Regulation of proteolysis by human deubiquitinating enzymes

Abstract

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USPs) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Keywords: Deubiquitinating enzyme; Poly-ubiquitin; Proteolysis; Regulation; Ubiquitin.

Figure 1

A given substrate (S) can exist in the free state or bound in a complex (C), each location containing its own distinct E3 and DUB. The strength of binding can be different for the free (black line) or ubiquitinated (green line) substrate. Depending on the value of these binding constants, ubiquitination can either recruit or release S from C. Ubiquitinated protein can be directly bound by the proteasome (red line) or shuttled to the proteasome (dashed blue line) if C is a ubiquitin receptor.

Figure 2

Ubiquitin binding sites within deubiquitinating enzymes. (A) Di-ubiquitin is depicted to exemplify the proximal ubiquitin (Ub1) and distal ubiquitin (Ub2). The proximal ubiquitin is the initial ubiquitin within a chain, while the distal ubiquitin is final ubiquitin within a chain. The proximal ubiquitin is tethered to the substrate in an anchored poly-Ub chain, or contains a free C-terminal carboxylate in an unanchored chain. (B) A general scenario depicting a thiol DUB catalyzing the hydrolysis of a di-ubiquitin chain. The nomenclature used to describe ubiquitin binding sites within DUBs stems from work on the serine protease papain and its recognition sites for peptide substrates. Like papain, the DUB active site must contact residues flanking the scissile bond, and the S1 and S1’ sites are attributed to the acyl-intermediate side and the leaving group side of the scissile bond respectively. The S1 site binds the ubiquitin containing carboxylate of the hydrolyzed (iso)peptide bond, and the S1’ site binds the ubiquitin containing the amine of the peptide bond. Thus S1 site binds to the distal ubiquitin within a chain, and the S1’ site binds the proximal ubiquitin. It should be noted the S1’ site in DUBs can be formed by residues outside the catalytic domain or S1‘ can reside within the catalytic domain. (C) IsoT/USP5 has four ubiquitin binding sites. IsoT/USP5 is one example of a DUB containing S2 and S3 binding sites which, in this case, are formed by two UBA domains that are inserted within the catalytic USP domain. IsoT/USP5 cannot hydrolyze substrate-anchored poly-Ub as its N-terminal ZnF-UBP domain forms direct interactions with the ubiquitin C-terminal carboxylate.

Figure 3

The Josephin domain (JD) contains multiple ubiquitin binding sites. (A) NMR solution structure of the Ataxin-3 Josephin domain with residues of the catalytic triad shown as spheres (PDB 2AGA). (B) An adopt an alternative conformation (PDB 1YZB). (C) Solution structure of Ataxin-3 JD non-covalently associated with two ubiquitin molecules (PDB 2JRI). One ubiquitin is bound at the S1 site and its C-terminal tail bisects the catalytic Cys-His pair, possibly a reflection of the product complex that follows hydrolysis of the isopeptide bond. A second ubiquitin is bound at the S2 site of the Josephin domain, and its C-terminus is juxtaposed with K48 of ubiquitin bound at the S1 site. (D) Crystal structure of the Ataxin-3L JD covalently bound to Ub-chloroethylamine (PDB 3O65). This structure reveals the Ataxin-3L JD binds ubiquitin at the S1 site using different residues and positions ubiquitin in an alternate conformation.

Figure 4

The OTUB1 deubiquitinase binds charged E2s and inhibits their transfer of ubiquitin to E3. (A) The human OTUB1 structure (PDB 2ZFY) with side chains of the catalytic triad (C91, H265, and D267) depicted as spheres. The structure was solved using an OTUB1 deletion lacking the N-terminal 39 residues, which form the N-terminal helix and the S1‘ site. (B) The structure of human OTUB1 bound to UbcH5bC85S-Ub (covalently linked to UbcH5b S85 by an oxyester bond) and free Ub (PDB 4DDI). The E2 UbcH5b and its linked Ub form contacts with the N-terminal helix, and this E2-conjugated Ub binds the S1’ site with K48 positioned towards the catalytic triad and the C-terminus of ubiquitin at the S1 site. (C) The structure of human/worm OTUB1 bound to Ubc13-UbG75C (covalently linked to Ubc13 C87 with dichloroacetone) and Ub-aldehyde (PDB 4DHJ). The hybrid OTUB1 contains the first 45 residues from human OTUB1 followed by the OTU domain from worm OTUB1. Ub aldehyde is covalently bound at the S1 site. OTUB1 binds both Ub-charged E2s in a similar fashion, with K48 of the E2-linked Ub positioned towards the active site and the C-terminus of Ub bound at the S1 site. The binding and arrangement of two ubiquitin molecules at the S1 and S1’ sites in these two structures mimics OTUB1’s natural substrate K48-linked poly-Ub.