Abstract
SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.
Highlights
SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier
The 125I-ubiquitin-PESTc- and 125IpNEDD8-gsPESTc-degrading activities were eluted at several peaks [19], but little or no 125I-pSUMO-1-gsPESTc-degrading activity was detected in all of the column fractions eluted with the salt gradient
We previously reported that ubiquitin-PESTc acts as a substrate for the sensitive and quantitative assays of various deubiquitinylating enzymes (DUBs) and found a set of novel DUBs in the chicken skeletal muscle [17] and bovine brain [19]
Summary
Toshiaki Suzuki‡§, Arata Ichiyama‡, Hisato Saitoh¶, Takayuki Kawakamiʈ, Masao Omataʈ, Chin Ha Chung**, Michio Kimura‡‡, Naoki Shimbara‡‡, and Keiji Tanaka§ §§. The apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp. The apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1 These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1precursor. The ubiquitin-related ligating system for yeast Smt3 [9, 10] and human SUMO-1 [11, 12] were found to be activated by a heterodimeric E1 and conjugated by Ubc as the E2-like enzyme in the ubiquitin pathway. The physiological significance of the newly identified SUMO-1 C-terminal hydrolase is discussed
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