Protein Quality Control In Alveolar Type 2 Cells: The Proteasome Is Essential For Control Of Aggregation‐Prone SP‐C Mutations

Abstract

Mutations in the distal C‐terminus of the Surfactant Protein C (SP‐C) proprotein (BRICHOS domain) result in aggregation‐prone isoforms and are associated with interstitial lung disease (ILD). The ubiquitin proteasome system (UPS) and macroautophagy (autophagy) are the main quality control mechanisms for aberrant proteins. The extent to which these proteostatic pathways are utilized by human alveolar type II cells (hAT2) is unknown. Treatment of primary hAT2 with rapamycin resulted in inhibition of S6 phosphorylation, conversion of LC3I/II, and quenching of vesicular EGFP‐LC3‐mCherry fluorescence consistent with the presence of inducible autophagy. Using GFP‐U1 and chymotrypsin‐like activity as readouts, hAT2 exhibited a high degree of basal proteasomal function which was inhibited by MG132 but also resulted in accumulation of LC3 and p62 suggesting cross‐talk between the pathways. Compared to wild type SP‐C, EGFP tagged clinical SP‐C BRICHOS mutants were expressed at significantly lower levels in transfected hAT2. UPS inhibition by MG132 caused accumulation of BRICHOS mutants while autophagy inhibition with 3‐methyladenine had minimal effects on mutant SP‐C levels. Exposure of hAT2 to cigarette smoke (CS) condensate inhibited proteasomal activity, induced markers of both ER stress (GRP78) and autophagy inhibition (p62), and caused cytosolic aggregation of BRICHOS mutants. These results indicate that the UPS represents a primary protein quality control system for the distal lung epithelia. The inhibitory effect of CS exposure on both systems supports a 2‐hit model of ILD of a susceptible epithelial cell phenotype plus exogenous cellular stress.