4.3 The presence of five neighboring CSN subunits targets nuclear DenA for degradation
The two deneddylases DenA/DEN1 and COP9 signalosome remove the ubiquitin-like protein Nedd8 from modified targets and are required for development in fungi as well as in insects, plants and mammals (Serino & Deng, 2003; Tomoda et al, 2004; Singer et al, 2014; Beckmann et al, 2015). In A. nidulans the two deneddylases are involved in different developmental pathways. DenA supports asexual development, which is promoted when the fungus reaches the soil surface and perceives light signals, whereas CSN is essential for sexual development (Christmann
DISCUSSION
et al, 2013; Beckmann et al, 2015). Both deneddylases physically interact which is conserved from fungi to human (Christmann et al, 2013). The nuclear CSN-DenA interaction might be part of a developmental control which delays asexual development and supports sexual differentiation and secondary metabolism e.g. in the soil and in darkness. The impact of the interrelationship between the two deneddylases was studied by monitoring DenA protein levels in the absence of single CSN subunits. It was found that three PCI subunits (CsnC, CsnG, CsnH) and the two MPN containing proteins (CsnE, CsnF) were required to destabilize DenA, whereas the other three CSN subunits (CsnA, CsnB, CsnD) did not affect DenA stability (Figure 15). These findings indicate that both deneddylases do not only interact with each other, but that in addition five CSN subunits are responsible to trigger the amount of nuclear DenA.
Previous studies identified proteins whose stability is affected by the complete or partial loss of CSN (Wei et al, 2008; Choi et al, 2015). Among them are CSN interacting proteins such as the cyclin-dependent kinase inhibitors p57 and p27, which negatively control cell cycle progression. The stability of p57 is negatively regulated by subunit CSN6 (Chen et al, 2012a) whereas the influence of CSN on p27 stability is still controversial. In vitro data demonstrate a CSN mediated inhibition of p27 degradation (Yang et al, 2002), whereas transient overexpression of the five mammalian CSN subunits CSN3, CSN5, CSN6, CSN7 and CSN8 results in p27 down-regulation (Tomoda et al, 2002; Choi et al, 2015). These five mammalian CSN components correlate with the same five fungal CSN subunits, which were identified in this study as supporters of DenA degradation in A. nidulans. Although scientific evidences suggest that beside the eight subunit CSN holocomplex also CSN subcomplexes can be formed (Wei et al, 2008; Sharon et al, 2009; Kotiguda et al, 2012; Dubiel et al, 2015), no “mini-CSN” complex consisting of those five CSN subunits was described so far. As DenA is stabilized in distinct csn deletion strains it is likely that DenA degradation is not automatically induced by a disassembled CSN complex but might depend on physical interactions with respective CSN subunits.
The crystal structure of the human CSN complex revealed that it is composed of two organized centers. The C-terminal -helices of every subunit create a large bundle and the six subunits with a PCI domain form a horseshoe-like structure with their
N-subunit composition, the five CSN N-subunits with destabilization effect on DenA are adjacent and probably form a common surface (Figure 32). This suggests that proteins such as DenA, p57 or p27 physically interact with these CSN subunits resulting in the degradation of the interacting protein.
Figure 32: DenA degradation mediated by the COP9 signalosome.
The model shows the involvement of CSN in DenA degradation. The active CSN holocomplex consists of eight CSN subunits and mediates deneddylation of Nedd8 conjugates. The five DenA destabilizing CSN subunits CsnC, CsnE, CsnF, CsnG and CsnH are closely aligned, suggesting the formation of a common DenA binding surface. Interaction of DenA with this binding site targets DenA for degradation via the 26S proteasome.
Cellular proteins can be degraded via two different mechanisms. Proteins in the extracellular lumen are taken up by receptor-mediated endocytosis. Enclosed by vesicles those proteins are transported to lysosomes where they are degraded. The lysosomal destruction pathway can be further divided into phagocytosis and autophagy, but they represent a rather unspecific protein degradation system (Eskelinen & Saftig, 2009; Flannagan et al, 2012). To sustain fundamental biological processes a precise protein turnover is required. The non-lysosomal pathway, the ubiquitin-proteasome system, represents the major process of highly selective protein destruction of intracellular substrates (Schrader et al, 2009; Ciechanover, 2015).
Based on the observation that human DenA/DEN1 was stabilized in a cyclohexamide
DISCUSSION
experiment by addition of the proteasome inhibitor MG132 (Christmann et al, 2013), it is likely to assume that the five CSN subunits with destabilization effect on DenA support degradation of DenA via the ubiquitin-proteasome system (Figure 32). This suggestion is further supported by the finding that DenA/DEN1 of A. thaliana is modified with ubiquitin, indicating a 26S proteasome mediated degradation (Mergner et al, 2015).
Increased DenA stability in a strain that lacks the gene encoding for the main interaction partner of DenA, CsnG, might correlate with the compensatory effect of high abundant DenA, which was observed in the same deletion strain. DenA might be stabilized in a csnG deletion strain, in order to be capable to at least partially counteract the absent CSN activity by compensating the deneddylation of CSN related targets. Such a mechanism might exist to maintain fundamental cellular processes such as development and stress response in CSN defective cells. The supporting deneddylation function of DenA under conditions where CSN is inactive may represent an aspect, which explains the necessity to encode two deneddylases.
The interference of both deneddylases suggests that DenA degradation is supported by CSN under conditions where asexual development is negligible and sexual differentiation needs to be promoted by the CSN complex.
4.4 A specific choreography of changing phosphorylation events at DenA