Linear Ubiquitin Chains: Cellular Functions and Strategies for Detection and Quantification

Abstract

Ubiquitination of proteins is a sophisticated post-translational modification implicated in the regulation of an ever-growing abundance of cellular processes. Recent insights into different layers of complexity have shaped the concept of the ubiquitin code. Key players in determining this code are the number of ubiquitin moieties attached to a substrate, the architecture of polyubiquitin chains, and post-translational modifications of ubiquitin itself. Ubiquitination can induce conformational changes of substrates and alter their interactive profile, resulting in the formation of signaling complexes. Here we focus on a distinct type of ubiquitination that is characterized by an inter-ubiquitin linkage through the N-terminal methionine, called M1-linked or linear ubiquitination. Formation, recognition, and disassembly of linear ubiquitin chains are highly specific processes that are implicated in immune signaling, cell death regulation and protein quality control. Consistent with their role in influencing signaling events, linear ubiquitin chains are formed in a transient and spatially regulated manner, making their detection and quantification challenging.

Keywords: HOIL; HOIP; LUBAC; OTULIN; PRM; SHARPIN; SRM; ubiquitin.

Figure 1

Enzymatic cascade of ubiquitination. Ubiquitin is transferred to the target protein by an enzymatic cascade. Ubiquitin is first bound by an ubiquitin-activating enzyme (E1) using one ATP molecule. The activated ubiquitin is then transferred to a conjugating enzyme (E2). Depending on the type of the E3 ubiquitin ligase that is involved in the ubiquitination process, ubiquitin is directly transferred from the E2 to the target protein with the ligase acting as specific bridging factor (RING ligases). Alternatively, the ubiquitin moiety is transferred to the E3 ligase (RBR and HECT ligases) via a transient thioester bond before it is attached to the target protein by an isopeptide bond.

Figure 2

The linear ubiquitination machinery. Linear ubiquitin chains are assembled by LUBAC, the linear ubiquitin chain assembly complex, comprising HOIL, HOIL-1, and SHARPIN (writers). This modification can be translated into a cellular effect by proteins that specifically interact with linear ubiquitin chains (readers). The linear ubiquitination signal can be removed by deubiquitinases that disassemble M1-linked ubiquitin chains (erasers).

Figure 3

Structural diversity of ubiquitin chains. In homotypic ubiquitin chains ubiquitin monomers are linked to one of seven lysine (K) residues or to the N-terminal methionine (M1). For example, in a K48-linked ubiquitin chain all ubiquitin monomers are linked to lysine 48 of the acceptor ubiquitin moiety. Different linkage types are characterized by specific conformations of the polyubiquitin chain, such as an open or closed conformation. The structure of the chain can be further modified by post-translational modifications of ubiquitin, here indicated by the phosphorylation of serine 65 in a linear ubiquitin chain. Ubiquitin: green; target protein: blue.

Figure 4

Domain structure of the LUBAC components. LUBAC consists of three core proteins, HOIP, HOIL-1, and SHARPIN. HOIP and HOIL-1 are RBR E3 ligases characterized by a RING - IBR (in between RING) - RING domain structure. The interaction between the UBL domains of HOIL-1 and SHARPIN with the UBA domain of HOIP releases the autoinhibition of HOIP. HOIP interacts via its PUB domain with the PIM domain of OTULIN, SPATA2, or p97/VCP.

Figure 5

Linear ubiquitination and NF-κB activation. Formation of linear ubiquitin chains is implicated in NF-κB activation induced by different pathways, for example via TNF or IL-1 receptors, Toll-like receptors or NOD2 receptors. All these pathways involve the formation of signaling complexes to which LUBAC is recruited via K63-linked ubiquitin. K63-linked polyubiquitin is generated by cIAP1/2 at the TNFR complex and by TRAF6 at the IL-1R and at TLRs. LUBAC then assembles M1-linked ubiquitin on K63-ubiquitinated NEMO (and other substrates within the pathway). Oligomerization of NEMO activates the associated kinases IKKα and IKKβ, required for NF-κB activation.

Figure 6

Linear ubiquitination of Huntingtin aggregates. Huntingtin (Htt) aggregates are covered by a ubiquitin coat, including K48- and K63-linked chains. HOIP is recruited to these aggregates in a p97/VCP-dependent manner and together with HOIL-1 and SHARPIN assembles M1-linked ubiquitin chains. Subsequent recruitment of proteins specifically interacting with linear ubiquitin, such as NEMO, remodels the interactive surface of the aggregates. Linear ubiquitination promotes proteasomal degradation of misfolded Htt species and may also increase the removal of aggregates by autophagy.

Figure 7

Heterotypic ubiquitin chains implicating M1-linked ubiquitin. Two examples for heterotypic ubiquitin chains containing M1-linked ubiquitin. Linear ubiquitin chains can branch off K63-linked chains via peptide formation between an N-terminal methionine of a ubiquitin molecule within the K63 polyubiquitin and the C-terminal glycine of the incoming ubiquitin (branched chain). Alternatively, the incoming ubiquitin can be added to the N-terminal methionine of the last ubiquitin of the K63-linked chain (mixed chain). G, glycine; K, lysine; M, methionine.

Figure 8

Selected and parallel reaction monitoring (SRM, PRM). The preferred method for the detection and quantification of ubiquitin chains is the use of SRM and PRM. The SRM method is bound to triple-quadrupole mass spectrometers indicated by four rods. The first quadrupole is used for the selection of ions, the second one for the fragmentation of the selected ionized peptide and the third one for the selection of a specific fragment ion. The fragment ions have to be selected one after the other in order to get a measurement for each of them. The PRM method replaces the last step with a scan of all ions in a high-resolution detector like an orbitrap. The selection of the ions is done in silico, so the best ions that show no interference can be selected without re-acquiring the spectra.

Figure 9

Generation of the ubiquitin chain specific peptides. The C-terminus of ubiquitin is bound to a lysine side chain of the previous ubiquitin. Ubiquitin contains a number of arginine residues that are recognized by the protease trypsin. By cutting after arginine 74 the last two amino acids of ubiquitin are remaining on the lysine side chain and create a peptide, which carries two glycines on the ε-amino group of the lysine. This prevents at the same time a digestion of the modified lysine. All key peptides for ubiquitin chains carry the two glycine residues on a specific lysine side chain except for linear ubiquitin, where the ubiquitin is fused head-to-tail. This particular key peptide carries the two glycine residues on the N-terminus.