Abstract
Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Animals
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Aptamers, Peptide / chemistry
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Aptamers, Peptide / metabolism*
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Cell Nucleus / metabolism
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Cells, Cultured
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Cytoplasm / metabolism
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Epithelial Cells / cytology*
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Epithelial Cells / drug effects
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Extracellular Signal-Regulated MAP Kinases / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins / chemistry
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Intracellular Signaling Peptides and Proteins / metabolism*
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Mesoderm / cytology*
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Mesoderm / drug effects
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Mice
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Molecular Sequence Data
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Multiprotein Complexes / metabolism
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Phosphorylation / drug effects
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Protein Binding
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Protein Transport / drug effects
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Proto-Oncogene Proteins c-akt / metabolism
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Serine Endopeptidases / chemistry
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Serine Endopeptidases / metabolism*
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Signal Transduction / drug effects*
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Smad Proteins / metabolism*
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Thioredoxins / chemistry
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Thioredoxins / metabolism
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Transforming Growth Factor beta / pharmacology*
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Transforming Growth Factor beta1
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p38 Mitogen-Activated Protein Kinases / metabolism
Substances
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Aptamers, Peptide
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Intracellular Signaling Peptides and Proteins
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Multiprotein Complexes
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Smad Proteins
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TGFB1 protein, human
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Tgfb1 protein, mouse
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Transforming Growth Factor beta
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Transforming Growth Factor beta1
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Thioredoxins
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Proto-Oncogene Proteins c-akt
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Extracellular Signal-Regulated MAP Kinases
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p38 Mitogen-Activated Protein Kinases
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ZFYVE16 protein, human
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Serine Endopeptidases