Canu N, Barbato C, Ciotti M T, Serafino A, Dus L, Calissano P
Dipartimento di Neuroscienze, Facoltà di Medicina e Chirurgia, Università di Tor Vergata, 00133 Roma, Italia.
J Neurosci. 2000 Jan 15;20(2):589-99. doi: 10.1523/JNEUROSCI.20-02-00589.2000.
We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pan-caspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases.
我们研究了泛素蛋白酶体系统在细胞外钾离子浓度降低所诱导的小脑颗粒神经元死亡过程中的潜在作用。蛋白酶体功能抑制剂在这一过程开始时给药可阻断细胞凋亡，但在2-3小时后添加则无此作用。同样的抑制剂还可阻止半胱天冬酶-3的活性以及钙蛋白酶-半胱天冬酶-3介导的tau蛋白加工过程，这表明蛋白酶体参与了半胱天冬酶激活的上游过程。尽管蛋白酶体似乎在程序性细胞死亡中起早期主要作用，但我们发现，随着凋亡的进展，在执行阶段，正常的泛素-蛋白酶体功能会出现紊乱，并且高水平的泛素化蛋白会在死亡细胞的细胞质中积累。这种积累与蛋白酶体胰凝乳蛋白酶和类胰蛋白酶活性的逐渐下降相关，在较低程度上也与类后酸性活性的下降相关。泛素化蛋白的胞质内积累和蛋白酶体功能的下降均可被泛半胱天冬酶抑制剂Z-VAD-fmk逆转。蛋白酶体功能的下降伴随着去泛素化活性的显著且逐渐下降，并且可能归因于此。蛋白酶体早期参与凋亡以及在此过程中泛素化蛋白积累这一发现，使颗粒神经元成为一个旨在将这些事件与神经退行性疾病相关联的模型系统。
