Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action
Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action
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DOI：
10.1016/j.exer.2009.10.014
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发表时间：
2010-02-01
影响因子：
3.4
通讯作者：
Kopitz, Juergen
中科院分区：
文献类型：
--
作者：
Krohne, Tim U.;Kaemmerer, Elke;Kopitz, Juergen
在年龄相关性黄斑变性（AMD）中，溶酶体容量降低可能导致脂褐素生成和视网膜色素上皮（RPE）的进行性功能障碍。我们以前证明，脂质过氧化相关的蛋白质修饰抑制溶酶体降解的感光细胞外节（POS）蛋白在RPE细胞。在此，我们研究脂质过氧化产物对关键RPE溶酶体蛋白酶活性的影响。在从原代人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产生剂量依赖性抑制作用，生物学相关浓度的IAM导致酶活性降低88- 94%。这种效应在培养的RPE细胞中得到证实。使用质谱，共价HNE和MDA加合物中检测到的活性中心区域的失活的组织蛋白酶。同样地，用HNE和MDA修饰的POS也引起了分离的溶酶体中组织蛋白酶B和L活性的剂量依赖性降低，此外，还抑制了天冬氨酸蛋白酶组织蛋白酶D。我们的研究结果表明，脂质过氧化产物在体外通过两种不同的作用机制干扰RPE溶酶体蛋白酶的活性：（i）HNE和MDA通过共价结合到活性中心直接抑制溶酶体半胱氨酸蛋白酶;（ii）HNE和MDA介导的蛋白质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，脂质过氧化产物可诱导老化RPE中的溶酶体功能障碍和脂褐素生成，从而有助于AMD的发病机制。(C)2009爱思唯尔有限公司保留所有权利。
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 I AM 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. (C) 2009 Elsevier Ltd. All rights reserved.