Recognition of Polyubiquitin Isoforms by the Multiple Ubiquitin Binding Modules of Isopeptidase T

Abstract

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpydriven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.

Ubiquitin-interacting Motifs Confer Full Catalytic Activity, but Not Ubiquitin Chain Substrate Specificity, to Deubiquitinating Enzyme USP37

“…Otherwise, binding of ubiquitin to the UIMs may allosterically activate the enzyme by inducing a conformational change of the catalytic domain. In the case of USP5, binding of monoubiquitin to the zinc finger domain, located adjacent to the catalytic core, elevates its DUB activity toward a synthetic substrate, ubiquitin-amidomethyl coumarin, suggesting that ubiquitin binding allosterically activates USP5 (21,22). We, however, did not observe an elevated activity of purified USP37 toward ubiquitin-amidomethyl coumarin in the presence of monoubiquitin (data not shown).…”

“…It has two tandem ubiquitin-associated (UBA) domains located at similar positions to UIM2 and UIM3 in USP37 (namely, positioned ϳ35 amino acids before the His box and spaced by a 26-amino acid sequence). The UBA domains in USP5 facilitate the binding of USP5 to ubiquitin oligomers (21). Therefore, although no sequence homology is found between the USP37 UIM region and the USP5 UBA region (data not shown), they possibly have the same biological function.…”

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

“…On the other hand, the yeast homolog of USP14, Ubp6, regulates the overall rate of proteolysis and appears critical in replenishing the free ubiquitin pool in yeast (6,76). Similarly, USP5 hydrolyzes anchorless ubiquitin chains (78,79) and appears to work downstream of Rpn11, an intrinsic DUB on the proteasome, to prevent the binding of free ubiquitin chains to the 19S, which would inhibit proteolysis. Together these data illustrate important roles for deubiquitylation in the aged muscle, probably ensuring a supply of ubiquitin for enhanced proteolysis but perhaps also serving additional regulatory functions.…”

Deubiquitylases From Genes to Organism

“…Asterisks mark two similar cases where the preferred accession shown is not the UniProt reference genome for D. rerio. selectivity for unanchored ubiquitin and is necessary for optimal catalytic activity (207), whilst in combination with other ubiquitin binding domains it contributes to a high avidity for tetra-ubiquitin (208). However, it is possible that the ZnF-UBP domain also presents a protein interaction module for the 72 other human proteins which possess COOH-terminal di-Gly motifs.…”