We report on a general strategy for engineering dominant negative mutations that, in principle, requires neither extensive structural or functional knowledge of the targeted protein. The approach consists of fusing the lysosomal protease cathepsin B (CB) to a subunit of a multimeric protein. The CB fusion polypeptide can proteolytically digest the multimer and/or detour the multimer from its usual subcellular destination to the lysosome. We first demonstrate the general validity of the approach with CB fusion to E. coli lacZ, encoding tetrameric beta-galactosidase. Cotransfection of NIH 3T3 cells with a vector expressing a CB-lacZ fusion inhibits the beta-galactosidase activity produced by transfection of lacZ alone. We infer that the dominant negative inhibition results from both direct proteolysis of the beta-galactosidase tetramer by the fusion subunit and detour of the tetramer to the lysosome. In a specific application of this strategy, we have fused CB to the dimeric bHLH skeletal muscle transcription factor MyoD. The CB-MyoD fusion protein localizes to the cytoplasm, presumably the lysosome, demonstrating the dominance of lysosomal localization to nuclear localization. The CB-MyoD fusion appears to divert homodimerizing native MyoD from its usual nuclear destination, consequently inhibiting MyoD-mediated transactivation and in vitro differentiation of C2C12 myoblasts. Surprisingly, the CB-MyoD fusion fails to interact with the bHLH heterodimerization partners, E12 and E47, suggesting preferential MyoD homodimer formation, at least in the prenuclear cellular compartments.
Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.
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F Q Li, A Coonrod, M Horwitz; Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.. J Cell Biol 15 November 1996; 135 (4): 1043–1057. doi: https://doi.org/10.1083/jcb.135.4.1043
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