Molecular aspects of copper and zinc promoted prion-prion interactions

描述(由申请人提供)：尽管付出了巨大的努力，但Pron蛋白的确切功能以及Pron积累和神经元损伤的机制仍不清楚。最近的研究表明，Prion自我识别可能是该蛋白正常功能和功能障碍的一个重要因素。Prion也被确认为一种金属蛋白，能与多种铜结合，可能还能结合锌。这些辅因子的结合似乎起到了一个开关的作用，诱导了Pron-Prion的相互作用。我们已经开发了源于Pron蛋白金属结合区的荧光标记多肽模型，允许在分子水平上研究Pron-Prion相互作用和金属结合。这些模型被固定在膜表面，以模拟普里恩蛋白的细胞环境。为了解决确定Pron-Prion相互作用的结构元件的特定目标，将使用脉冲梯度核磁共振和荧光技术研究多肽模型作为添加铜和锌的函数。第二个具体目标是利用圆二色谱、电子顺磁共振和金属催化的氧化反应，结合串联质谱学来确定铜的结合机制和不同载铜状态之间的相关性。所有的实验研究都将通过一套平行的分子模型研究来加强。这项提案中的AIMS的结果将为深入了解蛋白内吞的信号机制以及它可能转导其他信号的方式提供洞察力。此外，这些研究有望提供有关Prion-Prion相互作用如何导致疾病导致Prion错误折叠的信息。这项研究项目的所有方面都是由本科生和硕士研究生进行的。他们应该精通多肽的合成、纯化和表征，以及各种尖端的仪器技术。他们还将学习数据分析和分子建模的计算方法。

DESCRIPTION (provided by applicant): Despite intense effort, the precise function of the prion protein remains unclear as well as the mechanism of prion accumulation and neuronal damage. Recent studies now indicate that prion self-recognition may be an important factor in both the normal function and misfunction of this protein. Prion has also been identified as a metalloprotein, binding multiple coppers and possibly zinc. The binding of these cofactors appears to act as a switch that induces prion-prion interactions. We have developed fluorescently labeled peptide models derived from the metal binding region of the prion protein that allow prion-prion interactions and metal binding to be investigated on the molecular level. These models are anchored to a membrane surface so as to mimic the prion protein's cellular environment. To address the specific aim of determining the structural elements responsible for prion-prion interactions, peptide models will be investigated as a function of added copper and zinc using pulsed-field gradient NMR and fluorescence techniques. The second specific aim of determining the copper binding mechanism and the relevance of different copper loaded states will be addressed using circular dichroism, electron paramagnetic resonance and metal catalyzed oxidation reactions in conjunction with tandem mass spectrometry. All experimental studies will be enhanced with a parallel set of molecular modeling studies. Results from the aims in this proposal will provide insight into the signaling mechanism for prion endocytosis and the means by which it might transduce other signals. In addition, these studies are anticipated to provide information on how prion-prion interactions may lead to disease causing prion mis-folding. All aspects of this research project are designed to be conducted by undergraduate and masters-level students. They should become proficient in peptide synthesis, purification and characterization plus a variety of cutting-edge instrumental techniques. They will also be introduced to computational methods of data analysis and molecular modeling.