Exploiting PrP for the diagnosis and treatment of protein aggregation diseases

描述(申请人提供)：神经退行性疾病是影响数千万美国人的主要公共卫生问题，令人信服的证据表明，特定蛋白质的寡聚和纤化是几乎所有此类疾病的共同方面。参与神经退行性变的两种最广泛研究的蛋白质是普里恩蛋白(PrP)和淀粉样蛋白b蛋白(Ab)。在争论中，出现了一些具有挑衅性的数据，这些数据表明PrP可能作为一种受体介导抗体毒性。虽然抗体与PrP结合的病理意义仍有争议，但完全一致的是PrP以高亲和力结合抗体寡聚体，PrP是抗体的亚化学计量比抑制剂 聚合。具有这种特性的试剂有可能用于阿尔茨海默病的诊断和治疗，但到目前为止，靶向和检测疾病相关蛋白寡聚体的努力在很大程度上依赖于抗体的使用。基于我们的论证，PrP在亲和力、特异性和特异性方面优于铅构象特异性抗体 对于抗聚集活性，我们建议利用PrP来产生高效的抗低聚物试剂。然而，PrP结合抗体低聚物并阻止抗体单体聚集的分子基础知之甚少。因此，我们建议鉴定PrP中与抗体识别有关的氨基酸，并对其进行修饰，以进一步增强PrP结合抗体寡聚体和抑制抗体聚集的能力。PrP中有两段序列与寡聚体结合有关，我们将集中精力研究这两段序列。关键残基的鉴定和PrP与抗体低聚物结合的进一步优化将通过在这两个位点引入“设计突变”的迭代过程来实现。在每一步，我们将测试诱变的PrP是否能与抗体低聚体结合或阻止抗体聚集。这种方法应该产生一种重组PrP衍生物--一种可用于定量和靶向有毒抗体低聚物的“超级结合剂”。然而，PrP衍生品的潜在用途可能不止是阿尔茨海默病。由于PrP已知可以结合某些设计的富含b-折叠的多肽的寡聚体形式，我们假设PrP将能够与其他蛋白质低聚体结合。因此，我们将研究PrP是否能与寡聚体结合和/或阻止与帕金森病相关的α-突触核蛋白和与额颞痴呆相关的tau的聚集。在这样做的过程中，我们将确定PrP的活性是抗体特异性的，还是延伸到其他关键的与疾病相关的蛋白寡聚体的特性。我们还将把两个PrP寡聚体结合位点的序列移植到一种免疫球蛋白中，以产生一种独特的嵌合抗体，并测试这种抗体是否能够识别和保护从人脑中分离出来的抗体寡聚体。最后，我们将产生额外的抗体，其中包含我们在PrP诱变研究中优化的PrP序列，并将这些抗体与α-突触核蛋白和tau寡聚体进行测试。通过这种方式，我们将产生新的工具来检测和中和与人类神经退化密切相关的有毒低聚物。

DESCRIPTION (provided by applicant): Neurodegenerative diseases are a major public health problem that affects tens of millions of Americans and compelling evidence indicates that oligomerization and fibrillization of specific proteins is a common facet of almost all such disorders. Two of the most widely studied proteins involved in neurodegeneration are the prion protein (PrP) and the amyloid b-protein (Ab). Amid controversy, provocative data have emerged which suggest that PrP may serve as a receptor that mediates Ab toxicity. While the pathological significance of Ab binding to PrP remains contentious, there is complete agreement that PrP binds Ab oligomers with high affinity and that PrP is a sub-stoichiometric inhibitor of Ab aggregation. Agents with such properties have potential for use in both the diagnosis and treatment of Alzheimer's disease, but until now efforts to target and detect disease- associated protein oligomers have largely depended on the use of antibodies. Based on our demonstration that PrP is superior to lead conformation-specific antibodies in terms of affinity, specificity and anti-aggregation activity, we propose to exploit PrP to generate highly potent anti-oligomer reagents. However, little is known about the molecular basis that underlies PrP's ability to bind Ab oligomers and prevent the aggregation of Ab monomer. Therefore, we propose to identify the amino acids in PrP that are involved in the recognition of Ab and to modify these to further enhance PrP's ability to bind Ab oligomers and inhibit Ab aggregation. Two stretches of sequence within PrP are implicated in oligomer binding and we will focus our efforts on these. Identification of the key residues and further optimization of PrP's binding to Ab oligomers will be achieved by an iterative process of introducing "design mutations" in these two sites. At each step we will test if the mutagenized PrP can bind to Ab oligomers or prevent Ab aggregation. This approach should produce a recombinant PrP derivative - a 'super-binder' that can be used for both the quantification and the targeting of toxic Ab oligomers. However, the potential uses of PrP derivatives may go beyond Alzheimer's disease. Since PrP is known to bind oligomeric forms of certain designed b-sheet-rich peptides, we hypothesize that PrP will be capable of binding to other protein oligomers. Consequently we will investigate if PrP can bind to oligomers and/or prevent the aggregation of a-synuclein which is associated with Parkinson's disease, and of tau which is associated with frontotemporal dementia. In so doing, we will determine whether PrP's activity is specific for Ab or is a property that extends to other key disease-implicated protein oligomers. We will also transplant sequences from the two PrP oligomer-binding sites into an IgG to produce a unique type of chimeric antibody and test if this antibody can recognize and protect against Ab oligomers isolated from human brain. Finally, we will generate additional antibodies containing PrP sequences we optimize in our PrP mutagenesis studies and test these against a-synuclein and tau oligomers. In this way we will generate new tools to detect and neutralize toxic oligomers centrally implicated in human neurodegeneration.