Grune Tilman, Merker Katrin, Jung Tobias, Sitte Nicolle, Davies Kelvin J A
Neuroscience Research Center, Medical Faculty (Charité), Humboldt University Berlin, D-10098 Berlin, Germany.
Free Radic Biol Med. 2005 Nov 1;39(9):1208-15. doi: 10.1016/j.freeradbiomed.2005.06.009.
Oxidized and cross-linked proteinacious materials (lipofuscin, age pigments, ceroid, etc.) have long been known to accumulate in aging and in age-related diseases, and some studies have suggested that age-dependent inhibition of the proteasome and/or lysosomal proteases may contribute to this phenomenon. Cell culture studies trying to model these aging effects have almost all been performed with proliferating (divisionally competent) cell lines. There is little information on nondividing (postmitotic) cells; yet age-related accumulation of oxidized and cross-linked protein aggregates is most marked in postmitotic tissues such as brain, heart, and skeletal muscles. The present investigation was undertaken to test whether oxidized and cross-linked proteins generally accumulate in nondividing, IMR-90 and MRC-5, human cell lines, and whether such accumulation is associated with diminished proteolytic capacities. Since both protein oxidation and declining proteolytic activities might play major roles in the age-associated accumulation of intracellular oxidized materials, we tested for protein carbonyl formation, proteasomal activities, and lysosomal cathepsin activities. For these studies, confluent, postmitotic IMR-90 and MRC-5 fibroblasts (at various population doubling levels) were cultured under hyperoxic conditions to facilitate age-related oxidative senescence. Our results reveal marked decreases in the activity of both the proteasomal system and the lysosomal proteases during senescence of nondividing fibroblasts, but the peptidyl-glutamyl-hydrolyzing activity of the proteasome was particularly inhibited. This decline in proteolytic capacity was accompanied by an increased accumulation of oxidized proteins.
氧化和交联的蛋白质材料（脂褐素、老年色素、蜡样质等）长期以来一直被认为会在衰老过程以及与年龄相关的疾病中积累，并且一些研究表明，蛋白酶体和/或溶酶体蛋白酶的年龄依赖性抑制可能导致了这一现象。试图模拟这些衰老效应的细胞培养研究几乎都是使用增殖性（具有分裂能力）细胞系进行的。关于非分裂（有丝分裂后）细胞的信息很少；然而，氧化和交联蛋白质聚集体的年龄相关积累在大脑、心脏和骨骼肌等有丝分裂后组织中最为明显。本研究旨在测试氧化和交联蛋白质是否普遍在非分裂的人IMR-90和MRC-5细胞系中积累，以及这种积累是否与蛋白水解能力的降低有关。由于蛋白质氧化和蛋白水解活性下降可能在细胞内氧化物质的年龄相关积累中起主要作用，我们检测了蛋白质羰基的形成、蛋白酶体活性和溶酶体组织蛋白酶活性。对于这些研究，将汇合的、有丝分裂后的IMR-90和MRC-5成纤维细胞（处于不同的群体倍增水平）在高氧条件下培养，以促进与年龄相关的氧化衰老。我们的结果显示，在非分裂成纤维细胞衰老过程中，蛋白酶体系统和溶酶体蛋白酶的活性均显著降低，但蛋白酶体的肽基-谷氨酰水解活性受到的抑制尤为明显。蛋白水解能力的这种下降伴随着氧化蛋白质积累的增加。
