Zhang Xinyu, Zhou Jilin, Fernandes Alexandre F, Sparrow Janet R, Pereira Paulo, Taylor Allen, Shang Fu
United States Department of Agriculture, Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA.
Invest Ophthalmol Vis Sci. 2008 Aug;49(8):3622-30. doi: 10.1167/iovs.07-1559. Epub 2008 Apr 11.
Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study was to investigate the effect of oxidative stress on the UPP in cultured human retina pigment epithelial cells.
To mimic physiological oxidative stress, ARPE-19 cells were exposed to continuously generated H2O(2) or A2E-mediated photooxidation. Proteasome activity was monitored using fluorogenic peptides as substrates. The ubiquitin conjugation activity and activities of E1 and E2 were determined by the thiolester assays. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting.
Exposure of ARPE-19 cells to 40 to 50 microM H2O(2) for 4 hours resulted in a 30% to 50% reduction in all three peptidase activities of the proteasome. Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40% to 60% reduction in proteasome activity. Loading of A2E or exposure to blue light alone had little effect on proteasome activity. In contrast, exposure of ARPE-19 to low levels of H2O(2) (10 microM) stimulated ubiquitin conjugation activity. Loading of A2E, with or without exposure to blue light, upregulated the levels of ubiquitin-activating enzyme and increased conjugation activity. Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold to threefold increase in levels of endogenous ubiquitin conjugates.
These data show that the proteasome in ARPE-19 is susceptible to oxidative inactivation, whereas activities of the ubiquitin-conjugating enzymes are more resistant to oxidative stress. Oxidative inactivation of the proteasome appears to be one of the mechanisms underlying stress-induced accumulation of ubiquitin conjugates in the cells.
泛素 - 蛋白酶体途径（UPP）功能障碍与多种年龄相关性退行性疾病相关。本研究的目的是探讨氧化应激对培养的人视网膜色素上皮细胞中UPP的影响。
为模拟生理性氧化应激，将ARPE - 19细胞暴露于持续产生的H2O₂或A2E介导的光氧化作用下。使用荧光肽作为底物监测蛋白酶体活性。通过硫酯测定法测定泛素缀合活性以及E1和E2的活性。通过蛋白质印迹法测定泛素和泛素缀合物的水平。
将ARPE - 19细胞暴露于40至50μM H2O₂ 4小时导致蛋白酶体的所有三种肽酶活性降低30％至50％。同样，将加载A2E的ARPE - 19细胞暴露于蓝光下导致蛋白酶体活性降低40％至60％。单独加载A2E或暴露于蓝光对蛋白酶体活性影响很小。相反，将ARPE - 19暴露于低水平的H2O₂（10μM）刺激泛素缀合活性。加载A2E，无论是否暴露于蓝光，都会上调泛素激活酶的水平并增加缀合活性。暴露于H2O₂或A2E介导的光氧化作用还导致内源性泛素缀合物水平增加两倍至三倍。
这些数据表明，ARPE - 19中的蛋白酶体易受氧化失活影响，而泛素缀合酶的活性对氧化应激更具抗性。蛋白酶体的氧化失活似乎是应激诱导细胞中泛素缀合物积累的潜在机制之一。
