Abstract
Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.
Highlights
Ubiquitin, or ubiquitin-like modifiers such as neural precursor cell expressed developmentally downregulated protein 8 (NEDD8), target both proteins and lipids to control their degradation, subcellular localization, macromolecular interactions, and enzymatic activity [1,2]
We observed increased amounts of intracellular and extracellular conditioned medium factor (CmfA) in cells treated with MLN4924, suggesting that cells may have attempted to compensate for the delayed aggregation by producing and secreting more CmfA. While these findings suggest that treatment with MLN4924 compromises cell–cell signaling during the early stages of development, we could not rule out the possibility that the increased amount of intracellular CmfA was due to impaired proteasome-mediated degradation
This study demonstrates how D. discoideum can be used as a model system to better understand the unicellular and multicellular processes regulated by neddylation
Summary
Ubiquitin-like modifiers such as neural precursor cell expressed developmentally downregulated protein 8 (NEDD8), target both proteins and lipids to control their degradation, subcellular localization, macromolecular interactions, and enzymatic activity [1,2]. NEDD8 is conjugated to a target protein at a C-terminal lysine through a process known as neddylation. Cullin proteins, which are components of the cullin–RING ligase (CRL) superfamily (the SCF complex is a part of this superfamily), are one of the main targets of neddylation in eukaryotic cells [5]. They serve as scaffolds for multi-subunit ubiquitin ligases that target proteins for ubiquitination [6]. The assembly of CRL complexes is modulated by cullin-associated and neddylation dissociated 1 (CAND1), which binds and sequesters cullin proteins to prevent CRL assembly [7,8]. While cullin proteins are the most well-studied targets of neddylation, non-cullin targets of neddylation have been reported in a variety of organisms, including Schizosaccharomyces pombe and Arabidopsis thaliana [10,11,12]
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