Seoane Rocío, Lama-Díaz Tomás, Romero Antonia María, El Motiam Ahmed, Martínez-Férriz Arantxa, Vidal Santiago, Bouzaher Yanis H, Blanquer María, Tolosa Rocío M, Castillo Mewa Juan, Rodríguez Manuel S, García-Sastre Adolfo, Xirodimas Dimitris, Sutherland James D, Barrio Rosa, Alepuz Paula, Blanco Miguel G, Farràs Rosa, Rivas Carmen
Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Cell Mol Biol Lett. 2024 Jan 16;29(1):15. doi: 10.1186/s11658-024-00533-5.
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.
真核生物翻译起始蛋白eIF5A是一种高度保守且必不可少的因子，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关键作用。不同的蛋白质组学研究表明，eIF5A可能是一种小泛素样修饰物（SUMO）底物，但eIF5A是否真的被SUMO化以及这种修饰与eIF5A活性的相关性仍不清楚。
使用体外SUMO化测定、从His6-SUMO2转染细胞中纯化组氨酸标签蛋白以及使用SUMO结合实体（SUBES）分离内源性SUMO化蛋白来评估SUMO化。通过定点诱变构建突变体。通过环己酰亚胺追踪测定法测量蛋白质稳定性。使用免疫荧光和细胞分级分离测定法确定蛋白质定位。分析了eIF5A1构建体对携带酵母EIF5A同源基因（HYP2）热敏突变体的酿酒酵母菌株生长的互补能力。通过免疫荧光测定了在热休克的酵母细胞中表达Pab1-GFP（一种应激颗粒标记物）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小室评估过表达不同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软件进行统计分析，采用不成对学生t检验或单因素或双因素方差分析（ANOVA）。
我们发现eIF5A在体外、转染细胞中和内源性条件下都被SUMO2修饰，揭示了其生理相关性。我们在eIF5A中鉴定了几个SUMO位点，发现SUMO化调节了eIF5A在哺乳动物细胞中的稳定性和定位。有趣的是，eIF5A的SUMO化对特定应激有反应，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化突变体无法完全抑制HYP2突变体的缺陷，并且eIF5A的SUMO化对于热休克诱导的应激颗粒形成和多核糖体解体都很重要。此外，eIF5A中SUMO化位点的突变消除了其在PANC-1细胞中的促迁移和促增殖活性。
SUMO2与eIF5A的缀合是一种应激诱导反应，与酵母细胞对热休克应激的适应有关，并且是促进胰腺导管腺癌细胞生长和迁移所必需的。
