Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?

Mutations in the gene encoding surfactant protein C (SP-C) cause interstitial lung disease (ILD), and glucocorticosteroid (GC) treatment is the most recognized therapy in children. We aimed to decipher the mechanisms behind successful GC treatment in twins carrying a BRICHOS c.566G > A (p.Cys189Tyr) mutation in the SP-C gene (SFTPC). METHODS: The twins underwent bronchoscopy before and after GC treatment and immunoblotting analysis of SP-C proprotein (proSP-C) and SP-C mature in bronchoalveolar fluid (BALF). Total RNA was extracted and analysed using quantitative real-time PCR assays. In A549 cells, the processing of mutated protein C189Y was studied by immunofluorescence and immunoblotting after heterologous expression of eukaryotic vectors containing wild type or C189Y mutant cDNA. RESULTS: Before treatment, BALF analysis identified an alteration of the proSP-C maturation process. Functional study of C189Y mutation in alveolar A549 cells showed that pro-SP-CC189Y was retained within the endoplasmic reticulum together with ABCA3. After 5 months of GC treatment with clinical benefit, the BALF analysis showed an improvement of proSP-C processing. SFTPC mRNA analysis in twins revealed a decrease in the expression of total SFTPC mRNA and a change in its splicing, leading to the expression of a second shorter proSP-C isoform. In A549 cells, the processing and the stability of this shorter wild-type proSP-C isoform was similar to that of the longer isoform, but the half-life of the mutated shorter isoform was decreased. These results suggest a direct effect of GC on proSP-C metabolism through reducing the SFTPC mRNA level and favouring the expression of a less stable protein isoform.

Monoclonal antibodies raised against Xenopus p53 interact with human p73.

The p53 tumor suppressor gene belongs to a multigene family that includes two paralogues, p63 and p73. The structure of the p63 and p73 genes is quite similar, but both have common activities with p53, such as DNA binding and transactivation. Both p53 and p73 bind to mdm2, but only p53 is degraded through the activity of mdm2. p63 neither binds to nor is degraded by mdm2 despite important conservation in the key interacting residues. Using a panel of monoclonal antibodies raised against human and Xenopus p53, we have been able to find several antibodies that cross-react strongly with human p73. These antibodies react both with exogenous p73 expressed in mammalian cells and with endogenous p73. Interestingly, all these antibodies react with the same epitope localized in the amino-terminus of p53, but have no cross-reaction with p63. This epitope corresponds to the exact mdm2 binding site to p53. These antibodies inhibit the interaction between either p53 or p73 and mdm2, and may be useful tools for the study of these proteins. Furthermore, our studies suggest that there exist specific spatial requirements for the interaction between p53 or p73 and mdm2.