T6 Understanding the cellular dysfunction caused by pathogenic surfactant protein c mutant i73t

Abstract

Introduction and Objectives Our understanding of the pathogenesis of interstitial pulmonary disease is limited largely to its late, fibrotic stages. Inherited forms offer the potential to understand earlier pathogenic events. Autosomal dominant mutations in surfactant protein C (SFTPC) cause some cases of familial pulmonary fibrosis; the most common mutation is I73T, which is said to be mistrafficked within the type 2 pneumocyte. We hypothesise that mistrafficking of SFTPC I73T is causal in triggering type 2 pneumocyte dysfunction in familial pulmonary fibrosis. This work aims to understand the mechanism of SFTPC I73T mistrafficking such that type 2 pneumocyte dysfunction in familial, and sporadic disease can be better understood. Methods We overexpressed GFP tagged wild-type (WT) and I73T SFTPC in HeLa cells to study their localisation and trafficking. Subcellular localisation and co-localisation with organelle markers was assessed using confocal microscopy and immunofluorescence. Western blotting was used to assess expression of SFTPC isoforms and GFP traps to study SFTPC interactors, including ubiquitin. Mass spectrometry was used to assess the interactome of WT and I73T SFTPC. Results In HeLa cells overexpressing GFP-SFTPC, we observed mistrafficking of SFTPC I73T; WT SFTPC is trafficked to the multivesicular body (MVB), while I73T mistrafficks to the cell surface and into tubular structures (figure 1A) which co-localise with a marker of recycling endosomes Rab8 (figure 1B). Correct trafficking of WT SFTPC to the MVB depends on ubiquitination of lysine-6 (K6). We see an absence of ubiquitination in the I73T mutant and a subcellular distribution similar to that seen in a SFTPC mutant (K6R) which cannot be ubiquitinated. Mass spectrometry revealed differential binding between WT and I73T SFTPC of a number of cell surface proteins. Conclusion We have shown that pathogenic SFTPC mutant I73T mistrafficks not only to the cell surface, but also into recycling endosomes. We propose that this occurs as a result of failure of ubiquitination and internalisation into the MVB. Trafficking defects appear to impact upon binding of WT vs I73T to cell surface markers, the functional significance of which is the focus of our ongoing work.