The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6–SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1–GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student’s t-test, or one-way or two-way analysis of variance (ANOVA). We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

中文翻译：

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SUMOylation 调节酵母细胞和胰腺导管腺癌细胞中的 eIF5A 活性

真核翻译起始蛋白eIF5A是一种高度保守的必需因子，在包括应激反应和癌症在内的不同生理和病理过程中发挥着关键作用。不同的蛋白质组学研究表明，eIF5A 可能是一种小型泛素样修饰剂 (SUMO) 底物，但 eIF5A 是否确实被 SUMO 化以及这种修饰与 eIF5A 活性的相关性如何仍不清楚。使用体外 SUMO 化测定、从 His6-SUMO2 转染细胞中纯化组氨酸标记蛋白以及使用 SUMO 结合实体 (SUBES) 分离内源 SUMO 化蛋白来评估 SUMO 化。突变体是通过定点诱变设计的。通过放线菌酮追踪测定来测量蛋白质稳定性。使用免疫荧光和细胞分级分析测定蛋白质定位。分析了 eIF5A1 构建体补充带有酵母 EIF5A 同源基因 (HYP2) 热敏突变体的酿酒酵母菌株生长的能力。在经受热休克的酵母细胞中，通过免疫荧光法测定了表达 Pab1-GFP（应激颗粒标记）的细胞中的多核糖体谱和应激颗粒的形成。分别使用结晶紫染色和 Transwell 插入评估过表达不同 eIF5A1 构建体的胰腺导管腺癌 PANC-1 细胞的细胞生长和迁移。使用 GraphPad 软件，使用未配对的学生 t 检验或单向或双向方差分析 (ANOVA) 进行统计分析。我们发现 eIF5A 在体外、转染细胞和内源条件下被 SUMO2 修饰，揭示了其生理相关性。我们鉴定了 eIF5A 中的几个 SUMO 位点，并发现 SUMO 化可调节哺乳动物细胞中 eIF5A 的稳定性和定位。有趣的是，eIF5A 的 SUMO 化对特定的应激有反应，表明它是一个受调控的过程。eIF5A的SUMO化在酵母中是保守的，eIF5A SUMO化突变体不能完全抑制HYP2突变体的缺陷，并且eIF5A的SUMO化对于热激诱导的应激颗粒的形成和多核糖体的解体都很重要。此外，eIF5A 中 SUMO 化位点的突变消除了其在 PANC-1 细胞中的前迁移和增殖活性。SUMO2 与 eIF5A 的结合是一种应激诱导的反应，涉及酵母细胞对热休克应激的适应，并且是促进胰腺导管腺癌细胞的生长和迁移所必需的。