| Autor: |
Pepinsky, Blake, Gong, Bang-Jian, Gao, Yan, Lehmann, Andreas, Ferrant, Janine, Amatucci, Joseph, Sun, Yaping, Bush, Martin, Walz, Thomas, Pederson, Nels, Cameron, Thomas, Wen, Dingyi |
| Zdroj: |
Biochemistry; 20240101, Issue: Preprints |
| Abstrakt: |
Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60–114, PDP60–114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60–114had no impact on activity. The specific activity of the GDF11/PDP60–114complex (EC50= 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60–114improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60–114can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60–114genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50= 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50= 2 nM) by protease treatment. Complex formation with PDP60–114represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes. |
| Databáze: |
Supplemental Index |
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