title: A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy.
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.
authors.affiliation: Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania;
authors.forename: Arie
authors.initials: A
authors.lastname: Hawkins
authors.affiliation: Department of Pediatrics; Monroe Carell Jr. Children's Hospital, Vanderbilt University, Nashville, Tennessee;
authors.forename: Susan H
authors.initials: SH
authors.lastname: Guttentag
authors.affiliation: Department of Pediatrics; University of Colorado School of Medicine, Denver, Colorado;
authors.forename: Robin
authors.initials: R
authors.lastname: Deterding
authors.affiliation: Department of Pathology and Lab Medicine; University of North Carolina, Chapel Hill, North Carolina;
authors.forename: William K
authors.initials: WK
authors.lastname: Funkhouser
authors.affiliation: Departments of Medicine and Pediatrics; University of North Carolina, Chapel Hill, North Carolina;
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Goralski
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania.
authors.forename: Shampa
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania; mulugeta@mail.med.upenn.edu.
authors.forename: Surafel
authors.initials: S
authors.lastname: Mulugeta
authors.affiliation: Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania;
authors.forename: Michael F
authors.initials: MF
authors.lastname: Beers
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000235:genetics; D019943:Amino Acid Substitution; D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D007223:Infant; D017563:Lung Diseases, Interstitial / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020125:Mutation, Missense; D057165:Proteostasis Deficiencies / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics / Q000378:metabolism*; D000071456:Sequestosome-1 Protein; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D000090802:rab7 GTP-Binding Proteins
chemical_list: C441524:ABCA3 protein, human; D018528:ATP-Binding Cassette Transporters; D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; C498698:GABARAPL2 protein, human; C477012:MAP1LC3A protein, human; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D037721:Pulmonary Surfactant-Associated Protein C; C489679:SFTPC protein, human; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D020691:rab GTP-Binding Proteins
keywords: alveolar epithelium; amphisome; autophagy; pulmonary fibrosis; surfactant protein