Structural characterization of human Uch37

Abstract

Uch37 is a deubiquitylating enzyme (DUB) that is functionally linked with multiple protein complexes and signal transduction pathways. Uch37 associates with the 26S proteasome1 through Rpn132 where it serves to remove distal ubiquitin moieties from polyubiquitylated proteins.1 Uch37′s proteasome associated activity was shown to liberate proteins from destruction.1 However, Uch37 may also specifically facilitate the destruction of inducible nitric oxide synthase and IκB-α at the proteasome.3 Wicks et al.4 established Uch37′s potential to modulate the transforming growth factor-β(TGF-β) signaling cascade, through its interaction with SMAD7. Yao et al.5 demonstrated that Uch37 also associates with the Ino80 chromatin-remodeling complex (Ino80 complex), which is involved in DNA repair and transcriptional regulation. Uch37′s importance in metazoan development was underscored recently as Uch37 knockouts in mice result in prenatal lethality, where mutant embryos had severe defects in brain development.6 Protein ubiquitylation is an ATP-dependent post-translational modification that serves to signal a wide variety of cellular processes in eukaryotes.7, 8 A protein cascade, generally comprising three enzymes, functions to activate, transport and specifically transfer ubiquitin to the targeted protein, culminating in an isopeptide linkage between the ε-amino group of a target protein's lysyl residue and the ubiquitin's terminal carboxylate.9 Monoubiquitination plays an important role in histone regulation, endocytosis, and viral budding.10 Further processing of the target protein may be accomplished by ubiquitylation of the protein on a different lysine, or through the formation of polyubiquitin chains,10 where the best-characterized outcome is destruction of the polyubiquitin-labeled protein in the proteasome.9 DUBs catalyze the removal of ubiquitin from proteins. This activity serves to reverse the effects of ubiquitination, permit ubiquitin recycling, or liberate free ubiquitin after translation.11 Uch37, is a cysteine protease from the ubiquitin C-terminal hydrolase (UCH) family.7 The catalytic domain of UCH enzymes has a central, six-stranded, antiparallel β-sheet that is flanked on each side by α-helices.12 Like picornain 3C, UCH family proteins contain a catalytic triad comprising the side chains of cysteine, histidine, and aspartate. In picornain 3C, the aspartate's carboxylate side chain acts to position histidine's imidazole group for general base catalysis, which activates cysteine for nucleophilic attack.13 UCH enzymes also contain a glutamine, which may serve to stabilize the tetrahedral transition state.11 The UCH family can be divided into two distinct groups, that is, enzymes solely composed of the globular UCH fold, or enzymes comprising the UCH domain and a C-terminal extension. The latter group can be subdivided based on the composition of the C-terminal extension.14 Uch37 carries a C-terminal extension. Until now, the structure of a full-length UCH from this structural class has not been reported. In this article, we report the X-ray crystal structure of full-length human Uch37 at 2.95 Å resolution. This model provides a structural rationale for the accessibility of Uch37′s C-terminus for Rpn13 binding. The globular catalytic domain of full-length Uch37 is similar to those of all UCHs characterized to date.12, 15-17 The C-terminal extension is elongated, predominantly helical and as predicted contains coiled coils.11 Chromatographic analysis of Uch37 demonstrated that Uch37′s oligomeric state varies as a function of concentration. Uch37′s specific activity is sensitive to Uch37 concentration in the absence of a nonspecific binding agent, but nearly constant and an order of magnitude higher in the presence of BSA. These data show that activity assays of isolated Uch37 at low protein concentration suffers from a serious systematic error and suggests that the effects of Uch37 binding proteins on Uch37′s activity from pr