Mistargeting and ER retention of CLN7 patient-associated nonsense and sequence deletion mutations as a novel cause for CLN7 disease.

IF 3.1 2区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Federica Valigi, Liana Uebler, Stephan Storch
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引用次数: 0

Abstract

CLN7 disease is a neurodegenerative lysosomal storage disorder caused by defects in MFSD8. We performed a comprehensive analysis of patient mutations causing CLN7 disease, variant late-infantile and non-syndromic adult phenotypes. Our analyses of protein expression and post-translational modifications, such as proteolytic cleavage and complex type N-linked oligosaccharide processing, along with double immunofluorescence analyses, demonstrated that the nonsense mutations p.Q206X, p.W456X, p.Q474X, and p.R482X, or the in-frame deletion mutation p.V109_I113del, resulted in decreased protein levels at steady state compared with wild type CLN7 and showed mistargeting and ER retention as the primary cause for loss of CLN7 function. We also investigated several missense mutations clustered in transmembrane domain 11 that affect conserved residues, which are believed to be important for CLN7 function. Analysis of protein levels, complex type N-glycosylation, proteolytic cleavage in lysosomes, and colocalization with lysosomal marker proteins in double immunofluorescence analyses showed that patient mutations p.T458L, p.R465Q, and p.R465W did not affect protein stability or correct lysosomal targeting of CLN7, indicating functional impairment. The missense mutation p.M454T resulted in increased cysteine protease-mediated turnover of mutant CLN7 in lysosomes. Using an assay to measure the generation of an enlarged endosome phenotype in cells overexpressing CLN7 carrying missense mutations, a loss of CLN7 function could not be detected. The effects of missense mutations in transmembrane domain 11 on CLN7 function remain to be investigated. In summary, our study revealed mistargeting and ER retention of nonsense and in-frame deletion mutations in MFSD8 as a cause of CLN7 disease, variant late-infantile phenotype.

CLN7患者相关无义和序列缺失突变的错靶和ER保留是CLN7疾病的新原因
CLN7病是一种由MFSD8缺陷引起的神经退行性溶酶体贮积疾病。我们对导致CLN7疾病的患者突变、婴儿晚期变异和非综合征型成人表型进行了全面分析。我们对蛋白质表达和翻译后修饰的分析,如蛋白水解裂解和复杂型n-连接寡糖加工,以及双免疫荧光分析表明,无义突变p.Q206X, p.W456X, p.Q474X和p.R482X,或框内缺失突变p.V109_I113del,与野生型CLN7相比,导致稳态下蛋白质水平下降,并表明错靶和内质网保留是CLN7功能丧失的主要原因。我们还研究了聚集在跨膜结构域11的几个错义突变,这些突变影响保守残基,这些残基被认为对CLN7的功能很重要。在双免疫荧光分析中,蛋白水平、复合型n -糖基化、溶酶体蛋白水解裂解以及与溶酶体标记蛋白共定位分析表明,患者p.T458L、p.R465Q和p.R465W突变不影响蛋白稳定性或正确的溶酶体靶向CLN7,表明功能受损。错义突变p.M454T导致半胱氨酸蛋白酶介导的溶酶体突变体CLN7的周转率增加。使用一种检测方法来测量在携带错义突变的过表达CLN7的细胞中产生的增大的内核体表型,无法检测到CLN7功能的丧失。跨膜结构域11错义突变对CLN7功能的影响仍有待研究。总之,我们的研究揭示了MFSD8中无义和框内缺失突变的错靶和ER保留是CLN7疾病、婴儿晚期表型变异的一个原因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Human molecular genetics
Human molecular genetics 生物-生化与分子生物学
CiteScore
6.90
自引率
2.90%
发文量
294
审稿时长
2-4 weeks
期刊介绍: Human Molecular Genetics concentrates on full-length research papers covering a wide range of topics in all aspects of human molecular genetics. These include: the molecular basis of human genetic disease developmental genetics cancer genetics neurogenetics chromosome and genome structure and function therapy of genetic disease stem cells in human genetic disease and therapy, including the application of iPS cells genome-wide association studies mouse and other models of human diseases functional genomics computational genomics In addition, the journal also publishes research on other model systems for the analysis of genes, especially when there is an obvious relevance to human genetics.
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