2. Academic Validation
  3. SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells

  • Cell Mol Biol Lett. 2024 Jan 16;29(1):15. doi: 10.1186/s11658-024-00533-5.
Rocío Seoane 1 2,  Tomás Lama-Díaz 1 3,  Antonia María Romero 4 5,  Ahmed El Motiam 1 6,  Arantxa Martínez-Férriz 7,  Santiago Vidal 1 2,  Yanis H Bouzaher 1,  María Blanquer 1,  Rocío M Tolosa 1,  Juan Castillo Mewa 8,  Manuel S Rodríguez 9,  Adolfo García-Sastre 2 10 11 12,  Dimitris Xirodimas 13,  James D Sutherland 14,  Rosa Barrio 14,  Paula Alepuz 4 15,  Miguel G Blanco 1 3,  Rosa Farràs 7,  Carmen Rivas 16 17
Affiliations

Affiliations

  • 1 Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain.
  • 2 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
  • 3 Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain.
  • 4 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universitat de València, Burjassot, 46100, Valencia, Spain.
  • 5 Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), C/ Américo Vespucio 24, Edificio Cabimer, 41092, Seville, Spain.
  • 6 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
  • 7 Centro de Investigación Príncipe Felipe, 46012, Valencia, Spain.
  • 8 Research Department in Genomics and Proteomics, Instituto Conmemorativo Gorgas de Estudios de la Salud, 0816-02593, Panamá, Republic of Panama.
  • 9 Laboratoire de Chimie de Coordination LCC-UPR 8241-CNRS, 31400, Toulouse, France.
  • 10 Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
  • 11 Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
  • 12 The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
  • 13 Montpellier Cell Biology Research Center (CRBM), CNRS-UMR 5237 Université de Montpellier, Montpellier, France.
  • 14 Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160, Derio, Spain.
  • 15 Instituto Bio TecMed, Universitat de València, Burjassot, 46100, Valencia, Spain.
  • 16 Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain. mcarmen.rivas@usc.es.
  • 17 Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología (CNB), CSIC, Darwin 3, 28049, Madrid, Spain. mcarmen.rivas@usc.es.
PMID: 38229033 DOI: 10.1186/s11658-024-00533-5
Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Keywords

Pancreatic ductal adenocarcinoma; SUMO2; Stress granules; Stress response; eIF5A.

Figures
Products
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-108702
    99.90%, SUMO-Activating Enzyme Inhibitor