TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene

The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.

泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）在蛋白质降解中的核心作用，在神经退行性疾病（含视网膜营养不良）的研究背景中已得到充分重视。TOPORS是一种兼具E3泛素连接酶与SUMO1连接酶活性的双重连接酶，属于UPS的组成组分。其酶促活性的部分底物，如DJ-1/PARK7与APOBEC2，分别对神经元稳态与视网膜稳态具有重要意义。TOPORS呈广谱表达，但目前已知其突变仅会引发常染色体显性遗传性视网膜色素变性。本研究通过对人类视网膜cDNA文库进行酵母双杂交（yeast two-hybrid, Y2H）筛选，旨在鉴定视网膜中TOPORS的相互作用蛋白伴侣，进而初步阐明TOPORS突变引发的视网膜特异性表型相关的潜在致病机制。此次筛选成功分离得到26S蛋白酶体调节亚基4（26 S protease regulatory subunit 4, P26s4/PSMC1）——一种对UPS介导的蛋白质稳态正常发挥功能不可或缺的ATP酶。通过对哺乳动物细胞提取物进行免疫共沉淀实验，验证了内源性TOPORS与P26s4蛋白之间的相互作用；并通过在细胞系与视网膜组织切片中开展免疫荧光共定位研究，对该相互作用进行了进一步表征。对hTERT-RPE1与661W细胞的实验结果显示，在培养细胞中TOPORS与P26s4共定位于中心体。对小鼠视网膜进行免疫荧光染色后发现，在视网膜色素上皮（retinal pigmented epithelium, RPE）层与感光细胞外节（photoreceptors outer segments, OS）的交界处，P26s4呈现强阳性反应。这一发现提示，除了通过UPS参与感光细胞与视网膜整体的蛋白质稳态调控之外，P26s4与TOPORS可能还在RPE的吞噬过程中发挥一定作用。