International audienceThe newly synthesized surfactant protein C precursor (proSP-C) is an integral endoplasmic reticulum (ER) membrane protein with a single metastable polyVal α-helical transmembrane domain that comprises two thirds of the mature peptide. More than 20 mutations in the ER-lumenal, C-terminal domain of proSP-C (CTC), are associated with interstitial lung disease (ILD), and some of the mutations cause intracellular accumulation of cytotoxic protein aggregates and a corresponding decrease in mature SP-C. Here it is shown that (i) human embryonic kidney cells expressing the ILD associated mutants proSP-CL188Q and proSP-CΔExon4 accumulate Congo red positive amyloid-like inclusions, while cells transfected with the mutant proSP-CI73T do not, (ii) transfection of CTC into cells expressing proSP-CL188Q results in a stable CTC/proSP-CL188Q complex, increased proSP-CL188Q half life and reduced formation of Congo red positive deposits, (iii) replacement of the metastable polyVal transmembrane segment with a stable polyLeu likewise prevents formation of amyloid-like proSP-CL188Q aggregates, and (iv) binding of recombinant CTC to non-helical SP-C blocks SP-C amyloid fibril formation. These data suggest that CTC can prevent the polyVal segment of proSP-C from promoting formation of amyloid-like deposits during biosynthesis, by binding to non-helical conformations. Mutations in the Brichos domain of proSP-C may lead to ILD via loss of CTC chaperone function

The BiP cochaperone ERdj4 removes misfolded proteins from the ER lumen by associating with ERAD machinery. Global deficiency of ERdj4 results in widespread constitutive ER stress, decreased survival, and metabolic derangements in mice. These findings indicate that the chaperone activity of ERdj4 is important for ER homeostasis in vivo.

We previously proposed a model of surfactant protein (SP)-C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc 2/2 mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S 12 PPDYS 17 completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase-SP-C propeptide (residues 1-35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S 12 PPDYS 17 mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2-mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.

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