Enabling Protein Degradation Drug Discovery

NEMO [GST-tagged]

 

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    Catalogue Number
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  • Name:
    NEMO [GST-tagged]
    Catalogue Number:
    66-1002-050
    Size:
    50 µg
    Price:
    £250
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  • Species
    human
  • Source
    E.coli
  • Quantity
    50 µg
  • Storage
    -70°C
  • Concentration
    0.5 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    74.9kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: AAD38081. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    Ubiquitin Binding Domain Activity: The ubiquitin chain binding domain activity of GST-NEMO was validated through its ability to capture poly-ubiquitylated IRAK1 from a lysate preparation derived from IL-1 stimulated HEK293 cells. GST-NEMO was pre-incubated with Glutathione Sepharose 4B for 20 minutes at 4°C followed by incubation for 2 hours at 4°C with 2mg IL-1 stimulated HEK293 cell lysate. The binding reaction was then centrifuged and the pellet analysed by SDS-PAGE/ Western blotting (Lane 1). This sample was compared alongside similarlyderived pull-downs from control reactions containing GST-NEMO wild-type versus mutant (D311N) incubated in thepresence of lysates derived from either IL-1 stimulated or nonstimulated HEK293 cells (Lanes 2-4). Ubiquitylated IRAK1 was identified by Western Blotting using an anti-IRAK1 antibody andsuch species were observed only in the pellet sample derived from a binding reaction containing wild-type GST-NEMO and IL-1 stimulated HEK293 cell lysate (Lane 1).

Ubiquitin signals are decoded in cells by at least 200 ubiquitin binding proteins, which interact with different types of polyubiquitin chains and ubiquitin-like modifiers. These interactions induce conformational changes that allow these proteins to transmit the ubiquitin signal to effector proteins (Dikic et al., 2009). NEMO (NFκB Essential Modifier) is the protypic member of a family of proteins that interact with Lys63-linked and linear polyubiquitin chains (Nanda et al.,2011). NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. It is thought that this phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli (Kensche et al., 2012). NEMO is an integral component of the canonical IκB kinase (IKK) complex and is essential for the activation of IKKα and IKKβ, the protein kinase components of the complex. Mutations that abrogate binding of polyubiquitin chains to NEMO do not activate the IKK complex(Ea et al., 2006; Wu et al.,2006) and cause a severe immunodeficiency disease and greatly increased susceptibility to infection by bacteria of  the tuberculosis family (Doffinger et al.,2001). NEMO also interacts with TANK, a component of the IKK-related kinases TBK1 and IKKε (Chariot et al., 2002).

The NEMO-TANK interaction is essential for effective cross-talk between thecanonical IKK complex and the IKK-related kinases which, if disrupted by the loss of TANK, leads to the hyperactivation of the innate immune system and to autoimmune disease (Clark et al., 2011; Kawagoe et al., 2009). NEMO is a powerful reagent for capturing the Lys63linked and linear polyubiquitin chains and their binding partners present in cell extracts. It is recommended that the  NEMO [D311N] mutant, which is unable to bind polyubiquitin chains, is used as a control in such experiments (Windheim  et al., 2008).
References:Chariot A, Leonardi A, Muller J, Bonif M, Brown K and Siebenlist U (2002) Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases. J Biol Chem 277, 37029-37036.
Clark K, Takeuchi O, Akira S and Cohen P (2011) The TRAFassociated protein TANK facilitates cross-talk within the IkappaB kinase family during Toll-like receptor signaling. Proc Natl Acad Sci U S A 108, 17093-17098.
Dikic I, Wakatsuki S and Walters KJ (2009) Ubiquitin-binding domains – from structures to functions. Nat Rev Mol Cell Biol 10, 659-671.
Doffinger R, Smahi A, Bessia C, Geissmann F, Feinberg J, Durandy A, et al. (2001) X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nature Genetics 27, 277-285.
Ea CK, Deng L, Xia ZP, Pineda G and Chen ZJ (2006) Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Molecular Cell 22, 245-257.
Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, et al. (2009) TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis. Nature Immunology 10, 965-972.
Kensche T, Tokunaga F, Ikeda F, Goto E, Iwai K and Dikic I (2012) Analysis of NF-kappaB essential modulator (NEMO) binding to linear and lysine-linked ubiquitin chains and its role in the activation of NF-kappaB. J Biol Chem. 287, 23626-34.
Nanda SK, Venigalla RK, Ordureau A, Patterson-Kane JC, Powell DW, Toth R, et al. (2011) Polyubiquitin binding to ABIN1 is required to prevent autoimmunity. J Exp Med 208, 1215-1228.
Windheim M, Stafford M, Peggie M and Cohen P (2008) Interleukin-1(IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase. Mol Cell Biol 28, 1783-1791.
Wu CJ, Conze DB, Li T, Srinivasula SM and Ashwell JD (2006)  Sensing of Lys 63-linked polyubiquitination by NEMO is a key  event in NF-kappaB activation [corrected]. Nature Cell Biology,8, 398-406.