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

Abstract

Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum, has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum, which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.

Keywords: COP9 signalosome; Dictyostelium discoideum; SCF complex; cullins; neddylation.

Figure 1

Neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is conserved across eukaryotes. Alignment of NEDD8 ortholog protein sequences in different eukaryote species, including those in plants (pink), amoebozoa (red), fungi (blue), and animals (orange). For plants, the upstream ubiquitin sequence was trimmed from the NEDD8 orthologs. Sequences were aligned on MEGA7 using the MUSCLE alignment and displayed using ESPript 3.0. Colored residues indicate physiochemical properties and conservation at a position. The C-terminal “RGG” proteolytic processing motif is marked by red arrowheads and the NEDD8 overhang sequence that is cleaved is indicated by the blue line.

Figure 2

An overview of the neddylation and deneddylation pathway. NEDD8 is processed into its matured form through both ubiquitin C-terminal hydrolase isozyme (UCH) and Sentrin-specific protease 8 (SENP8) proteolytic cleavage. NEDD8 is passed through a “baton” mechanism, where NEDD8 is adenylated and activated in an adenosine triphosphate (ATP)-dependent mechanism by E1, which is a heterodimer of NEDD8-activating enzyme E1 regulatory subunit (NAE1) and ubiquitin-activating enzyme 3 (UBA3). NEDD8 binds specifically to UBA3 within E1. UBA3 binds both ubiquitin-conjugating enzyme (UBE) 2F (UBE2F) and ubiquitin-conjugating enzyme E2M (UBE2M) in E2 and transfers NEDD8 to both proteins. NEDD8 is finally attached to a substrate (i.e., cullins and non-neddylated proteins) by E3, which includes ring box 1 (RBX1), ring box 2 (RBX2), and other potential E3 ligases. In E3, NEDD8 loaded into UBE2M associates with RBX1 and defective in cullin neddylation 1 (DCUN1D1). NEDD8 loaded into UBE2F associates with RBX2, but the involvement of DCUN1D1 is unknown. Substrate neddylation displaces cullin-associated NEDD8-dissociated protein 1 (CAND1), which serves to regulate cullin-RING E3 ubiquitin ligases (CRL) assembly. NEDD8 is removed from the substrate via deneddylation, which involves the COP9 signalosome (CSN) complex and SENP8/DEN1. The Dictyostelium discoideum orthologs are displayed as red text beside the respective human protein. AMP, adenosine monophosphate; ATP, adenosine triphosphate; CAND1, cullin-associated NEDD8-dissociated protein 1; CSN, COP9 signalosome; CUL, cullin; E1, E1-NAE1 complex; E2, E2-conjugation complex; E3/CRL, E3-cullin-RING complex; NAE1, NEDD8 activating enzyme E1 subunit 1; NEDD8, neural precursor cell expressed developmentally downregulated protein 8; PPi, pyrophosphate; RBX, ring box; SENP8, sentrin-specific protease 8; UBA3, ubiquitin-activating enzyme 3; UBE, ubiquitin-conjugating enzyme; UCH, ubiquitin C-terminal hydrolase; and UCHL, ubiquitin C-terminal hydrolase isozyme.

Figure 3

(A) The Dictyostelium discoideum life cycle. The asexual life cycle of D. discoideum occurs within 24 h. D. discoideum cells are unicellular during growth and divide by mitosis. Starvation triggers the onset of development. Starved amoebae undergo cyclic adenosine monophosphate (cAMP)-mediated chemotactic aggregation to form a multicellular aggregate, followed by compact mound, which then undergoes a series of morphological changes to form a finger that falls on the surface to generate a motile slug. Cells within the slug then terminally differentiate during culmination to form a fruiting body composed of a mass of viable spores that sit atop a slender stalk. In the laboratory, the multicellular developmental program is induced by depositing cells on non-nutrient agar plates. Scale bar (0 h) = 200 μm. Scale bar (4–24 h) = 2 mm. Images are not drawn to scale. (B) Expression profiles of cullin genes, genes encoding CSN subunits, and genes involved in Nedd8 processing, activation, and conjugation during the D. discoideum life cycle. Transcript data were derived from RNA-Seq data obtained from dictyExpress (http://www.dictyexpress.biolab.si) and replotted using GraphPad Prism 8. Expression profiles were examined for genes that encode proteins involved in Nedd8 processing (nedd8, senp8, and uch1), E1 (nae1 and ube1C), E2 (ube2M), E3 (rbx1, DDB_G0290025), and the COP9 signalosome (csn1-8). Expression profiles for genes that encode cullins (culA-culE) are also shown. csn, COP9 signalosome; cul, cullin; nae1, Nedd8-activating enzyme E1 subunit 1; nedd8, neural precursor cell expressed developmentally downregulated protein 8; rbx1, ring box 1/regulator of cullins 1; senp8, sentrin-specific protease 8; ube, ubiquitin-activating enzyme; and uch, ubiquitin C-terminal hydrolase.

Figure 4

Alignment of cullins from Dictyostelium discoideum and human. Sequences were aligned on MEGA7 using the MUSCLE alignment and displayed using ESPript 3.0. Colored residues indicate physiochemical properties and conservation at a position. Arrowhead marks the lysine motif where the NEDD8 protein is conjugated within the conserved motif.

Figure 5

Skp1-Cullin-F-box (SCF) complex interactome reveals components of the neddylation pathway and subunits of the CSN. Sheikh et al. (2015) identified proteins that interact with D. discoideum Skp1, FbxD, and CulE after performing co-immunoprecipitations for each protein. Interactors were identified using LC–MS/MS. Connecting lines indicate the interactions between the proteins. Mohanty et al. (2001) reported an interaction between a Dictyostelium discoideum cullin (presumably CulA) and FbxA. In the dashed circle, CSN subunit interactions with one another are shown. This was determined in a yeast two-hybrid screen by Rosel and Kimmel (2006). Cand1, cullin-associated Nedd8-dissociated protein 1; Csn, COP9 signalosome; Cul, cullin; Fbx, F-box protein; Nedd8, neural precursor cell expressed developmentally downregulated protein 8; Rbx1, ring box 1/regulator of cullins 1; and Skp1, S phase kinase-associated protein 1.

Figure 6

The roles of cullins, neddylation, and the CSN during the Dictyostelium discoideum life cycle. (A) During growth, MLN4924 inhibits cell proliferation and folic acid-mediated chemotaxis. Loss of csn5 also inhibits cell proliferation. FbxD binds CfaD, which plays a role in quorum sensing. (B) During multicellular development, MLN4924 and loss of culA inhibit cAMP-mediated chemotaxis, which delays aggregation. MLN4924 also affects the intracellular and extracellular levels of CmfA, which plays a role in quorum sensing during aggregation. culA⁻ and culB⁻ cells form multi-tipped mounds during development, which is characteristic of D. discoideum mutants with defects in the autophagy pathway. MLN4924 inhibits slug and fruiting body formation. CulA, CulB, Skp1, FbxA, and FbxD collectively regulate fruiting body formation. CfaD, counting factor-associated protein A; CmfA, conditioned media factor A; Csn5 and COP9 signalosome complex subunit 5; Cul, cullin; Fbx, F-box protein; and Skp1, S phase kinase-associated protein 1.