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: Arie Hawkins, Susan H Guttentag, Robin Deterding, William K Funkhouser, Jennifer L Goralski, Shampa Chatterjee, Surafel Mulugeta, Michael F Beers
• Journal: American journal of physiology. Lung cellular and molecular physiology (Am J Physiol Lung Cell Mol Physiol) Vol. 308 Issue 1 Pg. L33-47 (Jan 01 2015) ISSN: 1522-1504 [Electronic] United States
• PMID: 25344067 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2015 the American Physiological Society.

Chemical References

• ABCA3 protein, human
• ATP-Binding Cassette Transporters
• Adaptor Proteins, Signal Transducing
• Autophagy-Related Protein 8 Family
• GABARAPL2 protein, human
• MAP1LC3A protein, human
• Microfilament Proteins
• Microtubule-Associated Proteins
• Pulmonary Surfactant-Associated Protein C
• SFTPC protein, human
• SQSTM1 protein, human
• Sequestosome-1 Protein
• rab7 GTP-Binding Proteins
• rab7 GTP-binding proteins, human
• Ubiquitin-Protein Ligases
• parkin protein
• rab GTP-Binding Proteins

Topics

• ATP-Binding Cassette Transporters (genetics, metabolism)
• Adaptor Proteins, Signal Transducing (biosynthesis, genetics)
• Amino Acid Substitution
• Autophagy
• Autophagy-Related Protein 8 Family
• Female
• Gene Expression Regulation (genetics)
• Genetic Diseases, Inborn (genetics, metabolism, pathology)
• HEK293 Cells
• Humans
• Infant
• Lung Diseases, Interstitial (genetics, metabolism, pathology)
• Lysosomes (genetics, metabolism, ultrastructure)
• Membrane Potential, Mitochondrial (genetics)
• Microfilament Proteins (biosynthesis, genetics)
• Microtubule-Associated Proteins (biosynthesis, genetics)
• Mitochondria (genetics, metabolism, ultrastructure)
• Mutation, Missense
• Proteostasis Deficiencies (genetics, metabolism, pathology)
• Pulmonary Surfactant-Associated Protein C (genetics, metabolism)
• Sequestosome-1 Protein
• Ubiquitin-Protein Ligases (biosynthesis, genetics)
• Vacuoles (genetics, metabolism, ultrastructure)
• rab GTP-Binding Proteins (biosynthesis, genetics)
• rab7 GTP-Binding Proteins