Jean-Philippe Nougayrède, C. Erec Stebbins, Ascel Samba-Louaka, Eric Oswald, Brenda A. Schulman, Claude Watrin, David M. Duda, Grégory Jubelin, Yun Hsu, Marie Penary, Rika Nobe, Frédéric Taieb, Unité de Microbiologie (MIC), Institut National de la Recherche Agronomique (INRA), Interactions hôtes-agents pathogènes [Toulouse] (IHAP), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Department of Structural Biology, Howard Hughes Medical Institute (HHMI)-St Jude Children's Research Hospital, Rockefeller University [New York], Institut de Recherche en Santé Digestive (IRSD ), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), St Jude Children's Research Hospital, Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), taieb, frederic, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and CHU Toulouse [Toulouse]
The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.