P.3.4 In vivo GNE interactions

Abstract

GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. We have previously shown that GNE interacts with alpha-actinin1 and alpha-actinin2 in vitro, either at different interaction sites on the GNE protein, or alternatively by changing the conformational state of GNE. In the current studies we have analyzed these interactions by the bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells and now demonstrate these interactions also in vivo. In addition, to elucidate the interaction domains in GNE, we have dissected the GNE molecule, first in its 2 main domains, epimerase and kinase. We have generated those TAG proteins in the insect cells/baculovirus system and performed pull down and immunoprecipitation assays, also in the presence of both alpha-actinins. Furthermore, interactions of these “half domain” proteins were also analyzed in living cells by the BiFC methodology. The potential of frequent mutations in GNE myopathy, such as the Middle Eastern founder mutation M712T, to disrupt such interactions will be shown. These studies could contribute new understandings on the mechanism of GNE in health and disease. GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. We have previously shown that GNE interacts with alpha-actinin1 and alpha-actinin2 in vitro, either at different interaction sites on the GNE protein, or alternatively by changing the conformational state of GNE. In the current studies we have analyzed these interactions by the bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells and now demonstrate these interactions also in vivo. In addition, to elucidate the interaction domains in GNE, we have dissected the GNE molecule, first in its 2 main domains, epimerase and kinase. We have generated those TAG proteins in the insect cells/baculovirus system and performed pull down and immunoprecipitation assays, also in the presence of both alpha-actinins. Furthermore, interactions of these “half domain” proteins were also analyzed in living cells by the BiFC methodology. The potential of frequent mutations in GNE myopathy, such as the Middle Eastern founder mutation M712T, to disrupt such interactions will be shown. These studies could contribute new understandings on the mechanism of GNE in health and disease.