Unidade de Biotecnologia e Recursos Genéticos, Instituto Nacional de Investigação Agrária e Veterinária, Quinta da Fonte Boa, Vale de Santarém, Portugal; CIISA, Faculdade de Medicina Veterinária (FMV), Universidade Técnica de Lisboa, Lisbon, Portugal.
Peptides. 2013 Nov;49:32-40. doi: 10.1016/j.peptides.2013.08.013. Epub 2013 Aug 23.
Prion protein (PrP(C)) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP(C) proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N-C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.
朊病毒蛋白（PrP(C)）的生物合成涉及一个多步骤的过程，包括翻译和翻译后修饰。虽然 PrP 已经被广泛研究，但对于同源物 Doppel（Dpl），我们的了解有限。在这项研究中，我们专注于真核生物蛋白质生物合成的一个重要步骤：信号识别颗粒（SRP）的靶向。以绵羊 Dpl（OvDpl(1-30)）肽作为模板，我们使用 CD 和 NMR 光谱研究了它在两种不同疏水环境中的行为。在三氟乙醇（TFE）和二己酰基-sn-甘油-3-磷酸胆碱（DHPC）中，OvDpl(1-30)肽都折叠成具有明确中心区域的α-螺旋构象，该中心区域从 Cys8 延伸到 Ser22。随后，将 NMR 结构包含在与 SRP54 蛋白（SRP54M）保守 M 结构域的计算对接复合物中，并与小鼠 Dpl 和牛 PrP(C) 蛋白的 N 端结构进行比较。这使得确定了 (i) 常见的预测 N 端/SRP54M 极性接触（Asp331、Gln335、Glu365 和 Lys432）和 (ii) 在 SRP54M 疏水性沟槽中，朊病毒样蛋白和 Dpl 肽之间不同的 N-C 取向，这与每个肽的静电势一致。这些发现为朊病毒样蛋白的生物合成提供了新的见解。此外，它们还展示了蛋白质构象开关在信号传导到内质膜中的作用，这是细胞周期中具有重要意义的关键事件。因此，它们对于研究朊病毒样以及其他蛋白质的生物学功能具有普遍适用性。
