Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Centre for Motor Neuron Disease Research, Macquarie University, 2 Technology Place, North Ryde, NSW 2109, Australia.
Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Dementia Research Centre, Macquarie University, 2 Technology Place, North Ryde, NSW 2109, Australia.
Hum Mol Genet. 2021 May 31;30(11):971-984. doi: 10.1093/hmg/ddab073.
Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.
先前，我们在 CCNF 中发现了导致家族性和散发性肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的错义突变。这些疾病的标志性特征包括不可溶性蛋白质聚集体的积累以及蛋白质的定位错误，例如反式激活反应 DNA 结合蛋白 43 kDa（TDP-43）。近年来，SFPQ（富含脯氨酸和谷氨酰胺的剪接因子）的失调也已成为 ALS/FTD 的病理学标志。CCNF 编码蛋白 cyclin F，它是一种 E3 泛素连接酶的底物识别成分。我们之前已经表明，CCNF 中的 ALS/FTD 相关突变会破坏整体蛋白质稳态，导致 K48 连接的泛素化蛋白质的积累以及自噬机制的缺陷。为了进一步研究 cyclin F 可能受到影响的过程，我们使用了一种称为生物素鉴定（BioID）的蛋白质邻近连接方法、标准免疫沉淀和质谱法来鉴定 cyclin F 的新相互作用伙伴，并推断可能受到 ALS/FTD 致病突变影响的进一步过程。结果表明，cyclin F 与参与 RNA 代谢的蛋白质以及先前与 ALS/FTD 相关的许多 RNA 结合蛋白密切相关，包括 SFPQ。值得注意的是，cyclin F（S621G）的过表达导致 SFPQ 在人胚肾（HEK293）细胞中的聚集和亚细胞分布改变，同时导致原代神经元中的降解改变。总体而言，我们的数据将 CCNF 中的 ALS/FTD 致病突变与 ALS/FTD 的汇聚病理学特征联系起来，并为蛋白降解系统缺陷与涉及 RNA 处理和代谢的蛋白质的病理性积累之间提供了联系。
