Proteome-wide Identification of WRN-Interacting Proteins in Untreated and Nuclease-Treated Samples

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale proteome analysis to determine protein partners of WRN. We expressed fluorescent tagged-WRN (eYFP-WRN) in human 293 embryonic kidney cells and detected interacting proteins by co-immunoprecipitation from cell extract. We identified by mass spectrometry 220 nuclear proteins that complexed with WRN. This number was reduced to 40 when broad-spectrum nucleases were added to the lysate. We consider these 40 proteins as directly interacting with WRN. Some of these proteins have previously been shown to interact with WRN, whereas most are new partners. Among the top 15 hits, we find the new interactors TMPO, HNRNPU, RPS3, RALY, RPS9 DDX21, and HNRNPM. These proteins are likely important components in understanding the function of WRN in preventing premature aging and deserve further investigation. We have confirmed endogenous WRN interaction with endogenous RPS3, a ribosomal protein with endonuclease activities involved in oxidative DNA damage recognition. Our results suggest that the use of nucleases during cell lysis severely restricts interacting protein partners and thus enhances specificity.

沃纳综合征（Werner syndrome, WS）以多种年龄相关性疾病的过早发作为典型特征。沃纳综合征患者体内存在功能缺陷的蛋白（WRN）是一种解旋酶/核酸外切酶，参与DNA修复、复制、端粒维持及转录调控。本研究通过大规模蛋白质组分析，旨在鉴定WRN的蛋白互作伴侣：我们在人293胚胎肾细胞中表达了荧光标记的WRN（eYFP-WRN），并通过细胞提取物的免疫共沉淀实验检测互作蛋白；经质谱分析鉴定出220种与WRN形成复合物的核蛋白。当向细胞裂解液中加入广谱核酸酶后，该数量降至40，我们将这40种蛋白视为与WRN直接互作的分子。其中部分蛋白此前已被证实可与WRN互作，但绝大多数为全新的互作伴侣。在排名前15的命中蛋白中，我们发现了全新的互作分子：TMPO、HNRNPU、RPS3、RALY、RPS9、DDX21及HNRNPM。这些蛋白或为解析WRN预防过早衰老功能的关键组分，值得开展进一步研究。我们已验证内源WRN与内源RPS3的互作关系：RPS3是一种兼具核酸内切酶活性的核糖体蛋白，参与氧化性DNA损伤的识别。本研究结果表明，细胞裂解过程中加入核酸酶会大幅限制可检测到的互作蛋白范围，从而提升实验特异性。