Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    ATG7 [6His-tagged]
    Catalogue Number:
    61-0008-050
    Size:
    50 µg
    Price:
    £325
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  • Species
    human
  • Source
    Insect sf21
  • 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
    81.34 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_006386. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E1 Thioester GABARAP Loading Assay: The activity of His-ATG7 was validated by loading GABARAP onto the active cysteine of His-ATG7. Incubation of the His-ATG7 enzyme in the presence of GABARAP and ATP at 30°C was compared at two time points, T0 and T10 minutes. Sensitivity of the GABARAP/His-ATG7 thioester bond to the reducing agent DTT was confirmed.

The enzymes of the autophagy pathway play a pivotal role in the degradation of cytoplasmic constituents and organelles. Structures known as autophagosomes sequester portions of the cytoplasm which are degraded by the lysosome and recycled back into the cell (Kuma et al. 2004). Three classes of enzymes are involved in autophagy; E1-like activating enzymes, E2-like conjugating enzymes and E3-like ligases. Ubiquitin-like proteins (ubl) Autophagy 12 (ATG12) and ATG8 are activated by ATG7 the E1-like activating enzyme. ATG12 and ATG8 are transferred to two E2-like conjugating enzymes ATG10 and ATG3 respectively. Cloning of the human ATG7 gene was first described by Yuan et al. (1999) and ATG7 shares 38% sequence identity to its yeast homologue Apg7. ATG7 can also activate ATG8 mammalian homologues, GABARAP and GATE-16. It forms a homodimer via the C-terminal region that is important for enzyme substrate interaction and E1-E2 complex formation (Komatsu et al. 2005). ATG7DFAP a mutant form of the ATG7 E1-like activating enzyme which lacks the (Phe-Asp-Pro) FAP motif has been shown to be unable to form an E2 substrate intermediate with ATG3 and the  ubl Microtubule-Associated Protein 1, Light Chain 3 (MAPLC3) (Tanida et al. 2012). ATG7 has been shown to be involved in a novel pathway in which the inhibition of caspase-8 results in autophagic death induced by receptor-interacting protein (RIP), Jun amino-terminal kinase, and beclin-1 (Yu et al. 2004).

References:

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, et al. (2005) Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169, 425-434.

Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432, 1032-1036.

Tanida I, Yamasaki M, Komatsu M, Ueno T (2012) The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation. Autophagy 8, 88-97.

Yu L, Alva A, Su H, Dutt P, Freundt E, et al. (2004) Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500-1502.

Yuan W, Stromhaug PE, Dunn WA, Jr. (1999) Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. Mol Biol Cell 10, 1353-1366.