Molecular Neuroscience Research Center, Shiga University of Medical Science, Shiga 520-2192, Japan.
Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Tokyo 187-8502, Japan.
J Biol Chem. 2010 May 14;285(20):14920-14931. doi: 10.1074/jbc.M109.038760. Epub 2010 Mar 18.
Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of gamma-secretase cleavage at the wing margins. gamma-Secretase is an intramembrane protease that is responsible for the generation of amyloid-beta (Abeta) peptides from the beta-amyloid precursor protein (APP). Here, we re-examined gamma-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Abeta and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased gamma-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of gamma-cleavage was also detected in cell-free assays. CRB2 interacted with the gamma-secretase complex, but was not a competitive substrate for gamma-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Abeta generation and mediated CRB2 binding with the gamma-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in gamma-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect gamma-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated gamma-secretase inhibition probably through prevention of the incorporation of CRB2 into the gamma-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the gamma-secretase complex.
果蝇 Crumbs 已被报道通过抑制翼缘处的γ-分泌酶切割来减弱 Notch 信号。γ-分泌酶是一种跨膜蛋白酶，负责从β-淀粉样前体蛋白 (APP) 生成淀粉样β (Abeta) 肽。在这里，我们重新研究了人 CRB2 对γ-分泌酶的抑制作用，CRB2 是人脑中最丰富的 Crumbs 同源物。转染的 CRB2 抑制了 HEK293 和 SH-SY5Y 细胞中 APP C 端片段的 APP 细胞内结构域的蛋白水解产生 Abeta 和 APP。相反，SH-SY5Y 细胞中内源性 CRB2 的敲低增加了 γ-分泌酶切割产物。在无细胞测定中也检测到 CRB2 对 γ-切割的抑制作用。CRB2 与 γ-分泌酶复合物相互作用，但不是 γ-切割的竞争性底物。CRB2 的跨膜结构域对于 Abeta 的产生抑制是必不可少的，并且介导了 CRB2 与 γ-分泌酶复合物的结合。此外，细胞质结构域似乎在 γ-分泌酶抑制中发挥支持作用，而参与细胞粘附复合物形成的两个蛋白结合基序的突变破坏则不影响 γ-分泌酶抑制。早老素-1 或 APH-1 的共过表达可能通过阻止 CRB2 掺入 γ-分泌酶复合物来消除 γ-分泌酶抑制。我们的结果表明，CRB2 作为一种抑制性结合蛋白发挥作用，参与成熟但无活性的 γ-分泌酶复合物的形成。
