DeSUMOylating isopeptidase: a second class of SUMO protease

Abstract

The modification of proteins by small ubiquitin-like modifier (SUMO) is crucial for the regulation of diverse cellular processes. Protein SUMOylation is reversed by isopeptidases, collectively known as deSUMOylases. Only one family of SUMO-specific proteases has been described so far: the sentrin-specific proteases (SENP). Here, we identify and characterize a new deSUMOylase, which we have named DeSI-1 (DeSumoylating Isopeptidase 1). We describe BZEL—a new transcriptional repressor—as substrate of DeSI-1. DeSI-1 catalyses the deSUMOylation, but not the deubiquitination, of BZEL. Furthermore, the SENP substrates PML and ΔNp63 are not deSUMOylated by DeSI-1, suggesting that SENP and DeSI enzymes recognize different sets of substrates. Together, these data identify a second class of SUMO proteases.

Figure 1

Identification of DeSI. (A) Schematic representation of the structure of DeSI-1 and DeSI-2. (B) Sequence alignment of DeSI-1 and DeSI-2. Amino acid identity is highlighted in black. The histidine and cysteine residues that form the catalytic site of cysteine protease are marked by asterisks. (C) Tissue expression pattern of DeSI-1 and DeSI-2. Murine tissue lysates were subjected to immunoblot analysis using rabbit anti-DeSI-1 or anti-DeSI-2 antibodies. Equal protein loading was shown by immunoblotting with anti-actin antibody. (D) Subcellular localization of DeSI-1 and DeSI-2. 293T cells were transfected with expression plasmids encoding Myc-tagged DeSI-1 or DeSI-2. Cells were visualized by staining with anti-Myc antibody along with Alexa 568-conjugated secondary antibody (red). (E) Homodimerization of DeSI-1 (left panel). The chromatogram shows that full-length DeSI-1 (Mr=20 kDa) was eluted as a ∼40-kDa protein from a Superdex 75 column. The arrowheads indicate the elution positions of the size marker proteins (right panel); 293T cells were transfected with the indicated combinations of expression plasmids encoding DeSI-1–FLAG and DeSI-1–Myc. Cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody and subsequently to immunoblot analysis with anti-Myc or anti-FLAG antibodies. DAPI, 4,6-diamidino-2-phenylindole; DeSI-1, DeSumoylating Isopeptidase 1; IB, immunoblot; WCE, whole-cell extract.

Figure 2

DeSI-1 acts as a desumoylase but not as a deubiquitinase of BZEL. (A) DeSI-1 associates with BZEL. Cells (293T) were transfected with the indicated combinations of expression plasmids encoding DeSI-1 and BZEL. Cell lysates were subjected to immunoprecipitation with the anti-Myc antibody and subsequently to immunoblot analysis with anti-FLAG (upper panel) or anti-Myc (middle panel) antibody. As a control for DeSI-1 expression, whole-cell extract (WCE) was analysed by immunoblotting with anti-FLAG antibody (lower panel). (B) DeSI-1 lacks the ability to deubiquitinate ubiquitin-modified BZEL. Cells (293T) were transfected with the indicated combinations of expression plasmids encoding DeSI-1, BZEL and/or ubiquitin. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody and subsequently to immunoblot analysis with anti-HA (upper panel) or anti-Myc (middle panel) antibody. (C) DeSI-1 desumoylates SUMO1-modified BZEL. Cells (293T) were transfected with the indicated combinations of expression plasmids encoding DeSI-1, BZEL and/or SUMO1. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody and subsequently to immunoblot analysis with anti-HA (upper panel) or anti-Myc (middle panel) antibody. (D) DeSI-1 desumoylates SUMO1-modified BZEL in a dose-dependent manner. The experiment was performed as described in C, except that cells were transected with increasing amounts of DeSI-1 (0, 4, 8 or 12 μg) or SENP1 (0, 2, 4, or 6 μg) expression plasmids. The expression levels of SENP1 and DeSI-1 in WCE were analysed by western blotting with anti-FLAG antibody (bottom two panels). (E) DeSI-1^C108S lacks desumoylase activity against SUMO-modified BZEL. Cells (293T) were transfected with the indicated combinations of expression plasmids encoding DeSI-1, DeSI-1^C108S, BZEL and/or SUMO1. Cell lysates were processed as described in B. (F) DeSI-1 desumoylates SUMO2-modified BZEL. The experiment was performed as described in C, expect that SUMO2 was coexpressed instead of SUMO1. (G) DeSI-1 desumoylates SUMO3-modified BZEL. The experiment was performed as described in C, except that SUMO3 was coexpressed instead of SUMO1. BZEL, BTB-ZF protein expressed in effector lymphocytes; DeSI-1, DeSumoylating Isopeptidase 1; HA, haemagglutinin; IB, immunoblot; IP, immunoprecipitation; SENP1, sentrin-specific proteases 1; SUMO1, small ubiquitin-like modifier 1.

Figure 3

In vitro desumoylation assay using recombinant DeSI-1 and BZEL from E. coli. (A) DeSI-1 desumoylates SUMO1-modified BZEL in vitro. A concentration of 10 μM of purified GST–BZEL fusion protein from E. coli was sumoylated in vitro. Subsequently, sumoylated BZEL was incubated with 0, 0.1 or 0.4 μM of DeSI-1or DeSI-1^C108S mutant purified from E. coli for 1 h at 37°C. After incubation, the reaction mixture was analysed by immunoblotting with anti-GST antibody. The arrow indicates the position of sumoylated BZEL. (B) DeSI-1 formed covalent adducts with SUMO1-VS but not with Ub–VS. Recombinant purified GST–DeSI-1, or DeSI-1^C108S mutant was incubated with 0, 1.5 and 3 μg each of SUMO1–VS or Ub–VS for 1 h at 37°C. After incubation, immunoblot analysis was performed with anti-GST antibody. (C) DeSI-1 lacks the SUMO-processing activity. A measure of 30 μg of SUMO precursors (SUMOs–His) was incubated for 1 h with 2 μg each of GST–DeSI-1, GST–DeSI-2 or GST–SENP1C purified from E. coli. Subsequently, the reaction mixtures were subjected to SDS–PAGE and stained with Coomassie blue. The size of the SUMO proteins generated by cleavage after the C-terminal diglycine motif is marked by an arrow. (D) DeSI-1 cleaves polymeric SUMO2/3 chains. A measure of 0.5 μg each of poly-SUMO2 or poly-SUMO3 chains was incubated with DeSI-1 or SENP6C at three different enzyme concentrations (0, 0.5 and 5 nM). Subsequently, the reaction mixtures were analysed by immunoblotting using anti-SUMO2/3 antibody. BZEL, BTB-ZF protein expressed in effector lymphocytes; DeSI-1, DeSumoylating Isopeptidase 1; E. coli, Escherichia coli; GST, glutathione S-transferase; His, histidine; IB, immunoblot; SENP1, sentrin-specific proteases 1; SUMO1, small ubiquitin-like modifier 1; Ub, ubiquitin; VS, vinyl sulphone.

Figure 4

PML and ΔNp63, substrates of SENP, are not desumoylated by DeSI-1. (A) 293T cells were transfected with the indicated combinations of expression plasmids encoding PML, SUMO1, DeSI-1 and/or SENP1. Cell lysates were subjected to immunoprecipitation with the anti-Myc antibody, followed by immunoblot analysis with anti-HA or anti-Myc antibodies. To control for the expression of DeSI-1 and SENP1, WCE was analysed by immunoblot (IB) analysis using the anti-FLAG antibody. (B) The experiment was performed as described in A, except that SuPr1 was expressed instead of SENP1, and the sumoylation of ΔNp63, instead of PML, was analysed. DeSI-1, DeSumoylating Isopeptidase 1; HA, haemagglutinin; IP, immunoprecipitation; SENP1, sentrin-specific proteases 1; SUMO1, small ubiquitin-like modifier 1; WCE, whole-cell extract.

Figure 5

DeSI-1 modulates the transcriptional repressor activity of BZEL. (A) Raji B cells were transfected with reporter genes driven by the blimp-1 promoter along with a combination of expression plasmids encoding BZEL (7 μg), DeSI-1 (7 μg) and DeSI-1^C108S (7 μg). As a control for the transfection efficiency, the cells were also cotransfected with pCMV-β-galactosidase control vector. Subsequently, cell lysates were assayed for luciferase activity, and the luciferase activity was normalized against β-galactosidase activity. Values are the averages of three independent experiments performed in duplicate. Error bars represent standard deviations. (B) A20 B cells were electroporated with reporter genes driven by the blimp-1 promoter along with a combination of expression plasmids encoding BZEL (2 μg), shDeSI-1–1 (10 μg), shDeSI-1–2 (10 μg) or control shRNA (10 μg). Subsequently, luciferase activity was determined as described in A. Values are the averages of three independent experiments performed in duplicate, and error bars representing standard deviations are shown. Data were also analysed by Student's t-test; *P<0.005 and **P<0.02. (C) Cell lysates from the experiment shown in B were subjected to immunoprecipitation (IP) with anti-BZEL antibody and subsequently to immunoblot (IB) analysis with anti-SUMO1 (upper panel) or anti-BZEL (lower panel) antibodies (left). As a control for DeSI-1 expression, whole-cell extracts (WCE) were analysed by immunoblotting with anti-DeSI-1 or anti-actin antibody (right). BZEL, BTB-ZF protein expressed in effector lymphocytes; DeSI-1, DeSumoylating Isopeptidase 1; shRNA, short hairpin RNA; SUMO1, small ubiquitin-like modifier 1.