Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells, and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau
Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells, and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau
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嘌呤霉素敏感氨肽酶参与培养细胞中 tau 蛋白的蛋白水解，以及与 17 号染色体 (FTDP-17) 突变 tau 相关的额颞叶痴呆和帕金森病的蛋白水解减弱
作者：
Kentarou Yanagi;Toshihisa Tanaka;Kiyoko Kato;G. Sadik;T. Morihara;T. Kudo;M. Takeda
在tau病中，tau蛋白被过度磷酸化、泛素化并在大脑中积累；然而，这种积累背后的机制仍不清楚。为了了解蛋白酶在tau蛋白代谢中的作用，在本研究中，我们评估了蛋白酶抑制剂在SH‐SY5Y人神经母细胞瘤细胞和转染tau基因的COS‐7细胞中的作用。当细胞用0.1-10µmol/L的乳酸蛋白酶和1.0-20µmol/L的MG‐132（蛋白酶体抑制剂）、0.1-10µmol/L的CA‐074Me（组织蛋白酶抑制剂）和0.1-2µmol/L的嘌呤霉素（嘌呤霉素敏感的氨基肽酶（PSA）抑制剂）处理24小时时，tau蛋白水平没有显著变化。然而，脉冲追踪实验表明，200 nmol/L嘌呤霉素处理后，SH - SY5Y细胞中tau蛋白的蛋白水解减弱。在SH - SY5Y细胞中，用短干扰（si） RNA对PSA进行处理以抑制PSA的表达，也观察到tau蛋白水平升高。这些数据表明PSA是一种在SH‐SY5Y细胞中主要催化tau蛋白的蛋白酶。我们还利用脉冲追踪实验研究了与17号染色体相关的额颞叶痴呆和帕金森病（FTDP - 17）含tau突变的蛋白质代谢。结果表明，转染突变tau基因的细胞在48小时后tau蛋白水解减弱。进一步的免疫细胞化学分析和亚细胞分离实验显示，突变并没有改变tau的细胞内分布，这表明tau对PSA的可及性受损不太可能是tau蛋白水解减少的原因。磷酸化依赖抗体的Western blotting结果显示，转染V337M、R406W和R406W突变tau基因的细胞中，tau蛋白Thr231、Ser396和Ser409位点的磷酸化水平分别升高。综上所述，这些数据表明FTDP‐17突变体tau蛋白水解的减弱可能是由于磷酸化水平的增加，从而导致对蛋白水解的抵抗。
In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH‐SY5Y human neuroblastoma cells and COS‐7 cells transfected with the tau gene. When cells were treated with 0.1–10 µmol/L of lactacystin and 1.0–20 µmol/L of MG‐132 (inhibitors of proteasome), 0.1–10 µmol/L of CA‐074Me (a cathepsin inhibitor), and 0.1–2 µmol/L of puromycin (a puromycin‐sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse‐chase experiments demonstrated that the proteolysis of tau protein in SH‐SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH‐SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH‐SY5Y cells. The protein metabolism of tau‐containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) was also investigated using pulse‐chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation‐dependent antibodies revealed that phosphorylation levels of tau at Thr231, Ser396, and Ser409 were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP‐17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.
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DOI：
10.1111/j.1432-1033.1995.009_1.x
发表时间：
1995-10-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
YANG, LS;KSIEZAKREDING, H
通讯作者：
KSIEZAKREDING, H
DOI：
10.1016/s0021-9258(17)42989-9
发表时间：
1984-04
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
G. Lindwall;R. Cole
通讯作者：
G. Lindwall;R. Cole
影响因子：
2.9
作者：
Sengupta, Soma;Horowitz, Peleg M.;Binder, Lester I.
通讯作者：
Binder, Lester I.
DOI：
10.1073/pnas.83.13.4913
发表时间：
1986-07-01
影响因子：
11.1
作者：
GRUNDKEIQBAL, I;IQBAL, K;BINDER, LI
通讯作者：
BINDER, LI
DOI：
10.1073/pnas.91.12.5562
发表时间：
1994-06-07
影响因子：
11.1
作者：
ALONSO, AD;ZAIDI, T;IQBAL, K
通讯作者：
IQBAL, K