The Somatostatin-28(1−12)-NPAMAP Sequence:  An Essential Helical-Promoting Motif Governing Prosomatostatin Processing at Mono- and Dibasic Sites

Abstract

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.

Processing of peptide and hormone precursors at the dibasic cleavage sites

“…7a, deletion of Pro residues (motif XAMAX) or shift of Pro -5 (motif PAMPA) increased both the helicity values per residue and the size of the domain containing the dibasic site, i.e., Som(Asn -6 -Asn ?5 ). Since Pro -5 is highly conserved in the primary sequence of prosomatostatin from various species, this amino acid residue additionally plays a role in the correct folding of the prosomatostatin processing domain [89]. These conclusions were supported by the results obtained for the processing of prosomatostatin mutants D[AMA] and [P -6 P -5 ].…”

“…Analysis of the processing efficiencies, observed with either the non-mutated precursor and prosomatostatin mutants in transfected Neuro2A cells ( Table 2), indicated that substitution of Pro -5 by an a-helix promoting amino acid residue ([A -5 ] mutant) abolished cleavage at the dibasic site [63,82]. In contrast, replacement of Pro -5 by a b-turn ''former'' residue-like ([G -5 ] mutant) or of the sequence Pro -5 -Arg-Glu-Arg -2 by non-homologous peptide stretches ([S -5 -N -3 ] and [Y -5 -G -3 ] mutants), known to organize as b-turn structures in proteins, did not affect prosomatostatin processing [89].…”

“…6, the S-28(1-12) sequence includes two Pro residues known to play a special role in structures [86] and functions [87] of proteins. Moreover, these Pro Table 2 Effects of various mutations on human prosomatostatin processing in transfected Neuro2A cells [89] Mutants…”

Structural Investigation of the HIV‐1 Envelope Glycoprotein gp160 Cleavage Site, 2: Relevance of an N‐Terminal Helix

“…Recently the role of a short helix-promoting sequence at the N-terminus of the multibasic cleavage site in governing the proteolytic processing was also emphasized for pro-somatostatin. [33] Structural studies are in progress on other HIV-1 gp160 cleavage site analogues to further investigate the structural factors that play a role in gp160-proteolytic enzyme recognition.…”

Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis

“…Prepro-SS has a sequence of hydrophobic aa at the N-terminus which is cleaved at the gly-ala junction at position -78 (from the N-terminus to the Cterminus). Pro-SS undergoes both monobasic (Arg -15 ) and dibasic (Arg -2 Lys -1 ) cleavages to release the two biofunctional hormones SS-28 and SS-14 (Funckes et al, 1983;Brakch et al, 2002) (Figure 1). …”