School of Life Science and Technology, Beijing Institute of Technology, Haidian District, Beijing 100081, China.
Curr Alzheimer Res. 2012 Jun;9(5):606-15. doi: 10.2174/156720512800618026.
Amyloid β protein (Aβ) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. Aβ is generated through proteolysis of β-amyloid precursor protein (APP) by two proteases: β-secretase and γ-secretase. BACE1, the β-secretase in vivo and the key rate-limiting enzyme that initiates the formation of Aβ, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at β site and Aβ production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential.
β淀粉样蛋白（Aβ）是阿尔茨海默病大脑中老年斑的主要成分，其聚集形式具有神经毒性。Aβ 通过β-淀粉样前体蛋白（APP）被两种蛋白酶：β-分泌酶和γ-分泌酶切割产生。BACE1 是体内的β-分泌酶，也是启动 Aβ 形成的关键限速酶，是治疗 AD 的有吸引力的药物靶点。我们之前的研究表明，BACE1 被泛素化，其降解和对 APP 切割过程的影响是由泛素蛋白酶体途径介导的。然而，具体的潜在机制仍未得到很好的定义。在本研究中，我们确定了负责 BACE1 蛋白体降解的特定结合位点。克隆了含有 1 到 3 个赖氨酸密码子的人 BACE1 cDNA 的 10 个片段，并用这些重组质粒转染 HEK293 细胞，并以特定的蛋白酶体抑制剂 lactacystin 处理。片段-3（Pro(149)-Leu(180))、-4（IIe(179)-Ser(230))和-8（Met(349)-Arg(400))的蛋白水平在 lactacystin 处理后显著增加，免疫细胞化学染色结果表明片段-3、-4 和-8 蛋白与泛素共定位。BACE1 的赖氨酸 203 和赖氨酸 382 点的定向突变消除了 BACE1 的蛋白体降解，并影响了 APP 在 β 位的加工和 Aβ 的产生。总之，我们的研究表明 BACE1 的赖氨酸 203 和赖氨酸 382 对其蛋白体降解至关重要，该结果可能有助于我们了解泛素蛋白酶体系统在 AD 发病机制中对 BACE1 和 APP 加工的调节，并为其药物潜力提供新的见解。
