Ubiquitin - a powerful posttranslational modifier

Ubiquitin is usually encoded by gene loci that represent head-to-tail fusions of many ubiquitin open reading frames (ORFs) or one ubiquitin ORF is fused to ribosomal protein encoding genes (Noventa-Jordão et al., 2000; Özkaynak et al., 1987; Wiborg et al., 1985). Humans contain a whole ubiquitin gene family (Wiborg et al., 1985). Fusion proteins containing up to nine ubiquitin head-to-tail repeats are described, but also genes encoding a single ubiquitin molecule are known (Wiborg et al., 1985). Ubiquitin is encoded as fusion protein of four head-to-tail repeats of ubiquitin by ubi4 or as fusion protein as N-terminal extension of ribosomal protein called carboxyl extension protein (CEP) by ubi1 in A. nidulans (Noventa-Jordão et al., 2000). Four loci encode different ubiquitin fusions, namely ubi1 - ubi4, in Saccharomyces cerevisiae (Özkaynak et al., 1987). The translated ubiquitin fusion proteins are processed by deubiquitinating enzymes and result in single ubiquitin proteins that constitute the free cellular ubiquitin pool (Grou et al., 2015).

Ubiquitin contains 76 amino acids and has a molecular weight of 8.5 kDa. The amino acid sequence is highly conserved among eukaryotic species, which indicates a conserved function (Figure 1). The characteristic di-glycine motif at its C-terminus is essential for the formation of isopeptide bonds between the ϵ-NH2 group of lysine residues of target proteins and the ubiquitin molecule itself. Ubiquitin can be attached as single molecule to one amino acid residue of the target protein (monoubiquitination), to multiple amino acid residues of a protein (multiubiquitination) or can be attached as polyubiquitin chain at one residue of a protein (polyubiquitination) (Ohtake and Tsuchiya, 2017; Pickart and Eddins, 2004). The isopeptide bond in a polyubiquitin chain is formed between one of ubiquitin’s seven lysine residues (K6,

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K11, K27, K29, K33, K48 and K63) or the initial methionine (M1) of one ubiquitin molecule and the di-glycine motif of the other molecule (Figure 1, 2; Spasser and Brik, 2012).

Modification of a protein with a single ubiquitin at a single lysine residue of the substrate has functions in endocytosis and DNA repair pathways (Hicke, 2001; Terrell et al., 1998; West and Bonner, 1980). Monoubiquitination of histones H2A or H2B influences transcription of genes in yeast and higher eukaryotes (Cao and Yan, 2012; Robzyk et al., 2000).

Figure 1: Ubiquitin is highly conserved between eukaryotes.

Multiple sequence alignments of single ubiquitin ORFs of Homo sapiens (Uniprot ID: P0CG48), Mus musculus (Uniprot ID: P0CG50), Saccharomyces cerevisiae (Uniprot ID: P0CG63) and Aspergillus nidulans (Uniprot ID: C8VLC7) were performed with the Clustal Omega alignment tool (Sievers et al., 2011). The seven lysine residues and the initial methionine are highlighted in blue. They can be used as a substrate for polyubiquitin chain formation. The C-terminal di-glycine motif is required for isopeptide bond formation with a lysine residue of a substrate protein or another ubiquitin molecule and depicted here in orange. The only three non-conserved amino acid residues are highlighted in green. The last row of the alignment represents the conservation key (Chenna et al., 2003). The “_*” indicates conserved amino acid residues among all the sequences used for this alignment. The “:” indicates a conservative mutation of the amino acid, meaning the exchange by another amino acid with similar chemical and physical properties. An empty space in the conservation key indicates non conserved residues.

The combinatorial possibilities generated by different ubiquitin linkages lead to a vast range of protein regulation through modification with polyubiquitin chains. Homotypic ubiquitin chains are always linked by only one lysine residue. The function of polyubiquitin chains linked through K6 or K27 remains unclear (Spasser and Brik, 2012). K11 linked chains were attributed to cell cycle signaling through tumor necrosis factor (TNF) and wingless-type (Wnt) signaling as well as to endoplasmatic reticulum-associated protein degradation (ERAD) (Bremm and Komander, 2011;

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Hay-Koren et al., 2011; Xu et al., 2009). K33 linked ubiquitin chains play a role in immune signaling, whereas K63 was linked to endocytosis, DNA damage response, selective autophagy and cell signaling cascades (Deng et al., 2000a; Lauwers et al., 2009; Spence et al., 1995; Tan et al., 2018; Yang et al., 2015). Linear ubiquitin chains linked through the initial methionine regulate NF-κB transcription factor family proteins (Ikeda, 2016; Rittinger and Ikeda, 2017). The complexity of ubiquitin modifications is multiplied by the fact that different lysine residues can be used for building up an ubiquitin chain. The function of these heterotypic or mixed ubiquitin chains remains still elusive. Another level of regulation is added by the posttranslational modifications of the ubiquitin chain itself due to phosphorylation, acetylation or sumoylation events (Ohtake and Tsuchiya, 2017; Sadowski et al., 2012; Spasser and Brik, 2012).

Figure 2: Localization of lysine residues in the three dimensional structure of ubiquitin.

Cartoon representation of the crystal structure of the human ubiquitin molecule comprising 76 amino acids modified from PDB 1UBQ (Vijay-Kumar et al., 1987). The protruding di-glycine motif at the C-terminus is highlighted in orange. The seven conserved lysine residues (Lys) and the initial methionine (Met), which can be used for polyubiquitin chain formation, are depicted in blue. The crystal structure was modified using the PyMOL 2.0 software.

So far, the best-studied polyubiquitin chains are connected through K48 (Spasser and Brik, 2012; Xu et al., 2009). Substrates marked with an ubiquitin chain consisting of at least four ubiquitin molecules linked through K48 are mainly targeted to the 26S proteasome for degradation (Finley et al., 1994; Glickman and Ciechanover, 2002; Thrower et al., 2000).

Proteasomal degradation is not restricted to K48 linked ubiquitin chains. Proteins modified with K11 were identified, which are substrates for proteasomal degradation as well (Xu et al., 2009).

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Linear ubiquitin chains linked through the initial methionine also induces the degradation by the 26S proteasome (Kirisako et al., 2006). Linear ubiquitin chains can bind to the 26S proteasome regulatory particle and inhibit its degradative function (Saeki et al., 2004). A protein complex named linear ubiquitin chain assembly complex (LUBAC) investigated in human cell lines creates linear ubiquitin chains by the formation of an isopeptide bond between the C-terminal glycine of one and the amino group of the N-terminal methionine of the other ubiquitin molecule (Kirisako et al., 2006). Ubiquitination in general is catalyzed by the orchestrated function of three enzymes: the E1 ubiquitin-activating, the E2 ubiquitin-conjugating and the E3 ubiquitin-ligating enzymes (Deshaies and Joazeiro, 2009; Finley et al., 2012).