da Costa C A, Ancolio K, Checler F
Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, Valbonne, France.
Mol Med. 1999 Mar;5(3):160-8.
Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS1 and PS2). Several lines of evidence indicate that PS1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome.
We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses.
We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated that two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of APP alpha and A beta elicited by CTF-PS1/PS2.
Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP maturation upstream to alpha-and beta/gamma-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular concentration of CTFs.
大多数早发性阿尔茨海默病是由位于两种同源蛋白早老素1和2（PS1和PS2）上的错义突变引起的。多项证据表明，PS1和PS2会经历各种转录后事件，包括内切蛋白水解切割，产生在体内积累的28 - 30kD的N端（NTF）和18 - 20kD的C端（CTF）片段。早老素的生物活性是由加工后的片段还是其全蛋白前体承担仍存在疑问。我们研究了CTF - PS1/PS2对β-淀粉样前体蛋白（β-APP）成熟的假定调控以及多催化复合物蛋白酶体对后两者蛋白的分解代谢过程。
我们用CTF - PS1或CTF - PS2 cDNA瞬时和稳定转染人胚肾293（HEK293）细胞。我们使用特异性多克隆抗体通过免疫沉淀法检测这些转染细胞中β淀粉样蛋白40（Aβ40）、β淀粉样蛋白42（Aβ42）和α-淀粉样前体蛋白（APPα）的产生。通过蛋白质印迹法检测一系列蛋白酶抑制剂对CTF - PS1/PS2免疫反应性的影响。最后，通过联合免疫沉淀和光密度分析评估蛋白酶体抑制剂对表达CTF的细胞产生β-APP片段的影响。
我们表明，在人细胞中瞬时和稳定转染CTF - PS1和CTF - PS2 cDNA会导致β-APP的成熟产物APPα和Aβ的分泌增加。此外，我们证明两种蛋白酶体抑制剂，乳胞素和Z - IE（Ot - Bu）A - 亮氨醛，可防止两种CTF的降解。因此，我们确定蛋白酶体抑制剂极大地增强了CTF - PS1/PS2引起的APPα和Aβ表型增加的产生。
我们的数据表明，PS1和PS2成熟的C端产物具有生物活性，特别是在α-和β/γ-分泌酶切割上游调控β-APP的成熟。该功能直接由调节CTF细胞内浓度的蛋白酶体控制。
