Role of BACE stabilization in Alzheimer's disease

描述(申请人提供)：阿尔茨海默病(AD)是影响530万美国人的最常见的痴呆症形式。在一小部分(&gt；1%)病例中，AD被遗传为常染色体显性遗传特征(家族性AD)，然而大多数病例是散发性的。阿尔茨海默病的一个关键神经病理事件是脑内Aβ的积聚，这是一种~4 kDa的多肽，由淀粉样前体蛋白(APP)在β-和β-分泌酶作用下的一系列蛋白分解而得。β-位点APP裂解酶(BACE1)是天冬氨酸蛋白水解酶中的一种膜依赖成员，已被鉴定为分泌酶。多项研究表明，阿尔茨海默病患者大脑中的BACE1蛋白水平和分泌酶活性增加。因此，至少在散发性病例中，BACE1的升高可能是增加A？并触发AD病理的第一步。我们的研究阐明了一种新的翻译后机制，即由BACE1相互作用的分子GGA3(Golgi-Located？-EAR-Containing ARF Binding Protein 3)介导的BACE1的翻译后调节机制。我们已经确定，GGA3的缺失可以稳定BACE1并增加分泌酶的活性。我们还发现，死后AD大脑中GGA3的水平降低，并与BACE1水平呈负相关。我们已经证明BACE1是通过溶酶体途径降解的，并且GGA3调节BACE1向溶酶体的输送。BACE1-C末端片段(CTF)包含一个特定的二亮氨酸(DXXLL)分选信号，该信号已被证明与GGA家族三个成员GGA1、2和3的VHS结构域结合。我们发现，出人意料的是，GGA3 VHS结构域与BACE1二亮氨酸基序的直接结合不是必需的。相反，GGA3与泛素的相互作用是调节BACE1水平的关键。相应地，我们发现BACE1在赖氨酸501处主要是单链和K63连接的多泛素。这一建议的中心假设是，BACE1降解的损害是阿尔茨海默病患者大脑中BACE1升高的潜在机制。这项建议的首要目标是确定GGA和泛素介导的BACE1调节在多大程度上代表了AD治疗的潜在靶点。因此，我们建议具体解决以下目标：1)确定BACE1泛素化在多大程度上通过蛋白酶体或溶酶体途径调节BACE1的运输、活性和降解；2)在体内确定GGA3的过度表达在多大程度上以泛素依赖的方式降低BACE1和A2的水平；3)确定GGA家族的另一个成员GGA1在多大程度上独立地调节BACE1和A？的水平，并在体外和体内与GGA3联合调节。

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is the most common form of dementia that affects 5.3 million Americans. In a small percentage (>1%) of cases AD is inherited as an autosomal dominant trait (Familial AD), however the majority of cases are sporadic. A key neuropathological event in AD is the cerebral accumulation of A¿, a ~4kDa peptide derived by serial proteolysis of the amyloid precursor protein (APP) by ¿- and ?-secretase. Beta-site APP-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as ¿-secretase. Several studies have shown that BACE1 protein levels and ¿-secretase activity are increased in AD brains. Thus, BACE1 elevation may be the first step in increasing A¿ and triggering AD pathology, at least in the sporadic cases. Our studies have elucidated a novel post-translational mechanism of regulation of BACE1 mediated by the BACE1-interacting molecule, GGA3 (Golgi-localized ?-ear-containing ARF binding protein 3). We have determined that GGA3 depletion stabilizes BACE1 and increases ¿-secretase activity. We also found that levels of GGA3 are decreased in post-mortem AD brains and are inversely correlated with BACE1 levels. We have shown that BACE1 is degraded via the lysosomal pathway and demonstrated that GGA3 regulates the delivery of BACE1 to the lysosomes. The BACE1-C-terminal fragment (CTF) contains a specific di-leucine (DXXLL) sorting signal that has been shown to bind the VHS domain of the three members of the GGA family of proteins, GGA1, 2, and 3. We have found that, unexpectedly, direct binding of GGA3 VHS domain to the BACE1 di-leucine motif is not necessary for this regulation. Instead, GGA3 interaction with ubiquitin is essential for regulating BACE1 levels. Accordingly, we have found that BACE1 is mainly mono- and K63-linked polyubiquitinated at lysine 501. The central hypothesis of this proposal is that the impairment of BACE1 degradation is the underlying mechanism of BACE1 elevation in the brains of subjects affected by AD. The overarching goal of this proposal is to determine the extent to which GGA- and ubiquitin-mediated regulation of BACE1 represent a potential target for the treatment of AD. Thus, we propose to specifically address the following aims: 1) To determine the extent to which BACE1 ubiquitination regulates BACE1 trafficking, activity and degradation via the proteasomal or lysosomal pathway; 2) To determine the extent to which over-expression of GGA3 reduces levels of BACE1 and A2 in a ubiquitin-dependent fashion in vivo; 3) To determine the extent to which GGA1, another member of the GGA family of proteins, regulates levels of BACE1 and A¿ independently and in association with GGA3 in vitro and in vivo.