Center for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.
Department of Biology, Texas A&M University, College Station, TX 3258, USA.
Dis Model Mech. 2018 Sep 13;11(9):dmm033449. doi: 10.1242/dmm.033449.
Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Str cells and ectopically expressed Str-GFP or Str-GFP in Str and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to Str At the cellular level, Str cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of Str-GFP in Str cells rescued all observed defects. In contrast, expression of Str-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system. This article has an associated First Person interview with the first author of the paper.
遗传性痉挛性截瘫（HSPs）具有遗传多样性，临床上表现为下肢无力和痉挛。已经证明，人类 Strumpellin（Str；也称为 WASHC5）的 N471D 和其他几个点突变，是 Wiskott-Aldrich 综合征蛋白和 SCAR 同源物（WASH）复合物的成员，会导致一种称为痉挛性截瘫 8 型（SPG8）的 HSP。为了研究野生型（WT）和 N417D Str 的分子功能，我们生成了 Str 细胞，并在 Str 和 WT 细胞中外源表达 Str-GFP 或 Str-GFP。两种蛋白的过表达显然导致了细胞分裂缺陷，因为我们观察到多核细胞明显增加。实时 PCR 分析显示 Str 细胞中 WASH 复合物亚基没有转录变化，但 Western blot 显示 SWIP 亚基减少了两倍。GFP-陷阱实验与质谱分析相结合，揭示了许多以前已知的以及新的 Str 相互作用蛋白，以及不再与 Str 结合的蛋白。在细胞水平上，Str 细胞显示出细胞生长、吞噬作用、巨胞饮作用、胞吐作用和溶酶体功能缺陷。Str-GFP 在 Str 细胞中的表达挽救了所有观察到的缺陷。相比之下，Str-GFP 的表达不能挽救溶酶体形态和不可消化物质的胞吐作用。我们的研究结果强调了 WASH 复合物及其核心亚基 Str 在溶酶体系统中的关键作用，并突出了 Str N471 残基对维持溶酶体形态和动力学的重要性。我们的数据表明，导致 SPG8 的 N471D 突变导致 Str 在溶酶体系统中的部分功能丧失。本文有该论文第一作者的相关第一人称采访。
