Grasso Domenico, Grasso Giulia, Guantieri Valeria, Impellizzeri Giuseppe, La Rosa Carmelo, Milardi Danilo, Micera Giovanni, Osz Katalin, Pappalardo Giuseppe, Rizzarelli Enrico, Sanna Daniele, Sóvágó Imre
Università di Catania, Dipartimento di Scienze Chimiche, Viale Andrea Doria 6, 95125 Catania, Italy.
Chemistry. 2005 Dec 23;12(2):537-47. doi: 10.1002/chem.200500534.
An abnormal interaction between copper and the prion protein is believed to play a pivotal role in the pathogenesis of prion diseases. Copper binding has been mainly attributed to the N-terminal domain of the prion protein, but this hypothesis has recently been challenged in some papers which suggest that the C-terminal domain might also compete for metal anchoring. In particular, the segment corresponding to the helix II region of the prion protein, namely PrP180-193, has been shown both to bind copper and to exhibit a copper-enhanced cytotoxicity, as well as to interact with artificial membranes. The present work is aimed at extending these results by choosing the most representative model of this domain and by determining its copper affinity. With this aim, the different role played by the electrostatic properties of the C- and N-termini of PrP180-193 (VNITIKQHTVTTTT) in determining its conformational behaviour, copper coordination and ability to perturb model membranes was investigated. Owing to the low solubility of PrP180-193, its copper affinity was evaluated by using the shorter PrPAc184-188NH2 (IKQHT) analogue as a model. ESI-MS, ESR, UV/Vis, and CD measurements were carried out on the copper(II)/PrPAc184-188NH2 and copper(II)/PrP180-193NH2 systems, and showed that PrPAc184-188NH2 is a reliable model for the metal interaction with the helix II domain. The affinity of copper(II) for the helix II fragment is higher than that for the octarepeat and PrP106-126 peptides. Finally, the different ability of PrP180-193 analogues to perturb the DPPC model membrane was assessed by DSC measurements. The possible biological consequences of these findings are also discussed briefly.
铜与朊病毒蛋白之间的异常相互作用被认为在朊病毒疾病的发病机制中起关键作用。铜结合主要归因于朊病毒蛋白的N端结构域，但这一假设最近在一些论文中受到了挑战，这些论文表明C端结构域也可能竞争金属锚定。特别是，与朊病毒蛋白螺旋II区域相对应的片段，即PrP180 - 193，已被证明既能结合铜，表现出铜增强的细胞毒性，又能与人工膜相互作用。本研究旨在通过选择该结构域最具代表性的模型并确定其铜亲和力来扩展这些结果。为此，研究了PrP180 - 193（VNITIKQHTVTTTT）的C端和N端静电性质在决定其构象行为、铜配位以及扰动模型膜能力方面所起的不同作用。由于PrP180 - 193的低溶解性，其铜亲和力通过使用较短的PrPAc184 - 188NH2（IKQHT）类似物作为模型进行评估。对铜（II）/PrPAc184 - 188NH2和铜（II）/PrP180 - 193NH2体系进行了电喷雾电离质谱（ESI - MS）、电子顺磁共振（ESR）、紫外/可见光谱（UV/Vis）和圆二色光谱（CD）测量，结果表明PrPAc184 - 188NH2是金属与螺旋II结构域相互作用的可靠模型。铜（II）对螺旋II片段的亲和力高于对八肽重复序列和PrP106 - 126肽段的亲和力。最后，通过差示扫描量热法（DSC）测量评估了PrP180 - 193类似物扰动二棕榈酰磷脂酰胆碱（DPPC）模型膜的不同能力。还简要讨论了这些发现可能的生物学后果。
