Abstract
Ubiquitin serves as a signal for a variety of cellular processes and its specific interaction with ubiquitin-binding domain (UBD) regulates key cellular events including protein degradation, cell-cycle control, DNA repair, and kinase activation. Several binding mechanisms for isolated UBDs have been reported in recent years. However, little is known about the mechanism through which proteins containing multiple-UBDs achieve specificity for a particular oligomer of polyUb. The NF-κB essential modulator (NEMO, also known IKKγ), which plays a key role in the NF-κB signaling pathway, belongs to the latter family of proteins since it contains two distal NOA (also known UBAN/CC2-LZ/NUB) and ZF UBDs, separated by an unstructured proline-rich linker of about 40 residues in length. Here, we show a new procedure for fast purification of this bipartite domain. We also describe the use of intrinsic fluorescence spectroscopy for quantitative investigations of ubiquitin interactions between two distal ubiquitin-binding domains of NEMO (NOA and ZF). This spectroscopic method has many advantages over other techniques like GST pulldown and Biacore’s SPR for monitoring avid interactions between two UBDs, especially when UBDs are located at significant distance from each other within the protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Behrends C, Harper JW (2011) Constructing and decoding unconventional ubiquitin chains. Nat Struct Mol Biol 18:520–528
Yamaoka S, Courtois G, Bessia C et al (1998) Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. Cell 93:1231–1240
Rahighi S, Ikeda F, Kawasaki M et al (2009) Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell 136:1098–1109
Dynek JN, Goncharov T, Dueber EC et al (2010) c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J 29:4198–4209
Ngadjeua F, Chiaravalli J, Traincard F et al (2013) Two-sided ubiquitin binding of NF-kappaB essential modulator (NEMO) zinc finger unveiled by a mutation associated with anhidrotic ectodermal dysplasia with immunodeficiency syndrome. J Biol Chem 288:33722–33737
Laplantine E, Fontan E, Chiaravalli J et al (2009) NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain. EMBO J 28:2885–2895
Kastritis PL, Bonvin AM (2013) On the binding affinity of macromolecular interactions: daring to ask why proteins interact. J R Soc Interface 10:20120835
Wienken CJ, Baaske P, Rothbauer U et al (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100–104
Hubeau M, Ngadjeua F, Puel A et al (2011) New mechanism of X-linked anhidrotic ectodermal dysplasia with immunodeficiency: impairment of ubiquitin binding despite normal folding of NEMO protein. Blood 118:926–935
Lo YC, Lin SC, Rospigliosi CC et al (2009) Structural basis for recognition of diubiquitins by NEMO. Mol Cell 33:602–615
Hofmann RM, Pickart CM (2001) In vitro assembly and recognition of Lys-63 polyubiquitin chains. J Biol Chem 276:27936–27943
Burgess RR (2009) Refolding solubilized inclusion body proteins. Methods Enzymol 463:259–282
Hartwig A, Schwerdtle T, Bal W (2010) Biophysical analysis of the interaction of toxic metal ions and oxidants with the zinc finger domain of XPA. Methods Mol Biol 649:399–410
Clarke DT (2011) Circular dichroism and its use in protein-folding studies. Methods Mol Biol 752:59–72
Vinolo E, Sebban H, Chaffotte A et al (2006) A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation. J Biol Chem 281:6334–6348
Acknowledgments
The authors thank Samuel Levy, MD, for his critical reading of the manuscript, and Prof. Alain Israël for fruitful discussions and continuous support. This work was supported, in whole or in part, by the BNP-Paribas Foundation and Institut de Recherches SERVIER (Croissy sur Seine, France).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Dubosclard, V., Fontan, E., Agou, F. (2015). Use of Fluorescence Spectroscopy for Quantitative Investigations of Ubiquitin Interactions with the Ubiquitin-Binding Domains of NEMO. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2422-6_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2421-9
Online ISBN: 978-1-4939-2422-6
eBook Packages: Springer Protocols