Insights in Post-Translational Modifications: Ubiquitin and SUMO

Abstract

Both ubiquitination and SUMOylation are dynamic post-translational modifications that regulate thousands of target proteins to control virtually every cellular process. Unfortunately, the detailed mechanisms of how all these cellular processes are regulated by both modifications remain unclear. Target proteins can be modified by one or several moieties, giving rise to polymers of different morphology. The conjugation cascades of both modifications comprise a few activating and conjugating enzymes but close to thousands of ligating enzymes (E3s) in the case of ubiquitination. As a result, these E3s give substrate specificity and can form polymers on a target protein. Polymers can be quickly modified forming branches or cleaving chains leading the target protein to its cellular fate. The recent development of mass spectrometry(MS) -based approaches has increased the understanding of ubiquitination and SUMOylation by finding essential modified targets in particular signaling pathways. Here, we perform a concise overview comprising from the basic mechanisms of both ubiquitination and SUMOylation to recent MS-based approaches aimed to find specific targets for particular E3 enzymes.

Keywords: E3 enzymes; SUMO; proteomics; ubiquitin.

Figure 1

Post-translational modifications of proteins. Chemical group modifications are shown in red, amino acid modifications in blue, complex molecules are in yellow, and additions of small proteins are displayed in green.

Figure 2

Ubiquitination cascade. Free and active ubiquitin (Ub) is conjugated to the activating enzyme (E1) in an ATP-dependent manner. Then, Ub is transferred to the conjugating enzyme (E2) to be finally covalently attached to the substrate protein assisted by the ligating enzyme (E3) which provides substrate specificity. Subsequently, Ub can be deconjugated from substrates by DeUBiqutinating enzymes (DUBs).

Figure 3

The E3 enzyme families and subclasses. RING E3 enzymes are shown as monomeric, homodimeric, heterodimeric, and forming multi-subunit complexes (CULLINs) E3 enzymes. HECT E3 enzymes are exhibited in three sub-families: NEDD4 Family, HERC Family, and Other HECT. RBR E3 Family is a 14 members family where two members are shown (PARKIN and HOIP), and the ubiquitination mechanism is displayed. The ubiquitination process is depicted. While the Ub transfer from the E2 to the substrate occur in a 2-step reaction for HECT and RBR families, there is a direct ubiquitin transfer from the E2 to the substrate in the RING E3 family.

Figure 4

Ubiquitination polymers. Ub moieties can modify proteins at one (mono ubiquitination) or several (multiple mono ubiquitination) Lys residues. Ub can form eight distinctive homotypic linkages, either through M1 (linear Ub chain) or 7 internal Lys residues (K6, K11, K27, K29, K33, K48, and K63 Ub chains). Additional complexity is achieved through the formation of heterotypic Ub chains, which contain multiple Ub linkages and adopt mixed or branched topology. Cellular functions associated to these ubiquitin polymers are displayed.

Figure 5

SUMOylation cascade. SUMO precursor matures by the action of a SENP that cleavages the SUMO C-terminal, leaving a diGly motive that forms a thioester bond with the activating enzyme E1 in an ATP dependent manner. Then, activated SUMO is transferred to the conjugating enzyme E2. Finally, the E2 conjugates SUMO to the acceptor lysine (usually in the consensus motive ψKxE) with or without the ligation enzyme E3 which confers substrate specificity. Additional rounds of this cascade form SUMO polymers that can be cleaved by specific SENPs.

Figure 6

Proteomics for E3 ligase target identification. (a) Hierarchical organization of the ubiquitination cascade. Emphasizing the difficulty of mapping substrate proteins for specific E3 enzymes. (b) Strategies for E3 ligase substrate identification divided into undirect and direct approaches. Within undirect methods, in red, the overexpression of an E3 ligase results in the increase in ubiquitination levels for putative substrates. Opposing, in blue, the depletion of an E3 ligase displays a decrease in ubiquitination levels of putative substrates. The direct approaches allow identification of specific-E3 ligase substrates where ligase trapping, NEDDylation approach, and UBAIT/TULIP methodology are shown.