Valeria Jaramillo-Martinez, Souad R. Sennoune, Elena B. Tikhonova, Andrey L. Karamyshev, Vadivel Ganapathy, Ina L. Urbatsch
{"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":null,"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":null,"pages":null},"PeriodicalIF":4.7000,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S002228362400442X","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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).
期刊介绍:
Journal of Molecular Biology (JMB) provides high quality, comprehensive and broad coverage in all areas of molecular biology. The journal publishes original scientific research papers that provide mechanistic and functional insights and report a significant advance to the field. The journal encourages the submission of multidisciplinary studies that use complementary experimental and computational approaches to address challenging biological questions.
Research areas include but are not limited to: Biomolecular interactions, signaling networks, systems biology; Cell cycle, cell growth, cell differentiation; Cell death, autophagy; Cell signaling and regulation; Chemical biology; Computational biology, in combination with experimental studies; DNA replication, repair, and recombination; Development, regenerative biology, mechanistic and functional studies of stem cells; Epigenetics, chromatin structure and function; Gene expression; Membrane processes, cell surface proteins and cell-cell interactions; Methodological advances, both experimental and theoretical, including databases; Microbiology, virology, and interactions with the host or environment; Microbiota mechanistic and functional studies; Nuclear organization; Post-translational modifications, proteomics; Processing and function of biologically important macromolecules and complexes; Molecular basis of disease; RNA processing, structure and functions of non-coding RNAs, transcription; Sorting, spatiotemporal organization, trafficking; Structural biology; Synthetic biology; Translation, protein folding, chaperones, protein degradation and quality control.