Abstract 1927: Molecular Characterization of Plakophilin-2 Mutations Suggests the Involvement of Proteolytic Degradation in the Disease Process of ARVC
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary myocardial disorder characterized by progressive loss of cardiomyocytes and fibro-fatty replacement. The armadillo protein plakophilin-2 (PKP2) has been identified as the most prevalent disease gene for ARVC. To get further insights in the underlying genetic mechanisms of these mutations and to investigate their influence on desmosomal integrity, protein stability and cell signalling in vitro we over-expressed different missense (C796R, S615F, K654Q) and frame-shift (C693fsX741) mutations in epithelial cell lines (A431, Hela-M2). In contrast to the wild-type protein and two unclassified variants (V587I, I531S), which showed the expected localization of PKP2 at the junctional plaque, the mutant proteins generated by missense and frame-shift mutations demonstrated a cytoplasmatic expression pattern with accumulation around the nucleus. However, the expression of mutant proteins did not disrupt the localization of other endogenous desmosomal components. Western blot analysis using antibodies detecting the C-terminal epitope of PKP2 demonstrated significantly lower levels of the mutant proteins. Interestingly, after treatment with inhibitors of calpain-proteases and the ubiquitin-proteasome-system we could show stabilized mutant proteins with amounts corresponding to the wild-type protein as well as increased cell numbers expressing the mutant protein. Assuming the involvement of a specific protein degradation process we expressed recombinant armadillo domains of PKP2 in E.coli with different mutations: C796R, S615F, C693fsX741, respectively. The mutant polypeptides were intrinsically unstable (wild-type protein: stable and soluble) and degraded in the same leaving fragments of arm-repeats analyzed by mass spectrometry indicating specific protease cleavage. Our data suggest that frame-shift and missense mutations produce unstable proteins in vitro, which might be removed rapidly from the cell by proteolytic degradation. Further investigations have to clarify the involvement of the UPS/Calpain system in this process as well as the influence of degraded protein fragments on desmosome pathology and cell signalling.