Role of BACE stabilization in Alzheimer's disease

描述（由申请人提供）：阿尔茨海默病（AD）是最常见的痴呆症，影响着530万美国人。在一小部分（约1%）病例中，AD是作为常染色体显性遗传的（家族性AD），但大多数病例是散发的。AD的一个关键神经病理事件是A¿的大脑积累，A¿是一种约4kDa的肽，由淀粉样前体蛋白（APP）被分泌酶和分泌酶连续蛋白水解而产生。β位点app切割酶（BACE1）是天冬氨酸蛋白酶的膜系成员，已被确定为分泌酶。几项研究表明，在AD大脑中，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升高可能是增加A¿和触发AD病理的第一步。我们的研究已经阐明了一种由BACE1相互作用分子GGA3(高尔基定位？-含耳ARF结合蛋白3)。我们已经确定GGA3的缺失稳定了BACE1并增加了分泌酶的活性。我们还发现，死后AD患者大脑中GGA3水平下降，与BACE1水平呈负相关。我们已经证明BACE1通过溶酶体途径被降解，并证明GGA3调节BACE1向溶酶体的传递。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家族蛋白GGA1、gg2和gg3的三个成员的VHS结构域结合。我们意外地发现，GGA3 VHS结构域与BACE1二亮氨酸基序的直接结合并不是这种调控所必需的。相反，GGA3与泛素的相互作用对于调节BACE1水平至关重要。因此，我们发现BACE1主要是单链和k63连接的赖氨酸501多泛素化。该研究的核心假设是，BACE1降解受损是AD患者大脑中BACE1升高的潜在机制。该提案的总体目标是确定GGA和泛素介导的BACE1调控在多大程度上代表了治疗AD的潜在靶标。因此，我们建议具体解决以下目标：1)确定BACE1泛素化通过蛋白酶体或溶酶体途径调节BACE1运输、活性和降解的程度；2)确定GGA3过表达在体内以泛素依赖的方式降低BACE1和A2水平的程度；3)确定GGA蛋白家族的另一成员GGA1在体外和体内独立调控BACE1和A¿水平以及与GGA3联合调控的程度。

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.