Deubiquitination

Given that Ub is covalently-linked to proteins destined to be degraded, it is a surprisingly long-lived protein in vivo. This is due to the removal of Ub from its conjugates by DUBs prior to proteolysis. This may represent a quality control mechanism that prevents the degradation of proteins that were inappropriately tagged for degradation. DUBs are responsible for processing inactive Ub precursors and for keeping the 26S proteasome free of unanchored Ub chains that compete for Ub-binding sites.

DUBs can be grouped into five families based on their conserved catalytic domains. Four of these families are thiol proteases and comprise the bulk of DUBs, while the fifth family is a small group of Ub specific metalloproteases.

Thiol protease DUBs contain a Cys-His-Asp/Asn catalytic triad in which the Asp/Asn functions to polarize and orient the His, while the His serves as a general acid/base by both priming the catalytic Cys for nucleophilic attack on the (iso)peptide carbonyl carbon and by donating a proton to the lysine epsilon-amino leaving group. The nucleophilic attack of the catalytic Cys on the carbonyl carbon produces a negatively charged transition state that is stabilized by an oxyanion hole composed of hydrogen bond donors. A Cys-carbonyl acyl intermediate ensues and is then hydrolyzed by nucleophilic attack of a water molecule to liberate a protein C-terminal carboxylate and regenerate the enzyme. Ub binding often causes structural rearrangements necessary for catalysis. Many DUBs are inactivated by oxidation of the catalytic cysteine to sulphenic acid (single bond SOH). This can be reversed by reduction with DTT or glutathione. The sulphenic acid can be irreversibly oxidized to sulphinic acid (single bond SO2H) or sulphonic acid (single bond SO3H).

Thiol proteases are reversibly inhibited by Ub C-terminal aldehyde, forming a thio-hemiacetal between the aldehyde group and the active site thiol.