Inhibiting Neddylation with MLN4924 Suppresses Growth and Delays Multicellular Development in Dictyostelium discoideum

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.

Altered protein secretion in Batten disease

“…Scale bars: 250 µm (Petri dishes), 1000 µm (nitrocellulose membrane). A modified version of this figure was previously published in Huber et al (2021) .…”

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum

“…Treatment of D. discoideum cells with MLN4924 delayed aggregation in a dose-dependent manner (Figure 6; Huber et al, 2021). Moreover, this delay persisted even after 24 h, where cells were still unable to form compact mounds (Huber et al, 2021). Similarly, loss of culA was shown to impact cAMP-mediated chemotaxis and delay aggregation (Figure 6; Mohanty et al, 2001).…”

“…In Opisthokonta, neddylation regulates cell cycle progression and consequently, cell proliferation (Lammer et al, 1998;Liakopoulos et al, 1998;Gan-Erdene et al, 2003;Mendoza et al, 2003;Wu et al, 2003;Shen et al, 2005;Chan et al, 2008;Shin et al, 2011). In D. discoideum, MLN4924 significantly reduces cell proliferation in a dose-dependent manner during the growth phase of the life cycle (Figure 6; Huber et al, 2021). In addition, counting factor-associated protein D (CfaD), which is a secreted quorum sensing protein that modulates cell proliferation, was detected in the FbxD interactome (Figure 6; Bakthavatsalam et al, 2008;Sheikh et al, 2015).…”

“…This expression profile overlaps with the expression profiles of most cullin genes in D. discoideum , which peak in expression during aggregation ( Figure 3B ). Unlike later diverging amorphea, the C-terminal extension is only one amino acid long for D. discoideum Nedd8 ( Figure 1 ; Huber et al, 2021 ). The D. discoideum genome also encodes a protein homologous to UCHL1 and UCHL3 in humans, Uch1 (encoded by uch1 ), and an ortholog of human SENP8, Senp8 (encoded by senp8 ; Figure 2 ; Table 1 ; Huber et al, 2021 ).…”

“…While the role of neddylation is well established for Opisthokonta (major clade containing both fungi and animals), until recently, it was unknown whether neddylation regulates the life cycle of organisms belonging to Amoebozoa (sister group to Opisthokonta; Lammer et al, 1998 ; Liakopoulos et al, 1998 ; Gan-Erdene et al, 2003 ; Mendoza et al, 2003 ; Wu et al, 2003 ; Shen et al, 2005 ; Chan et al, 2008 ; Shin et al, 2011 ). Recent work used the well-established NAE1 inhibitor, MLN4924 (Pevonedistat), to reveal the roles of neddylation during D. discoideum growth and multicellular development ( Huber et al, 2021 ). In Opisthokonta, neddylation regulates cell cycle progression and consequently, cell proliferation ( Lammer et al, 1998 ; Liakopoulos et al, 1998 ; Gan-Erdene et al, 2003 ; Mendoza et al, 2003 ; Wu et al, 2003 ; Shen et al, 2005 ; Chan et al, 2008 ; Shin et al, 2011 ).…”