Abstract
The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.
Highlights
cycle inhibiting factor (CIF) converts NEDD8 glutamine 40 to glutamate, inactivating cullin-RING ubiquitin ligases (CRLs)
Because in vitro biochemical studies have demonstrated that cullin neddylation enhances the activity of the human ubiquitin-conjugating enzyme CDC34 [33], we hypothesized that its replacement for the essential yeast Cdc34 homologue may render cells dependent upon NEDD8
By evaluating the effects of CIF and its product NEDD8 Q40E in yeast that are conditionally NEDD8-dependent, we have uncovered a mechanism by which NEDD8 deamidation by CIF disrupts normal CRL homeostasis to inactivate these enzymes, thereby effectively inhibiting cell proliferation (Fig. 10)
Summary
CIF converts NEDD8 glutamine 40 to glutamate, inactivating cullin-RING ubiquitin ligases (CRLs). The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation These have been attributed to blocking the functions of cullinRING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. The covalent modification of a conserved cullin lysine by a single NEDD8 molecule is associated with structural rearrangements within the CRL catalytic core [8] This conformational change has been proposed to underlie the observed effects of NEDD8 on enhancing CRL substrate ubiquitination [8, 9]. Induces cytopathic effects such as cell cycle arrest, focal adhesion formation, and stress fiber accumulation and delays apoptosis [21,22,23,24] These correlate to inactivation of a subset of CRLs and have been attributed to CIF-mediated conversion of glutamine 40 of NEDD8 to glutamic acid (Q40E) [25, 26]. We use a molecular genetics approach to explore the effects of CIF on cullin neddylation dynamics in yeast and uncover a mechanism by which CIF modulates CRL homeostasis through enhanced deneddylation of NEDD8 Q40E by the CSN
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