Some unanchored polyubiquitin chains are synthesised by the sequential reactions of two enzymes: E1 (the ubiquitin activating enzyme) and E2 (the ubiquitin conjugating enzyme). Pioneering work from Cecile Pickart showed that E1 and E2-25K synthesize unanchored homogeneous Lys48-linked chains, while ligation of similar chains linked through Lys63 requires the UBC (ubiquitin C)13/MMS2 heterodimer as the E2 [17
]. Many other isolated E2s synthesize multiple types of linkages. The length and linkage type of polyubiquitin chains can also be modulated by a third enzyme, the E3 ubiquitin ligase. In most cases, this E3 ligase is required for assembly of a polyubiquitin chain on a protein acceptor.
Chains can also be remodelled or disassembled by various deubiquitinating enzymes, proteases that cleave the (iso)peptide bond between ubiquitins or ubiquitin and a target protein. Deubiquitinating enzymes can show considerable specificity. For instance, isopeptidase T (USP5 [ubiquitin-specific protease 5]) only acts efficiently on free polyubiquitin chains but shows little linkage selectivity [18
]. Proteasomal deubiquitinating enzymes require association with the proteasome for maximal efficiency although the precise chain specificity is still unclear. Structural analysis of several deubiquitinases explains why TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage, Cezanne is specific for Lys11 linked chains [19
], CYLD prefers Lys63-linked chains [20
], and OTU1/Otubain is selective for long Lys48-linked chains [21
]. In many other cases the deubiquitinating enzyme's specificity is either minimal or has not been determined. Thus, the type of polyubiquitin chain that is accumulated will depend on the specificity of the enzymes that synthesize the chain (E2/E3) and disassemble it (deubiquitinating enzymes).
Indeed, because of the sequential action of various deubiquitinases (deubiquitinating enzymes) and/or conjugating enzymes/ligases, the length and linkage composition of a given chain can be dynamic [23
]. For instance, the promiscuous UBCH5 family of E2s combines with either of two E3 ligases, CIAP (a cellular inhibitor of apoptosis) or APC (the anaphase promoting complex), to monoubiquitinate their respective substrates [24
]. Subsequently Lys11-linked chains are elaborated by these E3s using the E2 enzyme UBE (ubiquitin-activating enzyme)2S. BRCA1/BARD1 autoubiquitination also shows this progression of priming (monoubiquitination) followed by elongation (polyubiquitination). Six different E2s can monoubiquitinate BRCA1, while different E2s are then used to elongate the chains with Lys63 (UBC13/MMS2), Lys48 (UBE2K), or Lys6 (UBCH5) linkages [28
]. Certain pairs of E2 and E3 can also synthesize branched chains [16
]. In another classic example of chain remodelling, A20 (a large enzyme with both deubiquitinating enzymes and E3 ligase activity) is thought to first trim K63-linked substrates of the NF-κB (nuclear transcription factor kappa B) pathway and then elaborate a K48-linked chain that results in the degradation of the substrate.