Involvement of a polyproline helix-like structure in the interaction of 7B2 with prohormone convertase 2.
The neuroendocrine protein 7B2 is known to be involved in the biosynthesis and activity of prohormone convertase 2 (PC2). Previous studies have demonstrated that while the carboxyl-terminal portion of 7B2 (residues 155-186) regulates the enzymatic activity of PC2, the amino terminus of the molecule (residues 1-151) is required for maturation of proPC2. In this study we employed four different experimental approaches (co-immunoprecipitation with proPC2, facilitation of pro-PC2 maturation, acquisition of enzymatic activity, and thermal protection assays) to identify structural elements of 7B2 important for bioactivity. Inspection of the sequence of 7B2 indicated potential involvement of a polyproline helix-like (PPII) structure, with similarities to those present within SH3 domain ligands, in the interaction of 7B2 with proPC2. Site-directed point mutagenesis of this proline-rich region confirmed the involvement of this area. Replacement of prolines in positions critical to helix formation (Pro90, Pro91, Pro93, and Pro95) either severely impaired or totally abolished 7B2 bioactivity, as gauged by the four assays described. In addition, constructs longer than residues 1-121 were still functional, whereas those shorter than residues 1-109 were not. Computer-assisted analysis predicts the presence of an alpha-helix structure between residues 107 and 123. We conclude that both the proline-rich region and the alpha-helix contribute to 7B2 activity. Polyproline-containing peptides have been shown to be involved in cytoplasmic protein-protein interactions; our results suggest that the polyproline helix motif may also be used to mediate protein-protein interactions within the secretory pathway.
Zhu
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
Lamango
N S
NS
Lindberg
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D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004795:Enzyme Stability; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051859:Neuroendocrine Secretory Protein 7B2; D010455:Peptides / Q000378:metabolism*; D010907:Pituitary Hormones / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics; D045706:Proprotein Convertase 2; D011485:Protein Binding; D011489:Protein Denaturation; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010455:Peptides; D010907:Pituitary Hormones; D011498:Protein Precursors; C011083:polyproline; D011392:Proline; D013381:Subtilisins; D045706:Proprotein Convertase 2