Ubiquitin Regulates GGA3-mediated Degradation of BACE1
Ubiquitin Regulates GGA3-mediated Degradation of BACE1
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DOI：
10.1074/jbc.m109.092742
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Tesco, Giuseppina
中科院分区：
文献类型：
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作者：
Kang, Eugene L.;Cameron, Andrew N.;Tesco, Giuseppina
BACE1(β-部位淀粉样前体蛋白裂解酶1)是天冬氨酸蛋白酶的一种膜连接成员，对产生β-淀粉样蛋白是必不可少的，β-淀粉样蛋白是一种有毒的多肽，积聚在阿尔茨海默病患者的大脑中。BACE1C-末端片段包含一个DXXLL基序，该基序与GGA1-3(高尔基体定位的含有伽马耳的ARF结合蛋白)的VHS(VPS27，HRS和STAM)结构域结合。GAs是一种运输分子，参与含有DXXLL信号的蛋白质从高尔基复合体到内体的运输。此外，GGA还与泛素结合，并将合成的和内体泛素化的货物与溶酶体结合。我们之前已经证明，由于溶酶体降解受损，GGA3的缺失会导致BACE1水平和活性的增加。在此，我们报道了在缺乏GGA3的H4神经胶质瘤细胞中，GGA3的异位表达挽救了BACE1的积累。因此，GGA3的过度表达降低了BACE1和α-淀粉样蛋白的水平。然后，我们证实了GGA3 VPS27、HRS和STAM结构域(N91A)或BACE1二亮氨酸基序(L499A/L500A)的突变能够消除它们的结合，并不影响异位表达的GGA3挽救缺乏GGA3的细胞中BACE1积聚的能力。相反，我们发现BACE1在赖氨酸501位泛素化，主要是单素化和Lys-63连接的多泛素化。最后，一个与泛素结合能力降低的GGA3突变体(GGA3L276A)在救援和过度表达实验中都无法调节BACE1的水平。这些发现表明，GGA3水平通过与泛素分选机制的相互作用，紧密地、反向地调节BACE1水平。
BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and alpha-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.