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. 2012 Jul:Chapter 10:Unit10.29.
doi: 10.1002/0471142727.mb1029s99.

Biochemical analysis of protein SUMOylation

Aileen Y Alontaga  1 , Ekaterina Bobkova, Yuan Chen
Affiliations

Biochemical analysis of protein SUMOylation

Aileen Y Alontaga et al. Curr Protoc Mol Biol. 2012 Jul.

Abstract

SUMOylation, the covalent attachment of Small Ubiquitin-like MOdifier (SUMO) polypeptides to other proteins, is among the most important post-translational modifications that regulate the functional properties of a large number of proteins. SUMOylation is broadly involved in cellular processes such as gene transcription, hormone response, signal transduction, DNA repair, and nuclear transport. SUMO modification has also been implicated in the pathogenesis of human diseases, such as cancer, neurodegenerative disorders, and viral infection. Attachment of a SUMO protein to another protein is carried out in multiple steps catalyzed by three enzymes. This unit describes and discusses the in vitro biochemical methods used for investigating each step of the SUMOylation process. In addition, a high-throughput screening protocol is included for the identification of inhibitors of SUMOylation.

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Figures

Figure 1
Figure 1
Schematic of the SUMOylation process with the protocols corresponding to the specific steps indicated. SUMOylation occurs in several steps. In the first step, SUMO (S) proteins are matured by SUMO specific proteases (SENPs). In the second step, E1 catalyzes the formation of SUMO adenylate in which the C-terminal –COOH group of SUMO is covalently linked to AMP. SUMO adenylate binds to E1 noncovalently. Then, SUMO is transferred to the active site Cys of E1, forming a thioester conjugate with E1. Both steps are reversible. In the third step, SUMO is transferred from E1 to E2, where it forms a thioester conjugate with the catalytic Cys residue of E2. This step is also reversible. In the final step, SUMO is ligated to target proteins by the formation of an isopeptide bond between its C-terminal –COOH group and the 3-amino group of a Lys residue on the target protein. This step generally requires an E3 ligase. The numbers in the yellow boxes represent the protocols outlined in this unit.
Figure 2
Figure 2
Outline of Basic and Alternate Protocols 1. Both assays are used to measure E1-catalyzed ATP:PPi exchange rate but the detection method for the radioactive ATP produced in the reactions are different. Steps 1–6 are the same for both protocols. In Basic Protocol 1, after the reaction is quenched, [32P]ATP is adsorbed by a charcoal slurry and detected using Cherenkov counter. In Alternate Protocol 1, the [32P]ATP is adsorbed in an activated charcoal paper and detected using a PhosphoImager.
Figure 3
Figure 3
Plots of E1-catalyzed ATP:PPi isotope exchange assay at different SUMO concentrations. The plots are generated based on the assays outlined in Basic and Alternate Protocols 1. (A) Plot of rate of ATP:PPi exchange (pmole/min) versus SUMO concentration (μm). (B) Double-reciprocal (Lineweaver-Burk) plot of (A) to linearized the data and determine the kinetic constants (K1/2 and Vmax).
Figure 4
Figure 4
ATP:AMP exchange assay using thin layer chromatography. This scheme illustrates the steps describes in Basic Protocol 2. The reaction mixture containing radioactive [14C]AMP was incubated at 37 ºC for 10 min and then quenched with 8 M urea. Three 4 μl aliquots were taken from the reaction mixture and loaded onto a PEI-cellulose TLC plate. The radioactive AMP and radioactive ATP were separated by a mixture of 0.5 M LiCl and 1 M formic acid (v/v) and imaged using a PhosphorImager. AMP and ATP were separated based from their retention factor (Rf). AMP has a higher Rf value compared to ATP.
Figure 5
Figure 5
E1•SUMO thioester formation assay. Half of the reaction mixture (see Basic Protocol 3) was added to 2X SDS sample buffer containing no DTT and loaded directly onto an SDS-PAGE gel (left lane), while the other half was mixed with 2X SDS sample buffer containing 100 mM DTT and heated at 95 °C for 5 min (right lane) before loading onto the gel. The sample on the right lane is a control to identify the thioester bond between the SAE2 subunit of E1 and SUMO. This assay can quickly detect dramatic changes of E1 activity, and determine the percentage of active E1.
Figure 6
Figure 6
Detection of Alexa680-labeled SUMO transfer from E1 to E2 (Ubc9). E1•SUMO thioester is formed initially as described in Basic Protocol 4. Then the reaction is quenched with EDTA, and E2 (Ubc9) was added to a final concentration of 0.2 μM. Aliquots of the reaction mixture were withdrawn at 0, 15, 30, 45 and 60 s after E2(Ubc9) addition and mixed with 2X SDS sample buffer (containing no DTT) with 8 M urea. Steps 8 and 9 should be performed at 4 ºC. Formation of the E2•SUMO complex was resolved by gel electrophoresis and visualized with the Odyssey infrared imaging system. Quantification of the transfer assays was carried out using ImageQuant 5.2 software.
Figure 7
Figure 7
E2 (Ubc9)•SUMO thioester formation assay. The reaction mixture (see Basic Protocol 5) was incubated at 37 °C for 0, 2 and 15 min. Reactions were quenched using 2X SDS buffer. The bands were resolved by SDS-gel electrophoresis and stained with SimplyBlue. This assay can provide a quick assessment of all SUMOylation steps prior to E2•SUMO thioester formation.
Figure 8
Figure 8
Temperature-dependent kinetic measurements of the E3 ligase (RanBP2). Assay conditions are described in Basic Protocol 7. (A) Immunoblots used to detect GST-Sp100-SUMO conjugation at four Sp100 concentrations (2.1, 4.2, 4.6 and 8.4 μM) and at different temperatures (25, 28, 31, and 373C). (B) Plots of [E]/Vo versus 1/[GST-Sp100] based on data from (A) are linear which is indicates that the conditions are appropriate for Michaelis-Menten kinetics. The Km/kcat ratios are measured from the slope of the double-reciprocal plots (Lineweaver-Burk), and correspond to the net transfer rate constant for SUMO transfer from E2 to substrates.
Figure 9
Figure 9
TR-FRET biochemical assay to monitor protein SUMOylation.
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