In D. discoideum, nedd8 is expressed throughout development but peaks in expression after 4 h of development (Figure 3B). 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).

The D. discoideum E1 complex is a heterodimer comprised of orthologs of mammalian NAE1 and UBA3 (Nae1 and Ube1C, respectively; Figure 2; Table 1). There is only one potential E2 encoded by ube2M (protein: Ube2M), which is similar in sequence to both UBE2M and UBE2F. The D. discoideum genome also encodes an ortholog of RBX1 and RBX2, Rbx1, that is proposed to function as an E3 in the organism, as well as an ortholog of DCUN1D1 (uncharacterized protein DDB0305617; Forman and Garrod, 1977). Finally, D. discoideum contains an ortholog of human CAND1 (Cand1). Together, the predicted neddylation pathway in D. discoideum shares similarities to the well-established pathway in metazoans.

In mammals, there are eight members of the cullin family (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, and CUL9; Table 2; Sarikas et al., 2011). The D. discoideum genome encodes five proteins (CulA, CulB, CulC, CulD, and CulE encoded by culA, culB, culC, culD, and culE, respectively) that all share sequence similarity with human cullins (Table 2). Moreover, Sheikh et al. (2015) compared characteristic sequence motifs in cullins to show that CUL1 is most similar to CulA, CulE, and CulB, CUL3 is most similar to CulC, and CUL4B is most similar to CulD. BLASTp searches also show that the D. discoideum ortholog of anaphase promoting complex subunit 2 (Anapc2) shares limited sequence similarity with human CUL1, CUL2, CUL3, CUL4A, and CUL4B (Table 2). In mammals, anaphase promoting complex functions as an E3 ubiquitin ligase that regulates cell cycle progression by mediating ubiquitination and subsequent degradation of target proteins (Tang et al., 2001; Jin et al., 2008). Since only CulE has previously been validated as a neddylated protein, we examined the sequences of other D. discoideum cullins to determine whether the neddylation motif is conserved (Robert and Gouet, 2014; Kumar et al., 2016). The alignment revealed that the cullin neddylation motif is highly conserved between cullins from D. discoideum and humans (Figure 4). While the residues upstream of the lysine are identical and conserved in D. discoideum cullins, there are differences in the two downstream residues. Adjacent to the lysine (+1 position), CulD has a threonine rather than adhering to the (MAS) amino acid sequence (methionine, alanine, or serine at the +1 position), indicating functional flexibility between the threonine and serine groups in D. discoideum. Similarly, CulE differs at the +2 position with a lysine residue that normally contains either an arginine, histidine, or glutamic acid residue [i.e., (RHE)]. Since there is a highly conserved neddylation motif present in each of CulA, CulB, CulC, CulD, and CulE, there is potential for Nedd8 to be conjugated to all D. discoideum cullins. Finally, the D. discoideum genome encodes orthologs of eight human CSN subunits (Table 1).

The D. discoideum cullin genes are differentially expressed during development and peak in expression at different times during the developmental program (Figure 3B). The expression of culB, culC, culD, and culE increase and reach peak levels during the first 8 h of development, followed by a decline throughout the remaining stages of development. Conversely, culA rises in expression throughout development and reaches its peak level after 20 h. Consistent with this, Mohanty et al. (2001) used an anti-CUL1 antibody to show that a cullin (presumed to be CulA) reaches peak levels after 16–20 h of development. Combined, these findings suggest that cullins may have specific roles during D. discoideum development.

In Sheikh et al. (2015) identified the SCF interactome in D. discoideum by expressing FLAG-tagged CulE in amoebae (Figure 5). In the interactome, proteins orthologous to components of the mammalian SCF complex were identified, such as homologs of SKP1 and three F-box proteins (FbxD, uncharacterized protein DDB0306343, and uncharacterized protein DDB0237864). Using an antibody directed against D. discoideum Skp1, another co-immunoprecipitation was performed that identified CulE as a Skp1-interactor via Western blotting (Sheikh et al., 2015). F-box proteins have been shown to interact with cullins in D. discoideum (Mohanty et al., 2001; Sheikh et al., 2015). For example, FbxD has been identified as a CulE interactor (discussed above; Sheikh et al., 2015; Figure 5). In addition, Mohanty et al. (2001) reported an in vivo interaction between CulA and FbxA using an antibody against human CUL1 that failed to detect a protein in culA⁻ cells via Western blotting. However, since the antibody was not specific for CulA, and there is strong sequence similarity between cullins in D. discoideum (and humans; Table 2; Sheikh et al., 2015), it is possible that another cullin was detected in the FbxA pull-down. Nonetheless, these observations suggest that the SCF complex associates with distinct cullins at different points in the life cycle to regulate specific processes during multicellular development. In addition, Skp1 modification affects the representation of F-box proteins in the Skp1 interactome suggesting that it influences the recruitment of F box proteins to the SCF complex (Sheikh et al., 2015).

The CulE interactome also revealed that known mechanisms of SCF regulation are conserved in D. discoideum (Enchev et al., 2015). For example, an ortholog of mammalian CAND1 (Cand1) was identified as an interactor suggesting that the mechanisms regulating CRL assembly and disassembly are similar in D. discoideum to what is observed in other eukaryotes (Figure 5). Furthermore, CulE was shown to interact with several proteins involved in neddylation and deneddylation such as Nedd8, Rbx1, and all the Csn subunits (Sheikh et al., 2015). As additional support for the neddylation of CulE, Western blotting for the CulE protein shows two distinct protein bands (Sheikh et al., 2015). In total, accumulated evidence strongly supports that not only is the neddylation machinery conserved in D. discoideum, but it also dynamically regulates the assembly of SCF complexes in D. discoideum by acting on cullins.

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). 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). These findings indicate a conserved role for neddylation in regulating cell proliferation and support recent work linking neddylation to the proliferation of cancer cells (Du et al., 2021; Zhang et al., 2021). However, whether MLN4924 specifically affects CRLs in D. discoideum remains to be determined since non-cullin targets of neddylation have been reported in several organisms including S. pombe, A. thaliana, and Trypanosoma brucei (Girdwood et al., 2012; Enchev et al., 2015; Mergner et al., 2015; Liao et al., 2017).

Deneddylation of cullins occurs through the actions of SENP8 and the CSN. Deneddylation by the CSN occurs via the CSN5 subunit, which is a metalloprotease (Cope et al., 2002; Echalier et al., 2013). For that reason, CSN5 has the highest selection pressure and is the most conserved of all the CSN subunits. Thus, in D. discoideum, Csn5 would be considered the essential subunit of the CSN required for the deneddylation of cullins. Consistent with this, loss of csn5 impairs cell proliferation in D. discoideum (Figure 6; Rosel and Kimmel, 2006). Since reduced neddylation and impaired deneddylation both impact cell proliferation D. discoideum, these findings indicate that cell proliferation in D. discoideum requires efficient cycling of neddylation and deneddylation.

D. discoideum cells use chemoattractants to sense nutrient levels within the environment. During the growth or feeding stage of the life cycle, D. discoideum cells sense and undergo chemotaxis towards folic acid, which is a biomolecule secreted by bacteria (Gerisch, 1982). Our group observed reduced chemotaxis toward folic acid when cells were treated with MLN4924 (Figure 6; Huber et al., 2021). Intriguingly, recent work supports a role for neddylation in regulating macrophage chemotaxis in chronic pancreatitis (Lin et al., 2021). Therefore, work in D. discoideum and humans indicates that neddylation plays an important regulatory role in chemotaxis. As a result, further probing of the chemotactic pathway in D. discoideum may improve our understanding of the mechanisms that CRLs use to regulate chemotaxis in humans.

During the early stages of multicellular development (0–10 h), D. discoideum amoebae aggregate by chemotaxis toward cyclic adenosine monophosphate (cAMP), which is produced and secreted by starving cells (Figure 3A; Almeida and Dilao, 2016; Nakajima and Sawai, 2016). Intriguingly, several cullin genes increase their expression during this stage of the life cycle (Figure 3B). Consistent with this, loss of culA delays aggregation (Figure 6; Mohanty et al., 2001). Using an assay that examines chemotaxis competence, Mohanty et al. (2001) showed that the response of culA⁻ cells to cAMP was suboptimal suggesting that CulA has an early role in development by influencing cAMP-mediated chemotaxis during aggregation (Noegel et al., 1986). As discussed above, the role of neddylation in regulating chemotaxis in D. discoideum is consistent with its role in regulating macrophage chemotaxis in chronic pancreatitis (Lin et al., 2021).

In D. discoideum, cAMP controls the actions of cAMP-dependent protein kinase A (PKA), which is a signaling kinase that regulates the expression of genes required for inducing aggregation, cell-type differentiation, and culmination (Loomis, 1998). Upstream of PKA activation, the cAMP phosphodiesterase, RegA, catalyzes the conversion of cAMP to 5′-adenosine monophosphate to prevent PKA activation (Shaulsky et al., 1996, 1998; Thomason et al., 1998). As a result of its regulatory role during aggregation, the level of RegA protein peaks at this stage of the developmental program (Mohanty et al., 2001). However, loss of culA or fbxA causes the level of RegA protein to remain high even after aggregation (Mohanty et al., 2001; Tekinay et al., 2003). When culA⁻ cells express a constitutively active PKA protein, the observed defects in aggregation and chemotaxis are absent (Mohanty et al., 2001). Since constitutively active PKA functions irrespective of cAMP levels, this supports the notion that abnormally high amounts of RegA interfere with cAMP levels to dysregulate PKA activity (Zhang et al., 2003). In conclusion, there is a significant role for CulA, and the SCF complex it functions with, in early development where it regulates the ubiquitination and degradation of RegA to maintain intracellular cAMP levels for PKA activation.

The SCF-dependent ubiquitination of RegA also appears to be dependent on extracellular signal-regulated kinase (ErkA), an ortholog of mammalian mitogen-activated protein kinase (MAPK). In D. discoideum, ErkA phosphorylates RegA to inhibit its activity and this phosphorylation could then prime RegA for SCF-mediated ubiquitination of RegA (Maeda et al., 2004; Kuburich et al., 2019). Additional work is required to determine if ErkA also directly regulates components of the SCF complex in D. discoideum. Intriguingly, ectopic expression of mouse Cul1 restored RegA degradation in culA⁻ cells suggesting that the functions of mouse CUL1 and D. discoideum CulA are conserved (Mohanty et al., 2001).

During D. discoideum development, cullins are differentially regulated and this is possibly linked to their roles in developmental processes at specific time points. Most cullin genes (except culA) peak in expression during the first 12 h of development when cells aggregate to form multicellular mounds (Figure 3B). In addition, genes involved in the conjugation of Nedd8 to cullins such as nedd8, uch1, nae1, ube1C, ube2M, and rbx1 all follow a similar pattern of expression to the cullin genes, where they peak in expression during the first 12 h of development and then decline in expression throughout the remaining stages (Figure 3B). These data suggest that transcriptional changes in genes associated with neddylation occur in tandem with changes in the expression of cullin genes. Since neddylation promotes CRL assembly, this would allow for timely assembly of CRLs early in development to mediate the initiation of development and aggregation. In addition, CRL assembly and disassembly are thought to occur at least as frequently as new substrate selection (Kleiger et al., 2009; Deol et al., 2019). Consistent with this, the CSN subunits in D. discoideum also peak in expression during the first 12 h of development (Figure 3B). Combined, these results indicate that CRL assembly and disassembly are regulated by cycles of neddylation and deneddylation and that this cycling plays an important role in regulating the early stages of D. discoideum development.

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). Since neddylation inhibition phenocopies this result, it suggests that SCF complex assembly is inhibited when neddylation does not occur, which is consistent with observations in human cells (Zheng et al., 2002; Dubiel et al., 2013). These results also reveal that by upregulating the expression of nedd8 and cullins during cell aggregation, D. discoideum utilizes neddylation as a mechanism to facilitate the timely assembly of the SCF complex. Therefore, the mechanism underlying how neddylation influences cell aggregation is likely tied to its regulation of the SCF complex. Finally, MLN4924 also affects the secretion of the quorum sensing protein conditioned medium factor A (CmfA) during aggregation (Figure 6; Huber et al., 2021). Upon starvation, amoebae begin secreting CmfA, which acts as a trigger for gene expression, as a high density of starved cells correlates to a high concentration of CmfA (Loomis, 2014). Once a CmfA threshold is reached, cells upregulate the expression of developmental genes, including spore coat protein (cotB) and cysteine protease D (cprD), and aggregate through cAMP-mediated chemotaxis (Loomis, 2014). Inhibition of neddylation by MLN4924 increases the intracellular and extracellular amounts of CmfA (Huber et al., 2021). While CmfA was not identified as an interactor of the D. discoideum SCF complex (Sheikh et al., 2015), the effect of MLN4924 on its intracellular and extracellular levels suggests it is regulated by neddylation.

A potential explanation for how cullins influence cell-type differentiation involves the process of autophagy. Autophagy is a metabolic pathway that degrades intracellular material through lysosomal digestion (Mizushima et al., 2008). Autophagy is a required pathway for D. discoideum development and influences cell differentiation (Otto et al., 2003). In mammalian cells, the roles of cullins in regulating autophagy are well-established and occur at different steps in the autophagy pathway (McEwan and Dikic, 2014; Mesquita et al., 2017). In general, cullins regulate the ubiquitination of proteins belonging to the autophagy machinery, mTOR activation, and the activation transcription factors.

There also appears to be a link between cullins and autophagy in D. discoideum. During the transition from mounds to slugs, mounds form a single tip (Figure 3A). However, culA⁻ and culB⁻ cells form multi-tipped mounds during development, which is characteristic of D. discoideum mutants with defects in the autophagy pathway (Figure 6; Mohanty et al., 2001; Wang and Kuspa, 2002; Otto et al., 2003; Mesquita et al., 2017). These results indicate that cullins may influence autophagy in D. discoideum thereby impacting cell differentiation. However, at present, this link remains to be experimentally determined for the D. discoideum cullins.

Chemical and physical cues from the environment (e.g., light, temperature, and ammonia) are critical for regulating D. discoideum development, an organism normally found in soils worldwide (Bonner and Lamont, 2005; Singleton et al., 2006; West et al., 2007; Mathavarajah et al., 2017). Accessible oxygen influences slug polarity and migration, cell differentiation, and other aspects of multicellular development (Sternfeld and Bonner, 1977; Sternfeld and David, 1981; Sternfeld, 1988; Sawada et al., 1998; Bonner, 2003; Biondo et al., 2021; Cochet-Escartin et al., 2021). However, the way this oxygen is sensed by D. discoideum differs from what occurs in metazoans. Before the evolution of metazoans, single-celled eukaryotes like D. discoideum and Toxoplasma gondii lacked the hypoxia inducible factor system for oxygen sensing (Liu, 2017). Instead, these protozoans relied on modifying the SCF complex to mediate oxygen sensing and the mechanism behind this has been well studied in D. discoideum (reviewed in West et al., 2010). In D. discoideum, regulation of the SCF complex allows for oxygen sensing and this occurs via post-translational modifications. Skp1 of the SCF complex is post-translationally modified at Pro143 through prolyl hydroxylation (the addition of a hydroxyl group) via the prolyl hydroxylase gene (phyA; West et al., 2010). Since phyA is a direct oxygen sensor, the presence of oxygen functions as the initial stimuli for Skp1 hydroxylation. Following hydroxylation, Skp1 is O-glycosylated through the actions of several glycosyltransferases including GlcNAc transferase (GntA), poly-glycosyltransferase (PgtA), and alpha-gal-transferase (AgtA; in that order; Teng-Umnuay et al., 1999; van der Wel et al., 2001; Ketcham et al., 2004). Glycosylation then promotes the association of Skp1 with F-box proteins and allows for the rapid assembly of the SCF complex to regulate culmination and spore formation, and perhaps other oxygen-dependent developmental pathways (Xu et al., 2012; Sheikh et al., 2015). In conclusion, this represents a novel post-translational modification of the SCF complex that is utilized for oxygen-dependent development in D. discoideum.

After aggregation, D. discoideum cells undergo cell-type differentiation to become pre-spore or pre-stalk cells, which each express cell-specific markers; extracellular matrix protein A (EcmA) for pre-stalk; and spore coat protein 60 (SP60/CotC) for pre-spore (Fosnaugh and Loomis, 1989; Morrison et al., 1994; Williams, 2006). When these markers were examined post-aggregation in culA⁻ cells, ecmA expression was absent and cotC expression was decreased (Mohanty et al., 2001). In addition, an altered ratio of pre-stalk to pre-spore cells was reported in fbxA⁻ cells and overexpression of FLAG-tagged FbxD has been shown to delay fruiting body formation (Nelson et al., 2000; Sheikh et al., 2015). Finally, inhibiting neddylation with MLN4924 was shown to impair fruiting body formation (Huber et al., 2021). Collectively, these findings indicate that CulA, FbxA, FbxD, and neddylation have roles in cell differentiation.

Cullin genes in D. discoideum have selective roles in cell differentiation. Unlike what was seen with loss of culA, culB⁻ cells express ecmA precociously and have a propensity to form pre-stalk cells (Wang and Kuspa, 2002). Moreover, culB⁻ cells that express constitutively active PKA differentiate into stalk cells prior to even reaching the mound stage of development (Wang and Kuspa, 2002). In addition, PKA activity is antagonistic with the loss of culB, worsening the phenotype, and contrasting work with culA⁻ cells (Wang and Kuspa, 2002). These results suggest that CulB has non-CRL differentiation functions or is utilized in another unique CRL complex involved in regulating differentiation in D. discoideum.

Previous work suggests that cell-type differentiation in D. discoideum may also be influenced by culD and culE. In a recent study examining the expression of genes in different cell-types after differentiation, culD and culE transcripts were both preferentially upregulated in spores and downregulated in stalk cells (Kin et al., 2018). Altered expression in specific cell types may occur to (1) directly facilitate the terminal differentiation (i.e., pre-stalk to stalk cell) or (2) allow for functions related to the distinct roles of the cell types. Future work examining these two cullin genes will help elucidate how culD and culE contribute to cell differentiation during multicellular development.