The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites.
Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.
Brakch
Noureddine
N
Division d'Hypertension, CHU Vaudois, CH-1011, Lausanne, Switzerland.
Lazar
Noureddine
N
Panchal
Maï
M
Allemandou
Flore
F
Boileau
Guy
G
Cohen
Paul
P
Rholam
Mohamed
M
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002942:Circular Dichroism; D005399:Fishes; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D013004:Somatostatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053561:Somatostatin-28; D017550:Spectroscopy, Fourier Transform Infrared; D014407:Tumor Cells, Cultured
D009842:Oligopeptides; D011498:Protein Precursors; D013004:Somatostatin; C028456:prosomatostatin; D053561:Somatostatin-28; D001120:Arginine; D008239:Lysine