Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.
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.
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Markey Molecular Medicine Center, Department of Medicine, University of Washington, Seattle 98195, USA.
Coonrod
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Horwitz
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D016475:3T3 Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002403:Cathepsins / Q000235:genetics*; D002454:Cell Differentiation / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D004792:Enzyme Precursors / Q000235:genetics*; D017930:Genes, Reporter / Q000502:physiology; D005816:Genetic Complementation Test; D007763:Lac Operon / Q000502:physiology; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology*; D051379:Mice; D016296:Mutagenesis / Q000502:physiology; D017570:MyoD Protein / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
D004268:DNA-Binding Proteins; D004792:Enzyme Precursors; D017570:MyoD Protein; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D001616:beta-Galactosidase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C057831:preprocathepsin B