School of Biological Sciences, National Institute of Science Education and Research (NISER) Bhubaneswar, HBNI, P.O. Bhimpur-Padanpur, Jatni, Khurda, Odisha, 752050, India.
Sri Sri Borda Hospital, Dhauli, Bhubaneswar, Odisha, 751002, India.
Exp Eye Res. 2019 Apr;181:197-207. doi: 10.1016/j.exer.2019.02.004. Epub 2019 Feb 7.
Pseudoexfoliation (PEX), an ocular disorder involving deposition of proteinaceous fibrils on the surface of anterior eye tissues, is a major contributing factor to worldwide glaucoma. Excessive production and accumulation of fibrillar materials in PEX could be an indication of proteostasis imbalance. This study aims at investigating the differential expression of various genes involved in unfolded protein response and ubiquitin proteasome pathway in pseudoexfoliation (PEX) patients compared to non-PEX controls using lens capsule tissue as the study material. The custom RT Profiler PCR array was used to identify a set of stress-related candidate genes that were differentially expressed in PEX. The expression of the highly deregulated genes was validated by qRT-PCR and subsequently their protein expression was checked through immunoblotting and immunostaining. Proteasome-Glo based assay and TUNEL assay were employed to detect specific proteasomal activity and apoptotic activity, respectively in the study subjects. Increased ER stress markers, Synoviolin1, Eukaryotic initiation factor 2-alpha kinase 3, DnaJ (Hsp40) homolog, subfamily B, member 11, Caspase 12, Heat shock 70 kDa protein 5, Heat shock 60 kDa protein 1 and Calnexin were observed in the lens capsule of PEX individuals compared to age-matched controls. On the other hand, increased ubiquitin B mRNA expression followed by significant downregulation of proteasome subunits; 26 S proteasome non-ATPase regulatory subunit 1, and proteasome subunit alpha-type 5 was found in pseudoexfoliation syndrome (PEXS) individuals. Decrease in chymotrypsin-like proteasome activity and increased apoptosis were also observed in PEX subjects. The present findings provide evidence for alterations in endoplasmic reticulum-related stress response and ubiquitin proteasome function in lens capsule of PEX individuals. Altogether, our study has identified deregulated expression of candidate genes in ER-UPR pathway and implicates proteasome impairment as a causative factor in PEX pathogenesis.
（PEX）是一种眼部疾病，涉及前眼部组织表面蛋白纤维的沉积，是全球青光眼的主要致病因素。PEX 中纤维状物质的过度产生和积累可能表明蛋白质平衡失调。本研究旨在使用晶状体囊组织作为研究材料，研究与非 PEX 对照相比，涉及未折叠蛋白反应和泛素蛋白酶体途径的各种基因在 PEX 患者中的差异表达。使用定制的 RT Profiler PCR 阵列来鉴定一组在 PEX 中差异表达的应激相关候选基因。通过 qRT-PCR 验证高度失调基因的表达，随后通过免疫印迹和免疫染色检查其蛋白表达。通过蛋白酶体-Glo 测定和 TUNEL 测定分别检测研究对象中的特定蛋白酶体活性和细胞凋亡活性。与年龄匹配的对照相比，在 PEX 个体的晶状体囊中观察到内质网应激标志物 Synoviolin1、真核起始因子 2-α激酶 3、DnaJ（Hsp40）同源物亚家族 B、成员 11、Caspase 12、热休克 70kDa 蛋白 5、热休克 60kDa 蛋白 1 和钙网蛋白的表达增加。另一方面，在 PEXS 个体中发现泛素 B mRNA 表达增加，随后蛋白酶体亚基 26S 蛋白酶体非 ATP 酶调节亚基 1 和蛋白酶体亚基 α 型 5 显著下调。在 PEX 个体中还观察到糜蛋白酶样蛋白酶体活性降低和细胞凋亡增加。本研究结果为 PEX 个体晶状体囊中内质网相关应激反应和泛素蛋白酶体功能改变提供了证据。总之，我们的研究鉴定了 ER-UPR 途径中候选基因的失调表达，并暗示蛋白酶体损伤是 PEX 发病机制的一个致病因素。
