Clarification of the mechanism underlying y-secretase cleavage by presenilin complexes

早老素是家族性阿尔茨海默病（FAD）的致病分子，是γ-分泌酶活性所必需的。γ-分泌酶介导淀粉样前体蛋白（APP）的膜内切割，导致称为淀粉样β（Aβ）的致病肽的产生。由于Aβ_（42/3）在AD的发病机制中起着重要作用，因此阐明这一机制一直是AD研究的热点。我们已经揭示，早老素通过与nicastrin和其它蛋白质一起形成大分子复合物来介导APP和Notch的γ-分泌酶切割（Nat Cell Biol，2001），并且另一种组分PEN-2增强γ-分泌酶底物的可及性（J Neurochem，2004）。早老素中引起FAD的突变通过改变γ-分泌酶活性增加了较长Aβ_<42/43>的产生，但其潜在机制尚不清楚。利用早老素1（PS1）的随机突变筛选，我们鉴定了包括R278 I-PS1和L435 H-PS1在内的五种独特突变体，其仅产生高水平的Aβ_，<43>但不支持生理性PS1内蛋白水解或Aβ_<40>产生（J Biol Chem，2005）。细胞生物学研究和药理学研究表明，PS1突变和γ-分泌酶抑制剂对特异性的影响通常是通过复合物的构象变化介导的。我们的研究结果表明，小分子可以被设计成诱导交替的PS复合物构象，这更有利于Aβ_<40>而不是致病性Aβ_&lt;42/43&gt;的产生。此外，我们报道早老素通过结合FKBP 38并减少线粒体中的抗凋亡Bcl-2而发挥促凋亡作用。此外，早老素的FAD突变增强了这种促凋亡活性，使神经元对脑中的凋亡刺激物敏感（AgNOMol Genet，出版中）。

Presenilins, causative molecules for familial Alzheimer's disease (FAD), are required for y-secretase activity. y- Secretase mediates intramembrane cleavage of amyloid precursor protein (APP), resulting in generation of pathogenic peptides named amyloid β(Aβ). The clarification of this mechanism is a central issue in AD research, because the longer Aβ ending at residue 42 or 43 (Aβ_<42/3>) are thought to play a critical role in the pathogenesis of AD. We have revealed that presenilins mediate y-secretase cleavage of APP and Notch by forming macromolecular complexes together with nicastrin and other proteins (Nat Cell Biol, 2001), and that another component PEN-2 enhances the accessibility of the y-secretase substrates (J Neurochem, 2004). FAD- causing mutations in the presenilins increase production of longer Aβ_<42/43> by altering y-secretase activity, although the underlying mechanism remains unknown. Using a random mutagenesis screen of presenilin 1 (PS1), we identified five unique mutants including R278I-PS1 and L435H-PS1, which exclusively generated a high level of Aβ_<43> but that did not support physiological PS1 endoproteolysis or Aβ_<40> generation (J Biol Chem, 2005). Cell biological investigation and pharmacological study using the inhibitors suggest that the effects of PS1 mutations and y-secretase inhibitors on the specificity are commonly mediated through a conformational change in the complex. Our results suggest that small molecules could be designed to induce alternate PS complex conformations that are more favourable to the generation of Aβ_<40> rather than pathogenic Aβ_<42/43>. Additionally, we reported that presenilins exert a pro-apoptotic effect by binding to FKBP38 and reducing anti- apoptotic Bcl-2 in the mitochondria. Furthermore, FAD mutations of presenilins enhance this pro-apoptotic activity, sensitizing neurons to apoptotic stimuli in brains (Hum Mol Genet, in press).