控机制,即前折叠素亚基 5 在维持蛋白酶体完整性方面发挥着关键作用,从而影响蛋白质的降解。
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引用次数: 0
Optogenetic Control of Condensates: Principles and Applications. 冷凝物的光遗传学控制:原理与应用。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-24 DOI: 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.

生物分子凝聚体出现在细胞生理和病理过程中,但凝聚体的具体作用或其动态往往难以确定。光遗传学提供了一个不断扩大的工具集来应对这些挑战,它提供了直接控制任意蛋白质凝集的工具,可以精确地控制它们在空间和时间上的形成、溶解和模式化。在这篇综述中,我们描述了光遗传学控制凝集的领域现状。我们调查了构成这一工具集基础的蛋白质及其衍生物,并讨论了区分它们的因素,以便为特定应用进行适当选择。我们还介绍了最近在基础研究和应用研究中使用光遗传缩聚技术的实例。最后,我们讨论了在设计用于光遗传凝聚的新蛋白质时需要考虑的重要设计因素,并展望了未来几年将进一步增强这一工具集的创新。
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引用次数: 0
Cryo-EM Structure of raiA ncRNA From Clostridium Reveals a New RNA 3D Fold 梭状芽孢杆菌 raiA ncRNA 的低温电子显微镜结构揭示了一种新的 RNA 3D 折叠。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-24 DOI: 10.1016/j.jmb.2024.168833
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.
随着全基因组序列分析技术的进步,人们发现了许多新型细菌非编码 RNA(ncRNA)。根据其大小、结构、功能和进化关系,这些 ncRNA 被分为不同的 RNA 家族和类别。其中一个 ncRNA 家族--raiA--在细菌的固氮菌门和放线菌门中非常丰富,而且在许多革兰氏阳性细菌中保存完好。在这项研究中,我们将低温电子显微镜单颗粒分析与计算建模和生物化学技术相结合,阐明了梭状芽孢杆菌 CAG 138 中 raiA 的结构特征。我们的研究结果揭示了 raiA 的球状三维折叠,提供了宝贵的结构见解。这项分析为今后研究 raiA 的功能特性铺平了道路,有可能揭示细菌 ncRNA 的新调控机制。
{"title":"Cryo-EM Structure of raiA ncRNA From Clostridium Reveals a New RNA 3D Fold","authors":"","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":null,"pages":null},"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}
引用次数: 0
Unraveling the Membrane Topology of TMEM151A: A Step Towards Understanding its Cellular Role 揭示 TMEM151A 的膜拓扑结构:了解其细胞作用的一步。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-23 DOI: 10.1016/j.jmb.2024.168834
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":"","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":null,"pages":null},"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}
引用次数: 0
Disruption of CAD Oligomerization by Pathogenic Variants 致病变体对 CAD 寡聚化的破坏。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-22 DOI: 10.1016/j.jmb.2024.168832
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.
CAD是启动嘧啶核苷酸从头生物合成所必需的多酶蛋白,可形成大型六聚体,其结构和功能尚不完全清楚。CAD缺陷会导致严重的神经代谢紊乱,而这种紊乱的诊断具有挑战性。我们开发了一种细胞功能检测方法来鉴定有缺陷的 CAD 变异体,在这项研究中,我们鉴定了 CAD 的二氢烟酸酶(DHO)和天冬氨酸转氨酶(ATC)结构域中的五个致病性错义突变。所有突变都损害了酶活性,其中两个突变明显破坏了 DHO 二聚体和 ATC 三聚体的形成。结合晶体结构和 AlphaFold 预测,我们对六聚体 CAD 复合物进行了建模,突出了 DHO 和 ATC 结构域在其组装中的核心作用。我们的研究结果提供了对 CAD 的稳定性、功能和组织的深入了解,揭示了 CAD 正确寡聚成超分子复合物是其发挥核苷酸合成功能的必要条件,而影响这种组装的突变具有潜在的致病性。
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引用次数: 0
Molecular Phenotypes Segregate Missense Mutations in SLC13A5 Epilepsy SLC13A5 癫痫错义突变的分子表型分离。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-22 DOI: 10.1016/j.jmb.2024.168820
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).
钠偶联柠檬酸盐转运体(NaCT,SLC13A5)通过内向 Na+ 梯度介导柠檬酸盐在质膜上的吸收。由于神经元和星形胶质细胞对柠檬酸盐的摄取能力受损,SLC13A5 基因突变会导致 25 型早期婴儿癫痫性脑病(EIEE25,SLC13A5 Epilepsy)。尽管临床上已发现了致病突变,但其潜在机制和治疗方法仍然难以捉摸。在这里,我们通过表型分析其蛋白细胞表面表达和柠檬酸盐转运功能,从机理上对六种常见的 SLC13A5 突变进行了分类。突变体 C50R、T142M 和 T227M 尽管在细胞表面表达正常,但却表现出柠檬酸盐转运功能受损。相比之下,突变体 G219R、S427L 和 L488P 的蛋白质总表达量较低,细胞表面没有成熟的糖基化蛋白质,蛋白质保留在内质网中,转运活性减弱。这种机理分类法将 SLC13A5 突变体分为两类,一类(C50R、T142M 和 T227M),另一类(G219R、S427L 和 L488P)。重要的是,突变体的 mRNA 水平与野生型相似,表明存在翻译后缺陷。II 类突变体显示出不成熟的核心-糖基化和缩短的半衰期,表明存在蛋白质折叠缺陷。这些实验从生化和分子水平全面了解了致病突变在 SLC13A5 癫痫中的缺陷,并揭示了 NaCT 的贩运途径。这两类突变需要根本不同的治疗方法,要么恢复突变蛋白的转运功能,使其能够到达细胞表面(第一类),要么采用能够纠正蛋白折叠缺陷的疗法,使其能够逃逸到细胞表面,从而恢复转运功能(第二类)。
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引用次数: 0
Engineered mRNAs With Stable Structures Minimize Double-stranded RNA Formation and Increase Protein Expression 具有稳定结构的工程 mRNA 可最大限度地减少双链 RNA 的形成,提高蛋白质的表达。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-19 DOI: 10.1016/j.jmb.2024.168822
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.
合成信息 RNA(mRNA)的治疗用途已在 COVID-19 疫苗中得到验证,并在开发传染病和肿瘤疫苗方面显示出巨大的潜力。然而,体外转录(IVT)过程中产生的双链 RNA(dsRNA)副产物会降低基于 mRNA 的疗法的疗效,并引发先天性免疫反应。消除dsRNA副产物的现有方法通常既繁琐又耗费人力。在这项研究中,我们发现松散的 mRNA 二级结构和序列中更多未配对的 U 碱基通常会导致在 IVT 过程中形成更多的 dsRNA 副产物。我们根据序列特征进一步开发了dsRNA副产物形成的预测模型,以指导 mRNA 序列的优化,从而帮助减少不必要的免疫反应,提高 mRNA 产物的蛋白质表达。总之,我们的研究为开发有效的 mRNA 疗法提供了新的线索和方法,从而最大限度地减少 dsRNA 副产物,提高蛋白质表达。
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引用次数: 0
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Journal of Molecular Biology
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