title: The Interacting Domains of PREP1 and p160 are Endowed with a Remarkable Structural Stability.
abstract: PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1 μM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization.
authors.affiliation: Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy.|Department of Environmental Biology, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Lorenzo
authors.affiliation: Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy.|Department of Pharmacy, University of Naples "Federico II", Naples, Italy.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Mascanzoni
authors.affiliation: Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Vitagliano
authors.affiliation: Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy.
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
authors.affiliation: Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy. nunzianna.doti@cnr.it.
authors.forename: Nunzianna
authors.initials: N
authors.lastname: Doti
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D018398:Homeodomain Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D018398:Homeodomain Proteins; C106682:PKNOX1 protein, human; D011994:Recombinant Proteins
keywords: Circular dichroism spectroscopy; PREP1; Protein domain design; Protein purification; Protein–protein interaction; Thermal stability; p160