Ubiquitin Proteasome System and Molecular Signatures of Alzheimer's Disease

需要工具来确定阿尔茨海默病(AD)的病因学，其签名出现在 疾病呈现。AD的早期是错误折叠的蛋白质的泛素化；那些没有被蛋白酶体降解的蛋白质可以 形成神经纤维缠结，如β-淀粉样蛋白或tau蛋白聚集体。泛素(Ub)介导的信号转导 蛋白质降解的核心作用，以及神经退化的第一步是泛素化模式的变化， 尤其是结合在蛋白质上的多Ub链。开发灵敏的方法来检测这些 泛素化标记将是AD药物发现和诊断的重大进展。形成Poly-Ub链 利用所有七种赖氨酸以及泛素的N-端，这表明这些链编码了很多 信息。每个连接，包括单链Ub，发出蛋白质特有的结果信号；Ub链 建筑是这个项目的主题。最具特征性的Ub类型是Lys48(K48)和Lys63(K63) 连接；K48和K11连接的链针对蛋白质被蛋白酶体降解，而K63连接的链 调节受体内吞作用、DNA修复、氧化应激等途径。用于其他方面的特定功能 目前正在研究它们之间的联系。理解特定多泛素化蛋白质的两个障碍 结构和功能是：1)虽然大多数蛋白质在某个时间点是泛素化的，但在任何给定的时间 泛素化部分可能只占细胞总蛋白的一小部分；以及2)在泛素化的 蛋白质，任何特定的联系都可能是罕见的。此外，很少有工具可用于特定检测或 具有特定多聚Ub连接的泛素化蛋白的纯化。在第一阶段，Ub结合域(UBD) 用150UBD蛋白质芯片鉴定选择性非典型多聚Ub(K6/K11/K27/K29/K33) 由M.D.安德森癌症中心的Mark Bedford博士开发。同样确定的是最高的UBD K48连接的多泛素化蛋白的选择性。此外，新的UBD的Kd值和特异性是 用SPR确定，实现了目标。在第二阶段，生命传感器将设计UBD域以改进 使用称为管(串联泛素结合实体)的UBD结构域的亲和力和选择性；这些包括 非选择性以及线性[M1]、K48和K63特定的管。贝德福德博士将继续他的合作 第二阶段。参与第二阶段的还有马里兰大学的大卫·富什曼博士，他开创了 溶液核磁共振方法研究多-Ub链。第二阶段项目需要建立多Ub链连接 利用基于结构的蛋白质工程和产生多Ub链的第一阶段确定的UBD的选择性- 选择性管，以提高亲和力，增强结合亲和力，并改善连接选择性。最后，这些 新型非典型聚Ub管将通过模拟蛋白质病来验证其在神经细胞中的应用。 。

Tools are needed to establish the etiology of Alzheimer’s disease (AD), whose signatures appear before the disease presents. Early in AD is ubiquitylation of misfolded proteins; those not degraded by the proteasome can form neurofibrillary tangles such as β-amyloid or tau protein aggregates. Ubiquitin (Ub)-mediated signaling plays a central role in protein degradation, and a first step in neurodegeneration is a change in ubiquitylation patterns, particularly, that of poly-Ub chains attached to proteins. Development of sensitive methods to detect these ubiquitylation signatures will be a major advance in AD drug discovery and diagnosis. Poly-Ub chains are formed utilizing all seven lysines, as well as the N-terminus of ubiquitin, suggesting that these chains encode much information. Each linkage, including mono-Ub, signals a characteristic outcome for the protein; Ub chain architecture is the subject of this project. The best characterized Ub types are Lys48 (K48) and Lys63 (K63) linkages; K48- and K11-linked chains target proteins for degradation by the proteasome while K63-linked chains regulate receptor endocytosis, DNA repair, oxidative stress and other pathways. Specific functions for other linkages are currently under study. Two impediments to understanding specific poly-ubiquitylated protein structures and functions are: 1) while most proteins at some point are ubiquitylated, at any given time the ubiquitylated fraction may be a small percentage of total cell protein; and 2) within the pool of ubiquitylated protein, any particular linkage may be rare. Further, very few tools are available for specific detection or purification of ubiquitylated proteins with specific poly-Ub linkages. In phase I, Ub Binding Domains (UBDs) selective for atypical poly-Ub (K6/K11/K27/K29/K33) were identified using a 150 UBD protein microarray chip developed by Dr Mark Bedford, M D Anderson Cancer Center. Also identified was the UBD with the highest selectivity for K48 linked poly-ubiquitylated proteins. In addition, KD values and specificity for novel UBDs were determined using SPR, fulfilling the aims. In Phase II LifeSensors will engineer the UBD domains to improve affinity and selectivity using UBD domains called TUBEs (Tandem Ubiquitin Binding Entities); these include nonselective as well as Linear [M1], K48- and K63-specific TUBEs. Dr. Bedford will continue his collaboration in Phase II. Also participating in Phase II will be Dr. David Fushman, University of Maryland, who has pioneered solution NMR methods to study poly-Ub chains. the Phase II project entails establishing the poly-Ub chain linkage selectivity of UBDs identified in Phase I using structure-based protein engineering and generating poly-Ub chain- selective TUBEs to improve affinity, enhance avidity of binding, and improve linkage selectivity. Finally, these novel atypical poly-Ub TUBEs will be validated for application in neuronal cells by simulating proteinopathies. .