Department of Human Genetics, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
Mol Cell Proteomics. 2010 Apr;9(4):705-18. doi: 10.1074/mcp.M800390-MCP200. Epub 2010 Jan 4.
Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons. Whereas recombinant and endogenous TDP-43 was primarily localized in the nucleus, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Western blot analysis of detergent-insoluble extracts showed an increase in high molecular weight immunoreactive species for TDP-S6 compared with TDP-43, consistent with ubiquitination or ubiquitin-like modifications. We used a multiplex stable isotope labeling with amino acids in cell culture approach to compare the detergent-insoluble proteome from mock-, TDP-43-, and TDP-S6-transfected cells. TDP-S6 overexpression caused a concomitant increase in both ubiquitin (Ub) and the small Ub-like modifier-2/3 (SUMO-2/3) within the insoluble proteome. Similarly, full-length TDP-43 overexpression also resulted in the elevation of SUMO-2/3. Immunofluorescence showed strong co-localization of endogenous Ub with both cytoplasmic and nuclear TDP-S6 inclusions, whereas SUMO-2/3 was co-localized mainly with the nuclear inclusions. Quantitative mass spectrometry further revealed that mixed Lys-48 and Lys-63 polyUb linkages were associated with the TDP insoluble fractions. Together our data indicate that expression of a TDP-43 splice variant lacking a C terminus recapitulates many of the cellular and biochemical features associated with disease pathology and that the interplay of ubiquitination and SUMOylation may have an important role in TDP-43 regulation.
反式作用应答（TAR）DNA 结合蛋白 43（TDP-43）是额颞叶变性和肌萎缩性侧索硬化症中泛素阳性包涵体的主要蛋白成分。虽然 TDP-43 是一种核 DNA/RNA 结合蛋白，但在病理条件下，已报道 TDP-43 重新分布到细胞质中，在细胞质中被切割并形成不溶性、泛素化和磷酸化的包涵体。在这里，我们提出了一个细胞模型，其中全长人 TDP-43 或缺乏 C 末端的剪接异构体（TDP-S6）在人细胞系和小鼠原代神经元中过表达。虽然重组和内源性 TDP-43 主要定位于核内，但较短的 TDP-S6 形成高度不溶性的细胞质和核内包涵体，类似于疾病特异性病理学。去污剂不溶性提取物的 Western blot 分析显示，与 TDP-43 相比，TDP-S6 的高分子量免疫反应性物质增加，这与泛素化或泛素样修饰一致。我们使用细胞培养中的多重稳定同位素标记与氨基酸的方法来比较模拟、TDP-43 和 TDP-S6 转染细胞的去污剂不溶性蛋白质组。TDP-S6 过表达导致不溶性蛋白质组中泛素（Ub）和小泛素样修饰物-2/3（SUMO-2/3）的含量同时增加。同样，全长 TDP-43 过表达也导致 SUMO-2/3 的升高。免疫荧光显示内源性 Ub 与细胞质和核 TDP-S6 包涵体强烈共定位，而 SUMO-2/3 主要与核包涵体共定位。定量质谱进一步表明，混合 Lys-48 和 Lys-63 多 Ub 连接与 TDP 不溶性部分有关。我们的数据表明，表达缺乏 C 末端的 TDP-43 剪接变体可重现与疾病病理学相关的许多细胞和生化特征，并且泛素化和 SUMO 化的相互作用可能在 TDP-43 调节中发挥重要作用。
