Post-translational regulation of ubiquitin signaling

Abstract

Ubiquitination regulates many essential cellular processes in eukaryotes. This post-translational modification (PTM) is typically achieved by E1, E2, and E3 enzymes that sequentially catalyze activation, conjugation, and ligation reactions, respectively, leading to covalent attachment of ubiquitin, usually to lysine residues of substrate proteins. Ubiquitin can also be successively linked to one of the seven lysine residues on ubiquitin to form distinctive forms of polyubiquitin chains, which, depending upon the lysine used and the length of the chains, dictate the fate of substrate proteins. Recent discoveries revealed that this ubiquitin code is further expanded by PTMs such as phosphorylation, acetylation, deamidation, and ADP-ribosylation, on ubiquitin, components of the ubiquitination machinery, or both. These PTMs provide additional regulatory nodes to integrate development or insulting signals with cellular homeostasis. Understanding the precise roles of these PTMs in the regulation of ubiquitin signaling will provide new insights into the mechanisms and treatment of various human diseases linked to ubiquitination, including neurodegenerative diseases, cancer, infection, and immune disorders.

Figure 1.

The chemical reactions and enzymes used in the canonical ubiquitination cascade. The structure of ubiquitin (Protein Data Bank accession number 1UBQ) with labeled landmark structural elements (including M1, the seven lysine residues, Arg₄₂, Ilu₄₄, and Gly₇₆) important for its functionality is shown (top). Note that the ribbon diagram has been oriented in two different angles to better view the relevant residues. The E1 enzyme uses ATP to activate ubiquitin by acyl-adenylation of its carboxyl terminus. Ubiquitin from the ubiquitin-AMP intermediate is transferred to the active site cysteine in E1 via the formation of a thioester bond between the carboxy-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group; AMP is concomitantly released (light purple background). The E2 ubiquitin-conjugating enzyme catalyzes the transfer of ubiquitin from E1-thio-Ub to the active site cysteine of the E2 via a trans(thio)esterification reaction (light green background). Depending on the E3 ubiquitin ligase used, ubiquitin on the E2-thio-Ub conjugate can be transferred to the protein substrate by at least two mechanisms. For members of the HECT and RBR family, ubiquitin is delivered to the active site cysteine of the E3 ligase before being transferred to the substrate. For E3 ligases in the RING family, ubiquitin is directly transferred from E2 to the substrate in a process facilitated by the E3 (yellow background). The major roles of the several distinct polyubiquitin chains formed at the primary methionine or one of the seven lysine residues are indicated (orange background). PPi, inorganic pyrophosphate.

Figure 2.

Representative PTMs of the E2 enzyme UBE2N. The structure of UBE2N (Protein Data Bank accession number 1JBB) is depicted by ribbon diagrams in which secondary structural elements such as α-helices, β-sheets, and links at different regions of the protein are shown in different colors. (A) Modification at Lys₉₂ by ISG15. (B) Deamidation at Gln₁₀₀ by OspI. (C) Ubiquitination at Lys₉₂ by MavC. The addition of the bulky ISG15 or ubiquitin at Lys₉₂, which is in close proximity with the active cysteine located at the 87th position, will sterically preclude the incoming ubiquitin from being linked to Cys₈₇, thus blocking the activity of the E2 enzyme.

Figure 3.

A schematic diagram of the E3 ubiquitin ligase Mdm2 and the sites receiving diverse PTMs. (A) Domain structure of Mdm2. S, serine; K, Lysine; P, phosphorylation; A, acetylation. Note the clustering of the modification sites at distinct domains. (B) The structure of the amino-terminal domain of Mdm2 (Protein Data Bank accession number 1Z1M; left) that harbors the phosphorylation site Ser₁₇ and the carboxyl terminus RING domain (Protein Data Bank accession number 5MNJ; right) that contains the Lys₄₄₆ SUMOylation site.

Figure 4.

Activation of the E3 ligase Parkin by PINK1-induced phosphorylation and phosphorylated ubiquitin. Under unstressed conditions, mature PINK1 is inserted into mitochondrial membranes via its amino-terminal domain and Parkin assumes an autoinhibitory conformation in the cytoplasm. Mitochondrial damage caused by events such as the loss of membrane potential causes PINK1 to accumulate on mitochondrial surface where it phosphorylates ubiquitin at Ser_65, the accumulation of phospho-Ub on the damaged mitochondria recruits Parkin. PINK1 also phosphorylates Parkin, leading to its complete activation. Fully activated Parkin ubiquitinates a number of mitochondrial proteins such as Miro-1 and GTPases mitofusins involved in mitochondria fusion, which eventually leads to elimination of damaged mitochondria by mitophagy. UBL, ubiquitin-like; IBR, R1-in-between-ring; MOM, mitochondrial outer membrane.

Figure 5.

Representative modifications of ubiquitin. The structure of ubiquitin (Protein Data Bank accession number 1UBQ) was shown in ribbon diagrams. Secondary structural features such as α-helices, β-sheets, and links at different regions of the protein are shown in different colors. (A) Phosphorylation can occur on multiple sites of ubiquitin, including Thr₇, Thr12, Thr1₄, Ser₂₀, Ser₅₇, Tyr₅₉, Ser₆₅, and Thr₆₆. Shown is the location of S₆₅, the best-studied phosphorylation on ubiquitin. (B) Acetylation of ubiquitin. Both Lys₈ and Lys₄₈ can be modified by acetylation, and shown is the modification on Lys₄₈. (C) Deamidation at Gln₄₀. (D) ADP-ribosylation at Gly_76. (E) ADP-ribosylation at Arg_42. (F) Phosphoribosylation at Arg₄₂.

Figure 6.

Ubiquitination catalyzed by ADP-ribosylation. (A) Domain structure of SdeA, a member of the SidE effector family. This protein contains a canonical DUB domain in its first 200 residues (Sheedlo et al., 2015), a PDE-like domain between residues 222 and 593, and a mono-ADP-ribosylation domain between residues 594 and 907; the function of the domain that comprises the last 500 residues is unknown. CTD, carboxy-terminal domain. (B) Biochemical reactions that lead to ubiquitination. The mART domain activates ubiquitin by transferring the ADP-ribosyl moiety from NAD⁺ to Arg₄₂ of ubiquitin. A PDE activity cleaves the phosphoanhydride bond in the reaction intermediate ADPR-Ub and transfers PR-Ub to serine residues of substrate proteins accompanied by the release of AMP.