Deubiquitinating enzymes reverse the ubiquitination process

Deubiquitination of proteins counteracts the CRL activity. The number of different deubiquitinating enzymes (DUBs) in humans is with 84 proteins quite high. Measured at the number of E3 ligases, the total number of DUBs is nearly one magnitude lower (Hutchins et al., 2013). Similar to the components of the ubiquitin-conjugating pathway, the number of E3 ligases or DUBs is correlated to the genome size of different organisms (Hutchins et al., 2013). DUBs are classified into six different families: the ubiquitin C-terminal hydrolases (UCH),

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Machado-Joseph domain (Josephin-domain) containing proteases (MJD), ovarian tumor proteases (OTU), ubiquitin-specific proteases (USP), the motif interacting with Ub-containing novel DUB family (MINDY) and JAMM domain metalloproteases. The first five families are cysteine proteases, whereas the JAMM motif containing DUBs are metalloproteases (Hanpude et al., 2015; Komander et al., 2009; Abdul Rehman et al., 2016).

Ubiquitin is not encoded as single gene, but transcribed as linear fusion protein consisting of several ubiquitin ORFs in a row or as fusion to ribosomal proteins (Noventa-Jordão et al., 2000;

Özkaynak et al., 1987; Wiborg et al., 1985). DUBs are required to make ubiquitin accessible by cleavage of the fusion proteins (Figure 6, Grou et al., 2015; Özkaynak et al., 1987; Wiborg et al., 1985). Similar to effects of ubiquitination, the removal of the PTM can change function, conformation, activity, stability or localization of target protein. Hence, DUB activity is involved in a number of cellular processes such as proteasomal degradation, endocytosis or immune signaling (Clague et al., 2012; Hicke and Dunn, 2003; Huang et al., 2009; Li et al., 2002; Mukai et al., 2010; Nicassio et al., 2007; Spasser and Brik, 2012; van der Horst et al., 2006). The ubiquitin chain needs to be removed from the protein prior to its degradation by the 26S proteasome. The ubiquitin chain is not degraded by the 26S proteasome as the unfolding of ubiquitin would require more energy than the cleavage of the ubiquitin chain by DUBs (de Poot et al., 2017; Worden et al., 2017). Additional DUBs are needed to recycle ubiquitin by cleavage of the resulting free ubiquitin chain into monomeric molecules that can be re-used for modification of substrates (Komander et al., 2009). The different functions of DUBs are depicted in Figure 6.

Many DUBs accomplish their function while interacting or being incorporated with or into other complexes (Ventii and Wilkinson, 2009). The proteasomal LID, which is structurally very similar to the COP9 signalosome, harbors a metalloprotease JAMM motif in its catalytically active subunit Rpn11. This deubiquitinase removes ubiquitin chains prior to substrate degradation through the proteasomal core complex when it is incorporated into the 19S regulatory particle (Worden et al., 2017; Yao and Cohen, 2002). Two more DUBs are associated to the 26S proteasome: Usp14 and Uch37 (de Poot et al., 2017). Usp14 preferably deubiquitinates proteins that carry more than one ubiquitin chain (Lee et al., 2016). The function of UCH37 is not well characterized, but it is proposed that it rather removes single ubiquitin moieties from chains than complete ubiquitin chains (Lam et al., 1997; de Poot et al., 2017; Yao et al., 2006). Usp15 deubiquitinates substrates while it interacts with the COP9 signalosome (Hetfeld et al., 2005;

Ventii and Wilkinson, 2009; Zhou et al., 2003).

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Figure 6: Diverse functions of deubiquitinating enzymes in the ubiquitin cycle.

DUBs provide free ubiquitin (Ub) molecules, which can be used for protein modification, by cleavage of the linear ubiquitin chains or processing of fusion proteins between ubiquitin and certain ribosomal proteins. The free cellular ubiquitin pool is then accessible for the activation by E1 enzymes, which start the ubiquitination cycle. DUBs regulate protein function, localization or conformation by removal of monoubiquitin from substrates (S). Furthermore, they can protect substrates from proteasomal degradation while removing single ubiquitin molecules from the distal end of the ubiquitin chain. DUBs are required for degradation of proteins by the 26S proteasome, because they have to remove the ubiquitin chain prior to degradation. This reaction is catalyzed by the intrinsic DUB subunit Rpn11 in the proteasomal LID, but can also be performed by additional proteasome associated DUBs. The cleaved ubiquitin chain needs to be dissected into single ubiquitin molecules to make them again accessible for new ubiquitination events. The blue arrows in the scheme indicate possible actions of different DUBs.

1.3.1 Ubiquitin-specific proteases

The largest DUB family are the USPs, which comprise 51 members in humans and 16 in S. cerevisiae (Hutchins et al., 2013). In S. cerevisiae Ubp1 was the first ubiquitin-specific protease that was characterized (Tobias and Varshavsky, 1991). USPs are cysteine proteases and catalyze the hydrolyzation of the isopeptide bond between ubiquitin molecules or between ubiquitin and substrate proteins by their catalytic triad consisting of a cysteine, a histidine and an

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aspartate/asparagine residue (Komander et al., 2009). The catalytic domain comprises approximately 350 amino acids and is located closely to the C-terminal part of the protein (Ye et al., 2009). The catalytic domain can be interrupted by different insertions and can comprise up to 800 amino acids (Ye et al., 2009). Structural analysis of the catalytic domain of human Usp7 and Usp2 revealed a hand like fold with fingers, palm and thumb (Hu et al., 2002; Renatus et al., 2006). Secondary structure predictions of other USPs in other organisms show a conserved pattern of α-helices and β-sheets strongly indicating a conserved fold for USP catalytic domains (Hu et al., 2002; Renatus et al., 2006). Many USP proteins contain at least two ubiquitin binding motifs: one for the distal and one for the proximal ubiquitin. Therefore, they are supposed to cleave preferably linkages between ubiquitin molecules rather than the isopeptide bonds between ubiquitin and the substrate (Ye et al., 2009). A common Cys-X-X-Cys motif was identified in the catalytic USP domain of humans, which was suggested to serve as zinc binding motif (Ye et al., 2009). The ability of zinc binding is shared by approximately 80 % of human and approximately 60 % of all S. cerevisiae USPs (Ye et al., 2009).

The ubiquitin-specific protease Usp15 carries two of these zinc binding motifs and co-purifies with the human COP9 signalosome (Hetfeld et al., 2005). The four cysteine residues comprising the zinc finger motif are located in the catalytic domain in between the residues that represent the catalytic triad. Mutations of only one cysteine codon in the motif revealed an inability of Usp15 to process polyubiquitin chains most probably due to a defect in binding to the ubiquitin chain (Hetfeld et al., 2005). Usp15 shows high sequence similarities to human Usp4 and Usp11, which constitute a small USP subfamily (Baker et al., 1999; Harper et al., 2011). All three proteins share at their N-terminus a domain present in ubiquitin-specific proteases (DUSP) followed by an ubiquitin-like domain (UBL), which are linked through a β-hairpin structure called DU finger. The function of this domain architecture is currently under investigation, but is speculated to play a role in protein-protein interactions (Harper et al., 2011). Usp4, Usp11 and Usp15 influence among others the transforming growth factor β (TGF-β) signaling pathway (Aggarwal and Massagué, 2012; Al-Salihi et al., 2012; Clague et al., 2013).