Department of Ophthalmology, University of Bonn, Bonn, Germany.
Exp Eye Res. 2010 Feb;90(2):261-6. doi: 10.1016/j.exer.2009.10.014. Epub 2009 Nov 4.
In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 muM resulting in a reduction of enzyme activities by 88-94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.
在年龄相关性黄斑变性（AMD）中，溶酶体容量减少可能导致脂褐素的生成和视网膜色素上皮（RPE）的进行性功能障碍。我们之前已经证明，脂质过氧化相关蛋白修饰会抑制 RPE 细胞中光感受器外节（POS）蛋白的溶酶体降解。在此，我们研究了脂质过氧化产物对关键 RPE 溶酶体蛋白酶活性的影响。在原代人 RPE 细胞分离的溶酶体中，脂质过氧化产物 4-羟基壬烯醛（HNE）和丙二醛（MDA）对半胱氨酸蛋白酶组织蛋白酶 B 和 L 表现出剂量依赖性抑制作用，生物相关浓度 1 μM 导致酶活性降低 88-94%。这一效应在培养的 RPE 细胞中得到了证实。使用质谱法，在失活组织蛋白酶的活性中心区域检测到共价 HNE 和 MDA 加合物。以前用 HNE 和 MDA 修饰的 POS 同样导致分离的溶酶体中组织蛋白酶 B 和 L 的活性呈剂量依赖性降低，此外还抑制了天冬氨酸蛋白酶组织蛋白酶 D。我们的结果表明，脂质过氧化产物在体外通过两种不同的作用机制干扰 RPE 溶酶体蛋白酶活性：（i）HNE 和 MDA 通过与活性中心的共价结合直接使溶酶体半胱氨酸蛋白酶失活；（ii）HNE 和 MDA 介导的蛋白修饰将蛋白水解底物转化为溶酶体蛋白酶的竞争性抑制剂。通过这些机制，脂质过氧化产物可能在衰老的 RPE 中诱导溶酶体功能障碍和脂褐素的生成，从而促进 AMD 的发病机制。
