A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy

Am J Physiol Lung Cell Mol Physiol. 2015 Jan 1;308(1):L33-47. doi: 10.1152/ajplung.00217.2014. Epub 2014 Oct 24.

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.

Keywords: alveolar epithelium; amphisome; autophagy; pulmonary fibrosis; surfactant protein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

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

Substances

  • ABCA3 protein, human
  • ATP-Binding Cassette Transporters
  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Protein 8 Family
  • GABARAPL2 protein, human
  • Microfilament Proteins
  • Microtubule-Associated Proteins
  • Pulmonary Surfactant-Associated Protein C
  • SFTPC protein, human
  • SQSTM1 protein, human
  • Sequestosome-1 Protein
  • light chain 3, human
  • rab7 protein
  • Ubiquitin-Protein Ligases
  • parkin protein
  • rab GTP-Binding Proteins