Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination.
Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination.
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DOI：
10.1371/journal.pone.0038658
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Seyfried NT
中科院分区：
文献类型：
--
作者：
Dammer EB;Fallini C;Gozal YM;Duong DM;Rossoll W;Xu P;Lah JJ;Levey AI;Peng J;Bassell GJ;Seyfried NT
TAR DNA结合蛋白43（TDP-43）是许多神经退行性疾病的泛素阳性包涵体中的主要成分，这些神经退行性疾病越来越多地被认为是TDP-43蛋白病。与TDP-43聚集相关的其他包涵体蛋白的身份仍然不清楚。在这项研究中，我们确定和定量35共聚集蛋白的HEK-293细胞，其中TDP-43或一个特别容易聚集的变体，TDP-S6，富集超表达后，耐洗涤剂部分，使用稳定同位素标记（SILAC）的内标物和液相色谱串联质谱（LC-MS/MS）。我们还寻找了差异翻译后修饰（PTM）的泛素化位点。泛素与TDP-43或TDP-S6偶联的四个位点通过二烷基化GST-TDP-43外部参比肽确认，其发生在RNA结合基序（RRM）1上或附近。在HEK-293细胞和原代运动神经元的细胞质颗粒结构中与不溶性TDP-S6共富集的含RRM蛋白，包括细胞质应激颗粒相关蛋白G3 BP、PABPC 1和eIF 4A 1。TDP-43与paraspeckle标志物RBM 14、PSF和NonO共聚集的蛋白质组学证据也通过蛋白质印迹和通过HEK-293细胞中的免疫细胞化学验证。在TDP过表达细胞的洗涤剂不溶性部分中，发现来自FUS/TLS和hnRNP的甲基化甘氨酸-甘氨酸-甘氨酸（RGG）RNA结合基序的肽增加。最后，TDP-43和TDP-S6洗涤剂不溶性物质减少所确定的泛素化位点的诱变，即使在氧化或蛋白水解应激。总之，这些发现定义了TDP-43的一些聚集伴侣，并表明TDP-43泛素化影响TDP-43寡聚化。
TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization.
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通讯作者：
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