A New 30-kDa Ubiquitin-related SUMO-1 Hydrolase from Bovine Brain

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-␣NH-HSTVG-SMHISPPEPESEEEEEHYC and/or GST-SUMO-1-35 SRanGAP1 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.

SUMO: of branched proteins and nuclear bodies

“…Since then, numerous additional enzymes have been isolated from yeasts and mammalian cells. (Li and Hochstrasser, 2000;Suzuki et al, 1999;Gong et al, 2000b;Nishida et al, 2000;Kim et al, 2000;Schwienhorst et al, 2000). These have been variously called SENPs (for`Sentrin-speci®c protease'; Gong et al, 2000b;Yeh et al, 2000), SUSPs (SUMOspeci®c protease; Kim et al, 2000) or SMT3IP1 (Nishida et al, 2000).…”

SENP1 mediates TNF-induced desumoylation and cytoplasmic translocation of HIPK1 to enhance ASK1-dependent apoptosis

“…5 Different members of these SUMO-specific proteases appear to localize in different cellular compartments where they regulate protein function by modifying the protein stability, cellular localization, and protein-protein interactions. 5,6,[8][9][10][11][12] However, it is not known how these SUMO-specific proteases are regulated.HIPK1 is one of three closely related serine/threonine protein kinases that are primarily localized in the nucleus where it is sumoylated. 13,14 The HIPKs were originally identified as nuclear protein kinases that function as co-repressors for various homeodomain-containing transcription factors.…”

“…4 Several members of SUMOspecific proteases have been reported in the mammalian system. [5][6][7][8][9][10] SENP1 is a protease that appears to deconjugate a large number of sumoylated proteins. 5 Different members of these SUMO-specific proteases appear to localize in different cellular compartments where they regulate protein function by modifying the protein stability, cellular localization, and protein-protein interactions.…”

“…5 Different members of these SUMO-specific proteases appear to localize in different cellular compartments where they regulate protein function by modifying the protein stability, cellular localization, and protein-protein interactions. 5,6,[8][9][10][11][12] However, it is not known how these SUMO-specific proteases are regulated.…”

Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2

“…This interaction is probably due to lower-affinity ionic interactions and made the purification of the sumoylated fraction of the LANA2 protein difficult. Taken together, these results demonstrated that LANA2 is sumoylated by SUMO1 and SUMO2 in vitro and by SUMO2 in vivo.To confirm that endogenous LANA2 protein is sumoylated in KSHV-infected cells, protein extracts from BC-1 cells prepared in the presence of N-ethylmaleimide, which has been previously shown to stabilize SUMO-modified proteins (Suzuki et al, 1999), were immunoprecipitated with anti-LANA2 antibody. Western-blot analysis of the immunoprecipitates using anti-LANA2 antibody revealed a major band of approximately 75 kDa, corresponding to LANA2, and two additional slower-migrating bands: one broad band of about 95-100 kDa that might correspond to the band detected in the in vitro sumoylation assay after incubation with SUMO1 and a light band of about 120 kDa (Fig.…”