G.P.54: 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. In order to unravel potential novel functions for GNE, we have analyzed interactions of GNE with other proteins by diverse methods. In these studies we have used bimolecular fluorescence complementation (BiFC) in living cells. By this method, we have validated in vivo interactions between GNE–GNE, between actinin and actinin, and between GNE and actinin in HEK293 cells transfected with the relevant expression vectors. To elucidate the interaction domains in GNE, we have applied the BiFC methodology with each of the 2 main domains of GNE, epimerase and kinase, in living cells, as well as with the mutations frequently found in GNE Myopathy patients, such as the Middle Eastern founder mutation M712T (designated M743T according to the novel recommended nomenclature). This method will be very valuable to elucidate the interaction domains (epimerase and kinase) between the GNE monomers and between GNE and actinins, as well as the potential of mutations to disrupt such interactions. These studies could contribute new understandings on the mechanism of GNE in health and disease.