Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties
Abstract
Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.
Key words
SPR Protein interactions SUMO-binding entities Affinity constants Avidity MultivalenceNotes
Acknowledgement
We are grateful with Sylviane Hoos for technical Assistance in SPR experiments. UCMB at Inbiomed was supported by the “Obra Social KUTXA” and the Diputación Foral de Gipuzkoa. The authors would like to acknowledge networking support by the PROTEOSTASIS action BM1307, supported by COST (European Cooperation in Science and Technology).
References
- 1.Eifler K, Vertegaal AC (2015) Mapping the SUMOylated landscape. FEBS J 282:3669–3680CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Da Silva-Ferrada E, Lopitz-Otsoa F, Lang V et al (2012) Strategies to identify recognition signals and targets of SUMOylation. Biochem Res Int. doi: 10.1155/2012/875148 PubMedPubMedCentralGoogle Scholar
- 3.Da Silva-Ferrada E, Xolalpa W, Lang V et al (2013) Analysis of SUMOylated proteins using SUMO-traps. Sci Rep. doi: 10.1038/srep01690 PubMedPubMedCentralGoogle Scholar
- 4.Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11:861–871CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Lang V, Aillet F, Da Silva-Ferrada E et al (2015) Analysis of PTEN ubiquitylation and SUMOylation using molecular traps. Methods 77–78:112–118CrossRefPubMedGoogle Scholar
- 6.Kerscher O (2007) SUMO junction-what’s your function? New insights through SUMO-interacting motifs. EMBO Rep 8:550–555CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Tatham MH, Geoffroy MC, Shen L et al (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10:538–546CrossRefPubMedGoogle Scholar
- 8.Keusekotten K, Bade VN, Meyer-Teschendorf K et al (2014) Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO. Biochem J 457:207–214CrossRefPubMedGoogle Scholar
- 9.de Mol NJ, Fischer MJE (2010) Surface plasmon resonance: methods and protocols. Methods Mol Biol 627. Humana. doi: 10.1007/978-1-60761-670-2
- 10.Hutsell SQ, Kimple RJ, Siderovski DP et al (2010) High affinity immobilization of proteins using biotin- and GST-based coupling strategies. In: Clifton NJ (ed) Methods Mol Biol 627:75–90. doi: 10.1007/978-1-60761-670-2_4
- 11.Hjerpe R, Aillet F, Lopitz-Otsoa F et al (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10:1250–1258CrossRefPubMedPubMedCentralGoogle Scholar