Title: A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy.
Authors: Hawkins, Arie; Guttentag, Susan H; Deterding, Robin; Funkhouser, William K; Goralski, Jennifer L; Chatterjee, Shampa; Mulugeta, Surafel; Beers, Michael F
Published In Am J Physiol Lung Cell Mol Physiol, (2015 Jan 01)
Abstract: 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, wild-type 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.
PubMed ID: 25344067
MeSH Terms: ATP-Binding Cassette Transporters/genetics; ATP-Binding Cassette Transporters/metabolism; Adaptor Proteins, Signal Transducing/biosynthesis; Adaptor Proteins, Signal Transducing/genetics; Amino Acid Substitution; Autophagy*; Autophagy-Related Protein 8 Family; Female; Gene Expression Regulation/genetics; Genetic Diseases, Inborn/genetics; Genetic Diseases, Inborn/metabolism*; Genetic Diseases, Inborn/pathology; HEK293 Cells; Humans; Infant; Lung Diseases, Interstitial/genetics; Lung Diseases, Interstitial/metabolism*; Lung Diseases, Interstitial/pathology; Lysosomes/genetics; Lysosomes/metabolism; Lysosomes/ultrastructure; Membrane Potential, Mitochondrial/genetics; Microfilament Proteins/biosynthesis; Microfilament Proteins/genetics; Microtubule-Associated Proteins/biosynthesis; Microtubule-Associated Proteins/genetics; Mitochondria/genetics; Mitochondria/metabolism; Mitochondria/ultrastructure; Mutation, Missense*; Proteostasis Deficiencies/genetics; Proteostasis Deficiencies/metabolism; Proteostasis Deficiencies/pathology; Pulmonary Surfactant-Associated Protein C/genetics; Pulmonary Surfactant-Associated Protein C/metabolism*; Sequestosome-1 Protein; Ubiquitin-Protein Ligases/biosynthesis; Ubiquitin-Protein Ligases/genetics; Vacuoles/genetics; Vacuoles/metabolism; Vacuoles/ultrastructure; rab GTP-Binding Proteins/biosynthesis; rab GTP-Binding Proteins/genetics