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. 2010 Sep 1;430(2):335-44.
doi: 10.1042/BJ20100504.

Distribution and paralogue specificity of mammalian deSUMOylating enzymes

Nagamalleswari Kolli  1 , Jowita Mikolajczyk, Marcin Drag, Debaditya Mukhopadhyay, Nela Moffatt, Mary Dasso, Guy Salvesen, Keith D Wilkinson
Affiliations

Distribution and paralogue specificity of mammalian deSUMOylating enzymes

Nagamalleswari Kolli et al. Biochem J. .

Abstract

The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA-SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogenous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

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Figures

Figure 1
Figure 1. Human SENP proteases
The white bars represent nonconserved N-terminal regions. The conserved catalytic domain is shown in grey. Note the insertions in the catalytic domains of SENPs 6 and 7 shown in black. Green circle indicates SUMO-binding Motifs.
Figure 2
Figure 2. Irreversible labeling of the catalytic domains of SENPs with HA-SUMO-VS
A–E) Recombinant cSENPs (cS) 1, 2, 5, 6, and 7 were reacted with HA-SUMO1-VS and HA-SUMO2-VS for 15 minutes at 25°C. Samples were run on 12% SDS-PAGE and visualized by Imperial protein stain. Asterisks show adduct formation with all cSENPs indicates reactivity with SUMO1-VS and SUMO2-VS. F) FLAG-cSENP3WT and FLAG-cSENP3C-A (active site mutation) were reacted with HA-SUMO1-VS and SUMO2-VS for 15 minutes at 25°C. The reactions were monitored by immunoblotting using anti-FLAG antibody. FLAG-cSENP3 WT formed adduct with both SUMO1-VS and SUMO2-VS, whereas FLAG-cSENP3CA did not form adducts. Asterisks indicate the vinylsulfone adducts.
Figure 3
Figure 3. Kinetic constants for cSENP catalyzed hydrolysis of SUMO-AMC Summary of steady state kinetic constants for the hydrolysis of SUMO1-AMC and SUMO2-AMC by the catalytic domains of human SENPs
A) (kcat/KM) ratio, SUMO2/SUMO1. B) kcat ratio, SUMO2/SUMO1
Figure 4
Figure 4. Paralog preference of endogenous SENPs
A–B) HEK293 lysate (30μg) was titrated with HA-SUMO2-VS and reacted for 15 minutes. Adduct formation was monitored by 12% SDS-PAGE and western blotting with anti-SENP3 and SENP5 antibodies in separate experiments. Lanes 5–6 of panel A indicates that all endogenous SENP3 in the lysate reacted with SUMO2-VS. Similarly in Figure 4B lanes 5–6 show that all endogenous SENP5 reacted with SUMO2-VS. C–H) HeLa cell lysate was incubated with HA-SUMO1-VS or HA-SUMO2-VS for 15 minutes. The incorporation of SUMO-VS into SUMO-specific proteases was monitored by 12% SDS-PAGE and immunoblotting with anti-SENPs 1, 2, 3, 5, 6 and 7 antibodies.
Figure 5
Figure 5. N-terminus of SENP1 and SENP2 contributes to SUMO paralog specificity
Full-length His6-SENP1 and His6-SENP2 recombinant proteins, purified by nickel resin, were labeled with HA-SUMO1 or HA-SUMO2-VS. Reactions were terminated with sample buffer and analyzed by western blotting with anti-HA antibody. Asterisks indicate probable degradation products.
Figure 6
Figure 6. SUMO2 specific SENPs are more abundant than SUMO1 specific SENPs in mammalian cell lysates
HA-SUMO1-VS or HA-SUMO2-VS (5ng) were reacted with HEK293, HeLa, LM TK−, A9 and Cos7 cell lysates. Reaction mixtures were analyzed by 12% SDS PAGE and western blotting with Anti-HA antibody. The expected migration for each adduct is indicated by the arrows.
Figure 7
Figure 7. Detection of SENPs in rabbit tissue extracts
Rabbit tissue extracts were prepared and incubated with HA-SUMO1-VS or HA-SUMO2-VS. Reactions were terminated and resolved by 12% SDS-PAGE and probed with anti-HA antibody. No SUMO-VS was added in the first lane in each panel. The arrows mark the positions of the observed SUMO-VS adducts. The asterisk marks non-specific covalent labeling with SUMO-VS.
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