title: Protein interaction studies point to new functions for Escherichia coli glyceraldehyde-3-phosphate dehydrogenase.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein-protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of Escherichia coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.
authors.affiliation: Departament de Bioquímica i Biología Molecular, Facultat de Farmàcia, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Av. Diagonal, 643, E-08028 Barcelona, Spain. elainefmelo@gmail.com
authors.forename: Elaine
authors.initials: E
authors.lastname: Ferreira
authors.forename: Rosa
authors.initials: R
authors.lastname: Giménez
authors.forename: Laura
authors.initials: L
authors.lastname: Aguilera
authors.forename: Karla
authors.initials: K
authors.lastname: Guzmán
authors.forename: Juan
authors.initials: J
authors.lastname: Aguilar
authors.forename: Josefa
authors.initials: J
authors.lastname: Badia
authors.forename: Laura
authors.initials: L
authors.lastname: Baldomà
mesh_terms: D002498:Centrifugation; D003432:Cross-Linking Reagents / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D015151:Immunoblotting; D013058:Mass Spectrometry; D011485:Protein Binding; D025941:Protein Interaction Mapping
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D005557:Formaldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases