Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions - PubMed (original) (raw)

doi: 10.1038/embor.2012.125. Epub 2012 Aug 10.

Georgia Chachami, Lukasz Kozaczkiewicz, Ulrike Winter, Nicolas Stankovic-Valentin, Petra Haas, Kay Hofmann, Henning Urlaub, Huib Ovaa, Joachim Wittbrodt, Erik Meulmeester, Frauke Melchior

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Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions

Sarah Schulz et al. EMBO Rep. 2012 Oct.

Abstract

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l--an essential but distant zebrafish homologue of USPL1--also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Identification of USPL1 as a SUMO isopeptidase candidate. (A) Model: suicide inhibitor Strep-TEV-HA-SUMO-vinylmethylester (SUMO-Vme) attacked by an isopeptidase. (B) SUMO-Vme is crosslinked to a known SUMO isopeptidase. The catalytic fragment GST-SENP1cat was incubated with SUMO1- or SUMO3-Vme. Reaction products were analysed by SDS–PAGE. (C) Sequential IP approach to identify new SUMO isopeptidases. (D) Analysis of peptide eluates (see C) by immunoblotting with anti-HA antibodies. (E) Schematic representation of USPL1 with catalytic domain and Cys–His–Asp triad. (F) Transfected YFP-USPL1 reacts with SUMO-Vme in HEK 293T extracts. Analysis was by immunoblotting with anti-GFP antibodies. (G) USPL1 requires its catalytic cysteine to react with SUMO-Vme. Recombinant USPL1cat (212–514) wt or C236S was incubated with SUMO1- or SUMO3-Vme. Reaction products were analysed by SDS–PAGE. cat, catalytic fragment; GFP, green fluorescence; Gly, glycine; GST, glutathione-_S_-transferase; HA, haemagglutinin epitope; IP, immunoprecipitation; Me, methyl; SENP1, SUMO/sentrin-specific protease 1; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; Strep, streptavidin; SUMO, small ubiquitin-related modifier; TEV, Tobacco Etch Virus protease cleavage site; USPL1, ubiquitin-specific protease like 1; Vme, vinylmethylester; WT, wild type; YFP, yellow fluorescence protein.

Figure 2

Figure 2

USPL1 is a SUMO-specific protease. (A) USPL1 binds SUMO, not ubiquitin. Recombinant USPL1cat and USP5 were incubated with immobilized ovalbumin (OVA), SUMO1, SUMO2 and ubiquitin. Bound proteins were analysed by SDS–PAGE. (B) USPL1cat C236S binds to immobilized SUMO1 and SUMO2. Experiment as in A. (C) USPL1cat cleaves SUMO-, but not ubiquitin-AMC. Ubiquitin-, SUMO1- and SUMO2-AMC (0.25 μM) were incubated with USPL1cat or USP5, and released AMC was detected by fluorescence. (D) USPL1cat matures SUMO. PreSUMO1 or preSUMO2 (8 μM) were incubated with 20 nM SENP1cat or USPL1cat (40, 17 and 860 nM) and samples were analysed by SDS–PAGE. (E) USPL1cat cleaves SUMO3 chains. SUMO3 chains of 12.5 μM were incubated with 5 nM SENP1cat, 5 nM USPL1cat or 150 nM USP5 for the indicated times. Reaction products were analysed by SDS–PAGE. (F,G) USPL1 cleaves sumoylated targets. (F) Two micrometre RanGAP1*SUMO1 or RanGAP1*SUMO2 were incubated with 4 nM USPL1cat for indicated times. Reactions were analysed by SDS–PAGE. (G) YFP-SP100*SUMO2 was incubated with 5 nM SENP1cat or USPL1cat (4.3, 8.6 or 43 nM). Reaction products were analysed by SDS–PAGE. (H) USPL1cat deconjugates SUMO targets in cells. HEK 293T cells were transfected with YFP empty vector, YFP-USPL1cat wt or C236S, and analysed after 24 h by immunoblotting. AMC, 7-amino-4-methylcoumarin; OVA, ovalbumin; preSUMO, precusor of small ubiquitin-like modifier; RanGAP1, Ran GTPase-activating protein 1; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; SP100, nuclear autoantigen Sp100; SUMO, small ubiquitin-related modifier; Ub, ubiquitin; USP5, ubiquitin-specific protease 5.

Figure 3

Figure 3

Zebrafish C13orf22l, a distant USPL1 homologue, is a SUMO-specific protease. (A) Alignment of human USPL1 and zebrafish C13orf22l highlighting conserved regions in the catalytic domain. * indicates residues of the catalytic triad. (B) C13orf22l reacts with SUMO3-Vme via its catalytic cysteine. Extract from untransfected, Flag-C13orf22l wt or C364S-transfected HEK 293T cells were labelled with SUMO1- and SUMO3-Vme, and analysed by immunoblotting. (C) Recombinant C13orf22lcat reacts with SUMO3-Vme. C13orf22lcat was incubated with SUMO1- or SUMO3-Vme. Reaction products were analysed by SDS–PAGE. (D) C13orf22lcat has little activity towards human SUMO propeptides. PreSUMO1 or preSUMO2 (8 μM) was incubated with 860 nM USPL1cat or 1 μM C13orf22lcat. Reaction products were visualized by SDS–PAGE. (E) C13orf22lcat cleaves human SUMO3 chains. SUMO3 chains (25 μM) were incubated with USPL1cat (8.6 nM) or C13orf22lcat (10, 50 or 250 nM). Reaction products were analysed by SDS–PAGE. (F) C13orf22lcat deconjugates human RanGAP1*SUMO2. RanGAP1*SUMO2 of 0.8 μM was incubated with USPL1cat (4.3, 21.5 nM) or C13orf22lcat (5, 25 nM). Products were analysed by SDS–PAGE. C13orf22l, zebrafish USPL1; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; SUMO, small ubiquitin-related modifier.

Figure 4

Figure 4

Identification of USPL1 residues required for SUMO binding and activity. (A) Alignment of USPL1 family members with human USP2 and USP14. * indicates catalytic cysteines. Mutations introduced in hUSPL1cat are indicated on top. (B) Top: His-GST-TEV-USPL1cat wt and indicated mutants (0.5 nM) were incubated with SUMO2-AMC (0.25 μM). Time-dependent release of AMC was detected by fluorescence. Bottom: SDS–PAGE of USPL1cat wt and mutants. (C) His-GST-TEV-USPL1cat wt and indicated mutants were applied to pulldown assays as described in Fig 2, using immobilized ovalbumin and SUMO2; lines separate sets of samples that were analysed on the same SDS gel. His, hexahistidine epitope tag; USP2, 5 and 14, ubiquitin-specific proteases 2, 5 and 14. AMC, 7-amino-4-methylcoumarin; OVA, ovalbumin; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; SUMO, small ubiquitin-related modifier; WT, wild type.

Figure 5

Figure 5

USPL1 is an essential Cajal body protein required for coilin localization and cell proliferation. (A) Affinity-purified anti-USPL1 recognize endogenous and transfected USPL1. Immunoblot analysis of HeLa cells transfected with nonspecific siRNA, siRNA against USPL1 (s2), pcDNA3.1-HA-USPL1 or pEYFP-USPL1. Tubulin was used as loading control. (B,C) Endogenous and transfected USPL1 colocalizes with coilin in Cajal bodies. (B) HeLa cells transfected with control siRNA or USPL1 siRNA s2 were stained with anti-USPL1 and anti-coilin. (C) HeLa cells transfected with pcDNA3.1-HA-USPL1 or pEYFP-USPL1 were stained with anti-HA and anti-coilin antibodies or observed directly (YFP). (D) Knockdown of USPL1 causes coilin mislocalization. HeLa cells were transfected with control siRNA or USPL1 siRNA, and stained with anti-coilin and Hoechst. Distribution of coilin was analysed in >200 cells per condition (three independent experiments). (E) Knockdown of USPL1 impairs HeLa cell proliferation. Analyses at indicated times were with a luminescent cell viability assay. Shown are means (±s.d.) of three independent experiments. (F) Transgenic c13orf22l zebrafish embryos show coilin mislocalization to the nucleolus. Labelling using anti-coilin sera (9EA2) on cryosections from the head of transgenic zebrafish mutants (c13orf22l hi3662Tg/hi3662Tg) and wt siblings (c13orf22l +/+, c13orf22l hi3662Tg/+) at 2 d.p.f. (G,H) Wt and inactive USPL1 variants rescue proliferation and coilin localization. Cells were transfected with USPL1 siRNA (s2), re-transfected with pcDNA3.1-HA-USPL1 variants and (G) analysed at indicated times with a colorimetric assay (normalized to control sample taken at the same times, error bars indicate s.d., five samples per time point), or (H) fixed and stained as indicated. Distribution of coilin was analysed in >200 cells per condition, in three independent experiments. DAPI, 4,6-diamidino-2-phenylindole; HA, haemagglutinin epitope; siRNA, short interfering RNA.

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