APP ubiquitination and implications for AB generation

描述（由申请人提供）：阿尔茨海默病（AD）是痴呆症的最常见原因，仅在美国就有大约530万人受到影响。然而，阿尔茨海默病的潜在细胞和分子病理生理机制在很大程度上仍然未知。阿尔茨海默病大脑的特点是存在淀粉样斑块和神经原纤维缠结。淀粉样蛋白β (Aβ)作为这些淀粉样斑块的主要成分的发现，加上在编码淀粉样前体蛋白（APP）的基因中发现AD引起的家族性突变，导致淀粉样蛋白级联假说，这表明Aβ是致病的罪魁祸首，减少Aβ负荷可以改善或预防AD症状。越来越多的证据表明，APP的细胞内转运途径对其在β-和γ-分泌酶的加工能力产生重大影响。在这里，我们将讨论通过内体系统控制APP分选的分子机制，因为最近的研究表明，内体膜可能是APP与其裂解酶β-和γ-分泌酶相互作用的主要场所。具体来说，我们已经获得了APP泛素化和运输所需的内体复合物（ESCRT）在淀粉样变性过程中的证据。ESCRT识别限制内体膜上的泛素化蛋白，并将它们分类到多泡内体内部的腔内囊泡（ILVs）中。我们已经确定了APP细胞结构域中的候选赖氨酸残基，这些残基经历泛素化并控制APP分类成ilv的能力。有趣的是，这些残基的突变导致a β40的产生选择性增加，a β40是a β更丰富、致病性更低的物种。虽然APP泛素化似乎是其运输的关键信号，但尚未确定作用于APP的E3泛素连接酶。F-box和富亮氨酸重复蛋白2 （FBL2）作为Skp1-Cullin-F box (SCF) E3连接酶复合物的一部分，是一个有希望的候选者。本提案的具体目标将是：(i)精确绘制APP的泛素化位点，并确定与该蛋白结合的泛素修饰类型；（ii）探讨FBL2在APP内体膜泛素化中的作用；（iii）确定APP泛素化缺乏对Aβ水平和淀粉样斑块生成的体内影响。我们期望本实验能够更好地理解APP通过内体系统的转运和加工机制，并为迫切需要的AD治疗找到潜在的新靶点。

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.