Ubiquitin Proteasome System and Molecular Signatures of Alzheimer's Disease

需要工具来建立阿尔茨海默病（AD）的病因学，其特征出现在阿尔茨海默病（AD）的发病之前。 疾病呈现。AD的早期是错误折叠蛋白质的泛素化;那些不被蛋白酶体降解的蛋白质可以被蛋白酶体降解。 形成神经元缠结，如β-淀粉样蛋白或tau蛋白聚集体。泛素（Ub）介导的信号转导作用 在蛋白质降解中起核心作用，神经变性的第一步是泛素化模式的变化， 特别是与蛋白质相连的poly-Ub链。开发灵敏的方法来检测这些 泛素化标记将是AD药物发现和诊断的主要进展。Poly-Ub链形成 利用所有七个赖氨酸，以及泛素的N-末端，这表明这些链编码了许多 信息.每一个连接，包括单-Ub，信号蛋白质的特征性结果; 建筑是这个项目的主题。最具特征的Ub类型是Lys 48（K48）和Lys 63（K63）。 连接; K48-和K11-连接的链靶向蛋白质以被蛋白酶体降解，而K63-连接的链 调节受体内吞、DNA修复、氧化应激等途径。其他具体职能 目前正在研究各种联系。理解特异性多聚泛素化蛋白的两个障碍 结构和功能是：1）虽然大多数蛋白质在某些时候是泛素化的，但在任何给定的时间， 泛素化部分可以是总细胞蛋白的一小部分;和2）在泛素化部分的库中， 蛋白质，任何特定的连接可能是罕见的。此外，很少有工具可用于特定的检测或诊断。 纯化具有特异性聚-Ub键的泛素化蛋白。在第一阶段，Ub结合结构域（UBD） 用150个UBD的蛋白质微阵列芯片鉴定非典型多聚Ub（K6/K11/K27/K29/K33）的选择性 由MD安德森癌症中心的马克贝德福德博士开发。还确定了UBD， 对K48连接的多聚泛素化蛋白的选择性。此外，新UBD的KD值和特异性是 使用SPR确定，实现目标。在第二阶段，LifeSensors将设计UBD域， 使用称为TUBE（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特异性TUBE。贝德福德博士将继续他的合作， 第二阶段。参加第二阶段的还有马里兰州大学的大卫福什曼博士，他开创了 溶液NMR方法来研究poly-Ub链。第二阶段工程需要建立多聚-Ub链连接 使用基于结构的蛋白质工程和产生多聚Ub链， 选择性TUBE，以提高亲和力，增强结合的亲合力，并提高连接选择性。最后这些 将通过模拟蛋白质病变来验证新型非典型聚Ub管在神经元细胞中的应用。 .

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. .