APP ubiquitination and implications for AB generation

描述(申请人提供)：阿尔茨海默病(AD)是痴呆症最常见的原因，仅在美国就估计有530万人受到影响。然而，AD的潜在细胞和分子病理生理学在很大程度上仍不清楚。阿尔茨海默病的特点是存在淀粉样斑块和神经原纤维缠结。淀粉样β蛋白(Aβ)作为这些淀粉样斑块的主要成分的发现，结合对导致AD的淀粉样前体蛋白(APP)编码基因的家族性突变的鉴定，导致了淀粉样级联假说，该假说认为Aβ是致病的罪魁祸首，降低Aβ负荷可以改善或预防AD症状。越来越多的证据表明，APP的胞内转运途径对其在生产Aβ过程中被γ-和β-分泌酶处理的能力有重要影响。在这里，我们将讨论通过内体系统控制APP分选的分子机制，因为最近的工作表明，APP可能是APP与其裂解酶β-和γ-分泌酶相互作用的主要场所。具体地说，我们已经获得了APP泛素化和淀粉样蛋白形成过程中运输所需的内体复合体(ESCRT)的证据。ESCRT识别限制内体膜上的泛素化蛋白，并将它们分类为多囊体内的管腔内小泡(ILV)。我们已经在APP细胞结构域中确定了经历泛素化的候选赖氨酸残基，并控制了APP分选ILV的能力。有趣的是，突变这些残基会选择性地增加Aβ40的生成，Aβ是一种含量更高、致病性更低的物种。尽管APP泛素化似乎是其贩运的关键信号，但作用于APP的E3泛素连接酶(S)尚未被识别。富含亮氨酸的F-box重复蛋白2(FBL2)是Skp1-cullin-F box(SCF)E3连接酶复合体的候选蛋白。这项建议的具体目的将是：(I)精确绘制APP泛素化位点的图谱，并确定与该蛋白结合的泛素修饰的类型(S)；(Ii)研究FBL2在APP内体膜泛素化中的作用；以及(Iii)确定APP泛素化缺陷对Aβ水平和淀粉样斑块生成的体内影响。我们预计，本提案中详述的实验将更好地了解通过内体系统管理APP贩运和加工的机制，并为迫切需要的AD疗法确定潜在的新靶点。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia, affecting an estimated 5.3 million individuals in the US alone. However, the underlying cellular and molecular pathophysiology of AD remains largely unknown. The AD brain is characterized by the presence of amyloid plaques and neurofibrillary tangles. The discovery of amyloid beta (Aβ) as the main component of these amyloid plaques, combined with the identification of AD-causing familial mutations in the gene encoding amyloid precursor protein (APP) led to the amyloid cascade hypothesis, which suggests that Aβ is the pathogenic culprit and that reducing Aβ load can improve or prevent AD symptoms. Growing evidence indicates that the intracellular trafficking routes of APP have a major impact on its ability to be processed by β- and γ-secretase, in the production of Aβ. Here, we will address the molecular mechanisms controlling the sorting of APP through the endosomal system since recent work indicates that the endosomal membrane is likely to be a primary locale where APP interacts with its cleavage enzymes, β- and γ-secretase. Specifically, we have obtained evidence implicating APP ubiquitination and the endosomal complex required for transport (ESCRT) in amyloidogenic processing. ESCRT recognizes ubiquitinated proteins on the limiting endosomal membrane and sorts them into intraluminal vesicles (ILVs) in the interior of multivesicular endosomes. We have identified candidate lysine residues in the APP cytodomain that undergo ubiquitination and control the ability of APP to sort into ILVs. Interestingly, mutating these residues leads to a selective increase in the generation of Aβ40, the more abundant and less pathogenic species of Aβ. Although APP ubiquitination appears to be a critical signal in its trafficking, the E3 ubiquitin ligase(s) acting on APP has not been identified. F-box and leucine-rich repeat protein 2 (FBL2) is a promising candidate as part of the Skp1-Cullin-F box (SCF) E3 ligase complex. The specific aims of this proposal will be: (i) To precisely map the ubiquitination sites of APP and determine the type(s) of ubiquitin modification bound to this protein; (ii) To investigate the role of FBL2 in APP ubiquitination at the endosomal membrane; and (iii) To determine the in vivo effects of APP ubiquitination deficiency on Aβ levels and amyloid plaque generation. We anticipate that the experiments detailed in this proposal will provide a better understanding of mechanisms regulating the trafficking and processing of APP through the endosomal system and identify potential new targets for urgently needed AD therapeutics.