Regulation of protein abundance by the COP9 signalosome

The timely coordinated synthesis and degradation of proteins in a cell is essential for accurate development. The COP9 signalosome regulates the activity of cullin RING ligases (CRLs) by removing the ubiquitin-like protein Nedd8 from the cullin subunit (Lyapina et al., 2001). CRLs modify target proteins with ubiquitin (Bosu and Kipreos, 2008; Glickman and Ciechanover, 2002). Polyubiquitinated proteins are substrates for degradation by the 26S proteasome (Glickman and Ciechanover, 2002). Hence, the COP9 signalosome is involved in protein half-life control. The embryonal lethality of csn mutants in higher eukaryotes indicates the importance of this protein complex for life (Dohmann et al., 2008; Lykke-Andersen et al., 2003). csn mutants in the reference organism A. nidulans are impaired in multicellular developmental programs but are still viable and allow the study of the functions of this protein complex (Beckmann et al., 2015;

Busch et al., 2003, 2007). As the COP9 signalosome is involved in protein turnover, the changes of the proteome in strains with a functional COP9 signalosome were compared to a ΔcsnE deletion strain, which lacks the catalytically active subunit.

In the scope of this study, stable isotope labeling for amino acids in cell culture (SILAC) was established to quantitatively monitor the changes of protein abundances in A. nidulans. To our current knowledge, no A. nidulans SILAC study has yet been published. Only one SILAC study, which was performed with Aspergillus spp. so far, used a wild type like A. flavus strain for top-down protein identification and quantification (Collier et al., 2013). Their challenge was to deal with the contamination of endogenous L-arginine, which is synthesized by the fungus itself.

121

L-lysine and L-arginine auxotrophic A. nidulans strains were generated to enable relative quantification of proteins without contaminations of endogenously synthesized amino acids in the present study. The L-lysine auxotrophic strain constructed in this study was used for the proteome comparison of fungal hyphae containing a defect or an intact COP9 signalosome. The A. nidulans genome encodes for more than 9,000 proteins (Galagan et al., 2005). Less than 10 % (745 proteins) of the fungal proteome were identified during LC/MS-MS measurements. In turn, 13 % of these identified proteins showed differential abundances in A. nidulans strains, which are defective in COP9 signalosome function even though the vegetative growth phenotype is similar in wild type and csn mutant strains.

Most of the proteins decreased in their abundance by CsnE have functions in primary metabolism; they are especially required for amino acid metabolism and vitamin biosynthesis (Table 11). Proteins become labeled with ubiquitin chains by CRLs and subsequently degraded by the 26S proteasome (Ciechanover et al., 2000).This leads to a constant supply with amino acids in the fungal cell. In A. nidulans strains harboring a csnE deletion, the function of CRLs is disturbed. This leads to changes or a reduction in the amino acid supply and in turn to the upregulation of enzymes that are involved in amino acid metabolism. Vegetative growth is characterized by the formation of a huge hyphal network in a short time, which enables the fungus to colonize different surfaces in a short time (Krijgsheld et al., 2011). For this fast growth, a constant supply of nutrients is essential to synthesize new proteins and vitamins for further growth. A dynamic process of protein biosynthesis and degradation seems to be required for hyphal growth. The COP9 signalosome with its catalytic active subunit CsnE regulates the protein turnover for enzymes that are important for metabolic processes.

The protein abundance of OrsE is increased in ΔcsnE (Table 11). It is a member of the orsellinic acid secondary metabolite gene cluster and harbors alcohol dehydrogenase activity (Sanchez et al., 2010). The csn mutant strains produce increased amounts of orsellinic acid and its derivatives (Nahlik et al., 2010). Other genes of this gene cluster are upregulated during the disturbed asexual or sexual development in csnE deletion strains (Nahlik et al., 2010). In contrast to the upregulation of protein abundance of OrsE observed in this study, the gene expression was reported to be moderately downregulated after 20 h vegetative growth in ΔcsnE (Nahlik et al., 2010). This can have several reasons: First of all, the chosen time point is not exactly the same and differs in 4 hours and also the used A. nidulans background strains differ.

Secondly, it is not very likely that CsnE influences gene expression directly, but rather protein abundances. Therefore, it is not mutually exclusive that orsE transcripts are downregulated,

122

whereas its protein abundance increases in the absence of CsnE during A. nidulans vegetative growth.

The protein abundance of peroxiredoxin, a protein required for the protection against oxidative stress is increased during vegetative, hyphal growth (Rhee et al., 2005). Contratry, the protein abundance of peroxiredoxin was reported before to be decreased in ΔcsnE mutants (Nahlik et al., 2010). It needs to be taken into consideration that the time point differs slightly and the used method for detecting changes in protein abundances were different in both studies. Anyhow, the differential abundance of proteins related to stress, especially oxidative stress response, is concomitant with the increased oxidative stress susceptibility of csn mutants (Nahlik et al., 2010).

The abundances of several developmental related proteins are increased by CsnE. Four different septins, AspA, -B, -C and -D were identified to be decreased in their abundance in ΔcsnE (Table 12). A. nidulans carries five different septin proteins (Hernández-Rodríguez et al., 2012; Momany et al., 2001). Septins are required for accurate hyphal growth and asexual development. Thereby, AspA-D are proposed to work together in a complex to control development, whereas AspE acts probably as single protein in a more specialized pathway (Momany et al., 2001). This is corroborated here by the fact that AspE is the only septin, which is not altered in its protein abundance by the lack of CsnE. Deletions of aspA or aspC genes lead to a formation of several germ tubes, early branching and altered septae positioning (Lindsey et al., 2010). The displacement of septae in csn deletion strains of A. nidulans and concomitant formation of short cells was observed previously (Busch et al., 2003). This might be ascribed to the decreased abundance of Septin proteins in a ΔcsnE strain. Furthermore, a number of other proteins predicted to be involved in the regulation of hyphal growth are positively influenced by CsnE. One of them is the glucose-methanol-choline (GMC) oxidoreductase A (Table 12). GmcA is essential for the induction of conidiophore formation (Etxebeste et al., 2012). Several other GTP binding proteins with a predicted role in cytoskeleton formation showed different protein abundances in ΔcsnE.

Taken together, this shows that the SILAC approach works in A. nidulans to quantify the abundances of proteins deriving from different cultures. Furthermore, the observed csn deletion phenotypes can be assigned to differentially regulated proteins in the absence of a functional COP9 signalosome. Due to the high conservation of the ubiquitin-proteasome system from fungi to mammals, new insights into CRL targets influenced by the COP9 signalome under different growth conditions or stress factors can be gained in future studies by applying this approach.

123

It needs to be taken into consideration that the relative quantification of protein abundances with SILAC in the scope of this study does not reflect a complete pattern of fungal proteins. The use of L-lysine as only isotopically-labeled amino acid leads to the fact that only L-lysine containing peptides can be used for quantification of protein abundances. The sample preparation for the required LC/MS-MS analyses was performed with the endopeptidase trypsin. It cleaves proteins after L-lysine or L-arginine residues into peptides (Simpson, 2006). According to the amino acid incorporation test, 60 % of all identified peptides contain L-lysine residues. This means that 40 % of the peptides cannot be used for relative quantification of protein abundances. A similar experiment using the L-arginine auxotrophic A. nidulans strain is required to get a more complete spectrum of regulated proteins in ΔcsnE. Furthermore, a label-swap experiment needs to be conducted to exclude the possibility that the detected differential protein abundances are artefacts of the labeling procedure. Furthermore, the SILAC approach should be applied during A. nidulans multicellular development. Quantitative changes in protein abundances during asexual and sexual development will reveal new insights into proteins that are regulated by CsnE. Analysis of the fungal transcriptome in ΔcsnE compared to wild type strains during multicellular fungal development revealed a long list of regulated candidates related to fungal development and secondary metabolism (Nahlik et al., 2010). However, as subunit of the COP9 signalosome CsnE is rather likely to affect protein abundances instead of gene transcription.

SILAC in A. nidulans provides an appropriate tool to track these differences also on the proteome level.