Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells.
Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells.
复制标题
DOI：
10.1111/j.1582-4934.2008.00577.x
复制
发表时间：
2009-09
影响因子：
5.3
通讯作者：
Kaarniranta K
中科院分区：
文献类型：
--
作者：
Ryhänen T;Hyttinen JM;Kopitz J;Rilla K;Kuusisto E;Mannermaa E;Viiri J;Holmberg CI;Immonen I;Meri S;Parkkinen J;Eskelinen EL;Uusitalo H;Salminen A;Kaarniranta K
老年性黄斑变性的发病机制包括慢性氧化应激、光感受器外节脱落的膜盘降解受损以及视网膜色素上皮(RPE)细胞中溶酶体脂褐素的积聚。据估计，细胞中的大部分蛋白分解发生在蛋白酶体中，但蛋白酶体和包括自噬在内的其他蛋白分解途径在RPE细胞中的重要性尚不清楚。在蛋白质降解之前，热休克蛋白(HSPs)作为分子伴侣发挥作用，试图对错误折叠的蛋白质进行重折叠，从而防止细胞质蛋白质聚集体的积累。在本研究中，我们评估了Hsp70分子伴侣以及蛋白酶体和溶酶体蛋白分解途径在人RPE细胞(ARPE-19)中的作用。用Western blotting和免疫荧光法分析HSP70和泛素蛋白的表达和定位。用激光共聚焦显微镜和透射电子显微镜观察细胞器的形态变化。利用RNA干扰和过表达技术调节HSP70水平。比色法测定细胞存活率。蛋白酶体抑制剂MG-132可诱导HSP70、泛素-蛋白结合物和溶酶体膜蛋白LAMP-2阳性的核周聚集。有趣的是，HSP70信使核糖核酸的缺失显著增加了与蛋白酶体抑制相关的细胞死亡。我们发现，溶酶体的积累是可逆的：蛋白酶体抑制的停止导致沉淀物的清除，人们认为这一机制包括自噬。分子伴侣Hsp70、蛋白酶体和自噬在人RPE细胞的蛋白质周转中具有重要的调节作用，因此可能为理解视网膜细胞的退化过程开辟新的途径。
The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.