A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy
A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy
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DOI：
10.1152/ajplung.00217.2014
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发表时间：
2015-01-01
影响因子：
4.9
通讯作者：
Beers, Michael F.
中科院分区：
文献类型：
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作者：
Hawkins, Arie;Guttentag, Susan H.;Beers, Michael F.
人表面活性蛋白C（hSP-C）基因（SFTPC）第73位密码子（I73 T）的苏氨酸突变为异亮氨酸，是与间质性肺病（ILD）相关的SFTPC突变的重要组成部分。产生稳定表达SP-C同种型的标记的初级翻译产物的细胞系，以测试hSP-C-I73 T在内体系统内的沉积促进关键细胞质量控制途径巨自噬的破坏的假设。通过荧光显微镜，野生型hSP-C（hSP-C-WT）与外源表达的人ATP结合盒A3类（hABCA 3）共定位，这是正常运输到溶酶体相关细胞器的指标。相反，hSP-C-I73 T从hABCA 3解离，但共定位于质膜以及内体网络。表达hSP-C-I73 T的细胞表现出胞质绿色荧光蛋白/微管相关蛋白1轻链3（LC 3）囊泡的大小和数量增加，其中一些囊泡与来自dsRed/hSP-C-I73 T基因的红色荧光蛋白共标记。通过透射电子显微镜，hSP-C-I73 T细胞含有异常大的自噬空泡，其中含有细胞器和蛋白质碎片，其表型模仿了SFTPC I73 T患者肺活检中肺泡2型细胞的超微结构变化。在生物化学上，hSP-C-I73 T细胞表现出Atg 8/LC 3、SQSTM 1/p62和Rab 7的表达增加，这与使用巴弗洛霉素A1和雷帕霉素的通量研究证实的自噬泡成熟的远端阻滞一致。在功能上，hSP-C-I73 T细胞表现出对聚集倾向的亨廷顿蛋白-1报告底物的降解能力受损。自噬依赖性蛋白质稳态的破坏伴随着线粒体生物量和帕金表达的增加以及线粒体膜电位的降低。我们的结论是，hSP-C-I73 T诱导获得性阻断巨自噬依赖性蛋白稳态和线粒体自噬，这可能有助于肺上皮细胞对ILD中所见的二次打击损伤的脆弱性增加。
Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C-I73T within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wildtype hSP-C (hSP-C-WT) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C-I73T was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C-I73T exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C-I73T. By transmission electron microscopy, hSP-C-I73T cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C-I73T cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C-I73T cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C-I73T induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.