Two Novel Ubiquitin-fold Modifier 1 (Ufm1)-specific Proteases, UfSP1 and UfSP2

Sung Hwan Kang,Gi Ryang Kim,Minu Seong,Sung Hee Baek,Jae Hong Seol,Ok Sun Bang,Huib Ovaa,Kanako Tatsumi,Masaaki Komatsu,Keiji Tanaka,Chin Ha Chung

doi:10.1074/jbc.m610590200

Abstract

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Highlights

deubiquitinating enzymes (DUBs) consist of five families that have distinct catalytic domain structures: the ubiquitin-specific protease family, the ubiquitin C-terminal hydrolase family, the ovarian tumor protease family, the Machado-Joseph disease protein family, and the Jab1/ MPN/Mov34-domain protease family [21,22,23,24,25]
To verify that the trypsin inhibitory activity is mediated by Ubiquitin-fold modifier 1 (Ufm1)-processing activity, His-GST-Ufm1-Ecotin was incubated with the same column fractions for 1 h at 37 °C, and the mixtures were subjected to SDS-PAGE followed by staining with Coomassie Blue R-250
We report the isolation, molecular cloning, and characterization of two novel Ufm1-specific proteases, named UfSP1 and UfSP2

Summary

Introduction

Two Ufm1-specific Proteases, UfSP1 and UfSP2 needs to be processed prior to conjugation to target proteins. Both enzymes could process the C-terminal extension of Ufm1 precursor, generating matured Ufm1 for conjugation to target proteins. For labeling UfSPs with FLAG-Ufm1-VME, enzyme samples were incubated with FLAG-Ufm1-VME for 2 h at 37 °C in 100 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA and 10% glycerol.

Results

Conclusion