Ubiquitin - Ubiquitin Chains - K63-Linked Chains

Ub modification is required for ATP-dependent (non-lysosomal) proteolysis by the proteasome, and also the regulation of activity of proteins in non-proteosomal pathways. The overall process of ubiquitin-dependent metabolism is a multi-enzymatic process requiring the successive activities of distinct conjugating (E1s, E2s , E3s) and deubiquitinating enzymes DUBS. The type and number of poly-ubiquitin chains that are conjugated to a target is highly regulated to generate distinct signals that affect different physiological processes. This versatility arises from the fact that not only can targets be mono-ubiquitinated or poly-ubiquitinated, but also that different types of poly-ubiquitin chains are formed. Proteins tagged with Ub are most often destined for degradation by the proteasome. However, mono-ubiquitination and poly-ubiquitination events also have non-proteasomal regulatory functions like targeting proteins to nucleus, cytoskeleton and endocytic machinery, or modulating enzymatic activity and protein-protein interactions.

Ub is synthesized intracellularly as an inactive precursor. The mature form is produced by the activity of DUBs which make specific cleavages at the C-teminus to expose the conserved di-glycine motif (G75-G76). Mutation or removal of these glycine residues results in an inactive Ub that is not a substrate for the conjugation enzymes. Much of the chemistry of Ub conjugation is confined to the conformationally flexible and accessible C-terminal Gly75-Gly76 residues, and the seven internal lysine residues which are critical for the formation of poly-ubiquitin chains. There are also important residues that form a hydrophobic patch on Ub which influences Ub structures and its interaction with itself and numerous other proteins that interact with Ub. The Ub C-terminal glycine residues are necessary for the activation of the Ub by its activating E1 enzyme. This activated Ub is then transferred to E2 and E3 enzymes via thioester chemistry to modify Ub or substrate proteins on specific lysine residues.

Multi-ubiquitin chains are built by formation of an isopeptide bond between Gly76 of one ubiquitin to the ε-NH2 group of one of the seven potential lysines (K6, K11, K27, K29, K33, K48 or K63) of the preceding ubiquitin. All lysine can participate in Ub chain formation, but five (K6, K11, K29, K48 and K63) are known to be major sites of initiation and these linkages exist in vivo as anchored and unanchored species. The solved structures of all known Ub chains are unique, strongly suggesting that the formation and hydrolysis of each linkage is catalyzed by a specific set of conjugation, enzymes and DUBs. In addition, for those linkage formation and hydrolytic processes that require interaction with other receptor or auxiliary proteins, a unique and specific protein would be required for each linkage type. Moreover, the different Ub structures possessed by Ub polymers likely play a role in targeting substrates to specific cellular pathways. Linkage-specific chains can either be assembled on the target protein one Ub at a time or transferred en masse as a pre-constructed, unanchored chain. In some cases the mechanism of assembly also involves E4 enzyme elongation factors. Residue K48 is a major site of chain initiation and K48 linkages are highly abundant, being the predominant signal for proteins destined for degradation by the proteasome. The other principle and relatively abundant poly-ubiquitin chain has K63 linkages. K63 linkages do not seem to play a role in protein turnover and have been implicated in receptor endocytosis and sorting, translation, DNA damage repair, the stress response and signaling through the TRAF pathway of NF-κB. Since K48- and K63-linked chains have such different targeting functions, they may have unique chain structures which result in selective recognition by regulatory components. Ub polymers with K6, K11 and K29 linkages are minor species in vivo and there is currently no evidence that K27 and K33 are used to form ubiquitin-ubiquitin isopeptide linkages. More recently, novel Ub chains have been identified and these include non-degradable “forked” chains with heterogeneous linkages.

Proteins with mutations at the seven Ub lysines (K6, K11, K27, K29, K33, K48 and K63) that can participate in chain formation are useful reagents for in vitro studies. These mutants are designed to implicate given lysine residues in poly-ubiquitin chain initiation and conjugation events. The lysine (K) to arginine (R) mutants renders Ub unable to form multi-ubiquitin chains via that specific lysine with other Ub molecules. However, these proteins can still be linked to the lysine residues on target proteins formed via the remaining Ub lysine residues that have not been mutated. The lysine (K) only mutants can only form poly-ubiquitin chains with other Ub molecules via that single lysine, since all other lysines are not present. These mutants are ideal for the reduction in poly-ubiquitin chain length or conjugation rates and determining if the chains have a specific linkage requirement. Importantly, since the C-terminal residues are intact, these proteins are fully functional for activation and thiolester formation by E1, E2 and E3 conjugating enzymes. These mutants can also be used in binding studies to determine affinities with proteins that contain Ub recognition domains. These reagents are valuable for the determination of structure-function requirements in chain synthesis, recognition or disassembly.

There are also non-lysine residues that are critical for Ub function and structure. There are three key residues (L8, I44 and V70) that form a repeating hydrophobic patch that mediates intra-chain interactions, and thus is necessary and important for chain recognition by various factors. Mutation of I44 affects the structure of this hydrophobic surface and affects the recognition of Ub chains by ubiquitin-binding domains such as: ubiquitin interacting motifs (UIMs), ubiquitin associated domains (UBAs), coupling of ubiquitin to ER degradation (CUE), Nlp14 zinc finger (NZF), ubiquitin E2 variant (UEV) or proteasome subunits such as S5a. There is another hydrophobic surface defined by Phe4 that is required specifically for non-proteasome-dependent functions such as endocytosis and internalization, which often involves mono-ubiquitination. D58 is a residue that has been identified as being important for binding and recognition by proteins that contain Ub binding domains (UBDs), and represents a new hydrophilic interaction surface on ubiquitin. This residue may be crucial for the interaction and recognition of poly-ubiquitin chains by the 26S proteasome and other enzymes involved in ubiquitination.

The chemical or enzymatic modification of Ub proteins at important functional sites results in several useful derivatives. Modifying the C-terminal glycine carboxyl of ubiquitin to an aldehyde Ub-H or vinylsulfone Ub-VS groups results in highly potent inhibitors of DUB enzymes. Alternatively, substrates for the kinetic analysis of DUBs are generated by synthetically conjugating fluorophores to the C-terminus of ubiquitin (Ub-AMC, Ub-AFC, Ub-R110). In addition, reductive methylation of the lysine amine groups prevents the formation of poly-ubiquitin chains via lysine linkages. Ubiquitin can also be coupled to agarose via its primary amines, leaving the C-terminus free and available to purify ubiquitin binding proteins. The N-terminus or lysine groups of Ub can be modified by small other haptens (fluorescein/rhodamine or biotin) for the sensitive detection by direct fluorescence or secondary reagents (eg. avidin or streptavidin reagents) respectively. These groups can also be used for the affinity purification of Ub conjugates or Ub binding proteins. Since the chemistry of ubiquitin conjugation involves its C-terminus, these modifications result in a fully functional molecule that is viable in conjugation reactions.