Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    TOM1 [GST-tagged]
    Catalogue Number:
    66-1015-050
    Size:
    50 µg
    Price:
    £250
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  • Species
    human
  • Source
    E. coli
  • Quantity
    50µg
  • 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
    ~81kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_001129204.1. 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 activity of GST-TOM1 and GST-TOLLIP were validated through their ability to capture poly ubiquitylated IRAK1 from a lysate preparation derived from IL-1 stimulated HEK293 cells. GST-TOM1 and GST-TOLLIP were 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 (Lanes 1 and 2). These samples were compared alongside GST-TOM1 and GST-TOLLIP binding reactions performed with lysates derived from non-stimulated HEK293 cells (Lanes 3 and 4). Ubiquitylated IRAK1 was identified by Western Blotting using an anti-IRAK1 antibody and such species were observed only in the pellet sample derived from a binding reaction containing wild-type GST-TOM1 or GST-TOLLIP and IL-1 stimulated HEK293 cell lysate (Lanes 1 and 3 respectively).

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). Cloning of the human Target Of Myb1 (TOM1) was first described by Seroussi et al. (1999). Human TOM1 shares 76% amino acid sequence identity with chicken TOM1 and 89% identity with mouse TOM1 (Seroussi, et al., 1999). The N-terminal domain of human TOM1 shares sequence similarity to the N-terminal domains of human Signal Transducing Adaptor Molecule (STAM) and Human Growth factor-regulated tyrosine kinase Substrate (HGS) (Seroussi, et al., 1999). TOM1 links polyubiquitin chains to Clathrin (Yamakami, et al., 2003). TOM1 has been shown to bind to human Toll-interacting protein (TOLLIP) via its GAT domain, TOM1 also interacts with Clathrin and when TOM1 and TOLLIP are co-expressed Clathrin is recruited to the endosome suggesting that they may modulate endosomal function (Katoh, et al., 2006). TOM1 directly associates with TOLLIP to form a complex, in which both TOM1 and TOLLIP are capable of directly binding polyubiquitin chains. It is thought that TOM1 is involved in the intracellular sorting of ubiquitylated proteins, analysis of the crystal structure of the TOM1-GAT domain with ubiquitin has revealed the presence of two ubiquitin binding domains (Akutsu, et al., 2005).  

References:  

Akutsu M, Kawasaki M, Katoh Y, Shiba T, Yamaguchi Y, Kato R, Kato K, Nakayama K, Wakatsuki S. (2005) Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain, FEBS letts, 579, 5385-5391.

Katoh Y, Imakagura H, Futatsumori M, Nakayama K. (2006) Recruitment of clathrin onto endosomes by the Tom1-Tollip complex, Biochem Biophys Res Comm 341, 143-149.

Seroussi E Kedra D, Kost-Alimova M, Sandberg-Nordqvist AC, Fransson I, Jacobs JF, Fu Y, Pan HQ, Roe BA, Imreh S, Dumanski JP. (1999) TOM1 genes map to human chromosome 22q13.1 and mouse chromosome 8C1 and encode proteins similar to the endosomal proteins HGS and STAM, Genomics, 57, 380-388.

Yamakami, M., Yoshimori, T. and Yokosawa, H. (2003) Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin, J Biol Chem, 278, 52865-52872.