Ubiquitin modifications

Abstract

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.

Figure 1

Modification sites on ubiquitin. Ubiquitin is shown as a cartoon under a semitransparent surface, and modifiable residues are shown in ball-and-stick representation with blue nitrogen and red oxygen atoms. (A) Structure of ubiquitin highlighting the eight sites of ubiquitination. (B) Six out of seven Lys residues on ubiquitin have been reported to be acetylated in proteomics datasets. An asterisk marks the seventh Lys, Lys29, for which acetylation has not been identified to date. (C) Identified phosphorylation sites of ubiquitin are displayed according to proteomic analysis. Red spheres indicate phosphorylatable hydroxyl groups on Ser/Thr and Tyr residues. The structure was rotated 180 degrees to show all phosphorylation sites. An asterisk on Thr9 indicates this site is ambiguously assigned.

Figure 2

New complexity in the ubiquitin code. (A) Conceptual representation of some of the possible ubiquitin-, Ubl (NEDD8, SUMO2/3)- and chemical modifications of ubiquitin. (B) Unanchored ubiquitin and ubiquitin chains, with or without modifications, can function as second messengers in cells.

Figure 3

Studying Ub modifications. Linkage-specific UBDs^,, Ubiquitin Chain Restriction (UbiCRest) analysis, linkage-specific antibodies^,,, a Ser65-phosphoUb antibody and mass spectrometry allow for the identification of chain types and ubiquitin modifications. Mass spectrometry enables the quantitation of all ubiquitin linkages in a sample. Relative quantitative techniques include tandem mass tag (TMT) labeling and stable isotope labeling by amino acids in cell culture (SILAC), while absolute quantification (AQUA) strategy determines the exact quantities of each ubiquitin chain type. Similar strategies are available to identify and quantify small molecule and Ubl modifications of ubiquitin.

Figure 4

Physiological roles associated with individual chain types. (A) A small selection of E2 or E3 enzymes that assemble and DUBs that disassemble ubiquitin chains with linkage preferences is indicated. Below, cartoons illustrate some of the (new) biological processes that particular linkage types have been linked with as discussed in the text. (B) APC/C is active during early mitosis and modifies cell cycle regulators such as Nek2A with Lys48/Lys11-linked branched polyubiquitin. In this process, UBE2C first assembles short chains on the substrates, and these are then elongated on each ubiquitin by Lys11-linked polymers. Lys48/Lys11 branched chains enhance proteasomal degradation. (C) Mixed or branched Lys63/Met1-linked chains serve as protein scaffolds at immune receptors, such as IL-1 receptors, to promote NF-κB signaling. (D) A viral E3 ligase initiates endocytic internalization of the MHC class I receptor through the attachment of mixed or branched Lys11/Lys63-linked ubiquitin chains.

Figure 5

A 'ubiquitin threshold' model for proteasomal degradation. Substrate ubiquitination can result in two general outcomes, cellular signaling or proteasomal degradation. Recent evidence supports a model in which multiple short chains (e.g., diubiquitins) or branched ubiquitin are better degradation signals as compared to a single Lys48-linked tetraubiquitin. These findings also suggest that non-degradative ubiquitin signals could be modified into degradative signals through addition of short and/or branched ubiquitin chains to substrates.

Figure 6

Ser65-phosphorylation of ubiquitin in mitophagy signaling. (A) Under normal growth conditions, the TIM/TOM complex continually imports PINK1 into mitochondria (step 1). Upon entry, PINK1 undergoes proteolytic processing by the protease PARL (step 2) and is exported and degraded by the N-end rule pathway (step 3). USP30 controls the basal levels of mitochondrial ubiquitination. (B) Loss of mitochondrial membrane potential inhibits PINK1 import and proteolytic cleavage (step 4), leading to insertion of its transmembrane domain into the outer mitochondrial membrane (OMM) (step 5). PINK1 phosphorylates ubiquitin on mitochondrial proteins such as mitofusins (step 6). (C) Ser65-phosphoUb recruits Parkin to damaged mitochondria (step 7), and Ser65-phosphoUb binding releases the Parkin Ubl domain and enables its phosphorylation by PINK1 (step 8). (D) Ser65-phosphoUb binding and phosphorylation activate Parkin which subsequently ubiquitinates OMM proteins, and the newly incorporated ubiquitins are further phosphorylated by PINK1 (step 9). Parkin-mediated ubiquitination of USP30 facilitates its proteasomal degradation (step 10). (E) Mitophagy receptors NDP52 and OPTN bind to ubiquitinated mitochondrial proteins via their UBDs (step 11). (F) NDP52 and OPTN recruit the autophagy machinery to mitochondria (step 12). The phagophore engulfs mitochondria and fuses with the lysosome to degrade and recycle its contents.

Figure 7

Structural and functional consequences of Ser65-phosphorylation of ubiquitin. (A) Ser65 phosphorylation generates a dynamic equilibrium between two ubiquitin conformations. A major conformation structurally resembles unmodified ubiquitin, but has altered electrostatic potential. The minor conformation has a retracted C-terminus induced by slippage of the β5-strand. The images show the differing region in stick-representation embedded in the remaining ubiquitin core under a surface. The phosphorylated Ser65 is indicated. (B) Ser65-phosphorylation of ubiquitin has neutral, loss-of-function and gain-of-function effects on components of the ubiquitin system. E1 and E2 charging is largely unaffected; however, E2 discharging and chain elongation mediated by a subset of E2 and E2/E3 chain assembly systems are inhibited. A substantial numbers of DUBs have reduced activity against Ser65-phosphoUb chains. Receptors recognizing Ser65-phosphoUb are unknown. Ser65-phosphoUb allosterically activates the E3 ligase Parkin and may activate kinase signaling towards Rab GTPases.

Figure 8

Insights into Parkin activation. (A) Structure of autoinhibited full-length Parkin (PDB ID: 4K95,). Domains are colored in green (Ubl), dark-blue (UPD, also known as RING0), blue (RING1), light-blue (IBR), cyan (RING2) and red (REP). The mechanisms of Parkin inhibition are listed in the schematic figure. (B) Complex structure of Parkin bound to Ser65-phosphoUb. Coloring as in A with Ser65-phosphoUb in orange. Conformational changes in RING1 and IBR domain form the Ser65-phosphoUb-binding site. The Ubl domain was not included in the crystallized construct. (C) Structure of a HOIP RBR module bound to ubiquitin-charged E2 (yellow/red) and an extra, 'activator' ubiquitin. The structures in B and C are shown side-by-side to point out the identical active RING1-IBR module, indicated in the cartoon. Importantly, RING2 is juxtaposed to the E2 active site to receive ubiquitin. (D) The model of HOIP in C may indicate what active Parkin could look like. In this model, the RING2-UPD interface has been opened, and RING2 now sits atop RING1/IBR to receive ubiquitin. The hydrophobic surface on the UPD that was occupied by RING2, could be covered by rebinding of the phosphorylated Ubl domain.