Ubiquitination is a post-translational modification in which the ubiquitin protein (Ub) is attached to an acceptor lysine residue in the substrate. Ub itself has seven lysine residues and an N-terminal methionine that can serve as acceptors for another Ub; thus, chains with different linkages and different functional outcomes may form. In particular, K48-linked chains target their substrates for proteasomal degradation. Ub conjugates can be removed by deubiquitinating enzymes (DUBs), of which five families have been identified in the human genome.

Kulathu and colleagues have now identified a new DUB family, named MINDY (for MIU-containing novel DUB family), that has selectivity toward long K48 Ub chains (Mol. Cell doi:10.1016/j.molcel.2016.05.009).

The authors started by investigating a previously uncharacterized protein, FAM63A, which bears tandem motifs interacting with Ub (MIUs; blue boxes in the domain-architecture schematics at top). A fragment containing the MIU motifs specifically binds K48 chains. FAM63A also contains a domain of unknown function, DUF544 (yellow box), and a conserved cysteine (red line), which together are part of a catalytic domain that displays cysteine protease activity with specificity toward K48 linkages. Other members of the family that were identified in humans and other eukaryotes on the basis of sequence similarity showed comparable properties in vitro.

Credit: Adapted with permission from Elsevier.

To understand how FAM63A (renamed MINDY-1) recognizes and cleaves K48 Ub chains, the authors solved the crystal structure of its catalytic domain, both alone (model, bottom left) and in a covalent complex with a modified Ub moiety. The structures reveal a new cysteine protease folding variant that has no structural similarity to other DUB families; they also indicate how substrate binding activates the enzyme.

Furthermore, detailed biochemical analysis showed that MINDY-1 trims K48-linked Ub chains from the distal end. This activity requires chains with at least four Ub moieties, because the tandem MIU motifs bind a K48 di-Ub, and the catalytic domain binds another K48 di-Ub and cleaves the intervening linkage (bottom right). Importantly, coimmunoprecipitation assays on cell lysates demonstrated that MINDY-1 recognizes K48-linked polyubiquitinated proteins in a manner that requires the MIU motifs.

The identification of a new family of DUBs paves the way for functional studies to bring their cellular roles to the fore.