Dunys Julie, Kawarai Toshitaka, Wilk Sherwin, St George-Hyslop Peter, Alves da Costa Cristine, Checler Frédéric
Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France.
Biochem J. 2006 Mar 1;394(Pt 2):501-9. doi: 10.1042/BJ20051197.
PS (presenilin)-dependent gamma-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the gamma-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.
依赖早老素（PS）的γ-分泌酶以高分子量复合物的形式存在，该复合物由与Nct（尼卡斯特林）、PEN2（早老素增强子2同源物）和APH1（前咽缺陷1同源物）相关的PS1或PS2组成。大量报告记录了这些蛋白质之间非常复杂的物理和功能相互作用，这些相互作用最终决定了γ-分泌酶的生物学活性，但很少有研究考察该复合物各组分的命运。我们发现，在HEK-293细胞和TSM1神经元细胞系中，蛋白酶体抑制剂ZIE和乳胞素增强了过表达的带有myc标签的APH1a和PEN2的免疫反应性，而多种蛋白酶抑制剂则没有影响。相比之下，蛋白酶体抑制剂完全无法影响HEK-293细胞、TSM1细胞和原代培养的皮质神经元中内源性APH1aL和PEN2的细胞表达。为了解释这一明显的差异，我们在体外通过含有内源性蛋白酶体的细胞提取物和纯化的20S蛋白酶体检测了带有myc标签的APH1a和PEN2的降解情况。令人惊讶的是，带有myc标签的APH1a和PEN2对内源性蛋白酶体和纯化的20S蛋白酶体的蛋白水解具有抗性。我们还发现，野生型成纤维细胞中内源性PEN2的表达明显高于PS和Nct缺陷的成纤维细胞，并且蛋白酶体抑制剂仅在这两种缺陷细胞系统中增强其表达。然而，同样地，纯化的20S蛋白酶体似乎无法切割PS缺陷成纤维细胞中存在的内源性PEN2。内源性APH1aL样免疫反应性的水平不受蛋白酶体抑制剂的影响，也不受PS缺陷的影响。总之，我们的结果表明，在HEK-293细胞、TSM1细胞和成纤维细胞的生理条件下，内源性PEN2和APH1aL不会经历蛋白酶体降解，并且在PS和Nct缺陷的成纤维细胞中PEN2的清除不是由20S蛋白酶体介导的。26S蛋白酶体是否参与缺陷成纤维细胞中PEN2的蛋白水解还有待确定。
