Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    LC3b [untagged]
    Catalogue Number:
    60-0112-500
    Size:
    500 µg
    Price:
    £175
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  • Species
    human
  • Source
    E.coli
  • Quantity
    500 µg
  • Storage
    -70°C
  • Concentration
    1 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~14.84 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_073729.1. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E1 Thioester LC3b Loading Assay: The activity of LC3b was validated by loading LC3b onto the active cysteine of His-ATG7. Incubation of the His-ATG7 enzyme in the presence of LC3b and ATP at 30°C was compared at two time points, T0 and T10 minutes.  Sensitivity of the LC3b/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 (E1s), E2-like conjugating enzymes (E2s) and ubiquitin-like proteins (ubls). Microtubule Associated Protein 1, Light Chain 3 beta, MAP1LC3b or LC3b is a member of the Ubl family and cloning of the human gene was first described by He et al. (2003). The C-terminal of LC3b is cleaved by ATG4B C-terminal to Gly120 in vitro and this has been shown to be required for the formation of intermediates with the ubiquitin-like activating enzyme ATG7 (Tanida et al. 2004). A proteomic analysis of the autophagosome interaction network was performed on a human cell line undergoing a basal level of autophagy. This study revealed that six of the ATG8 homologues (including LC3b) interacted with a cohort of 67 proteins in which there was frequent involvement with a conserved ATG8 surface region shown previously to interact with LC3 interacting regions in partner proteins (Behrends et al. 2010).
References:

Behrends C, Sowa ME, Gygi SP, Harper JW (2010) Network organization of the human autophagy system. Nature 466, 68-76.

He H, Dang Y, Dai F, Guo Z, Wu J, et al. (2003) Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B. J Biol Chem 278, 29278-29287.

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, Ueno T, Kominami E (2004) Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes. J Biol Chem 279, 47704-47710.