Zhu X, Lamango N S, Lindberg I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
J Biol Chem. 1996 Sep 20;271(38):23582-7. doi: 10.1074/jbc.271.38.23582.
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.
神经内分泌蛋白7B2已知参与激素原转化酶2（PC2）的生物合成和活性调节。先前的研究表明，虽然7B2的羧基末端部分（第155 - 186位氨基酸残基）调节PC2的酶活性，但该分子的氨基末端（第1 - 151位氨基酸残基）是激素原PC2成熟所必需的。在本研究中，我们采用了四种不同的实验方法（与激素原PC2共免疫沉淀、促进激素原PC2成熟、获得酶活性以及热保护分析）来鉴定7B2中对生物活性重要的结构元件。对7B2序列的检查表明，一种类似于多聚脯氨酸螺旋（PPII）的结构可能参与了7B2与激素原PC2的相互作用，这种结构与SH3结构域配体中的结构相似。对这个富含脯氨酸区域进行定点诱变证实了该区域的参与作用。通过上述四种分析方法检测发现，替换对螺旋形成至关重要位置的脯氨酸（Pro90、Pro91、Pro93和Pro95），要么严重损害要么完全消除了7B2的生物活性。此外，长于第1 - 121位氨基酸残基的构建体仍具有功能，而短于第1 - 109位氨基酸残基的构建体则没有功能。计算机辅助分析预测在第107和123位氨基酸残基之间存在α-螺旋结构。我们得出结论，富含脯氨酸区域和α-螺旋都对7B2的活性有贡献。含多聚脯氨酸的肽已被证明参与细胞质中的蛋白质-蛋白质相互作用；我们的结果表明，多聚脯氨酸螺旋基序也可能用于介导分泌途径中的蛋白质-蛋白质相互作用。
