Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    CHIP [untagged]
    Catalogue Number:
    63-0003-020
    Size:
    20 µg
    Price:
    £175
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  • Species
    Human
  • Source
    E. coli expression
  • Quantity
    20 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
    ~35 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_005852. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E3 Ligase Assay: The activity of CHIP was validated by its ability to form free polyubiquitin chains in the presence of the E2 enzymes UBE2N, UBE2V1 and ubiquitin (Windheim et al., 2008). Incubation of CHIP enzyme in the presence of ubiquitin, UBE2N, UBE2V1, and ATP at 30°C was compared at two time points, T0 and T60 minutes. The presence of ubiquitin oligomers was observed.

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasomal degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). C-Terminus of Hsc70 Interacting Protein (CHIP) is a member of the E3 protein ligase family and cloning of the human gene was first described by Ballinger et al. (1999). Human CHIP shares 97% and 53% amino acid identity with its mouse and Drosophila homologues respectively with the highest conservation in the 94 residues of the C-terminus. The intrinsic E3 ligase activity of CHIP is conferred though a Ubox domain at the C-terminus of the protein. CHIP interacts with the UBE2D E2 enzyme family targeting the Heat Shock Cognate protein-70 (HSC70) for ubiquitylation (Jiang et al., 2001). Accumulation of PAELR a substrate for the E3 ligase Parkin occurs in the stressed endoplasmic reticulum (ER) causing neurodegeneration. Positive regulation of Parkin activity has been shown to occur through the dissociation of CHIP in complex with Parkin, HSP70 and PAELR in the ER, facilitating Parkin mediated PAELR ubiquitylation (Imai et al., 2002). CHIP co-localises with α-synuclein in Lewy bodies and mediates alpha-synuclein degradation by both the proteasomal and lysosomal pathways (Shin et al., 2005). Cystic fibrosis arises from the misfolding and premature degradation of Cystic Fibrosis Transconductance Regulator (CFTR) carrying the deletion Phe508 (delF508). A cytosolic CHIP/Hsc70 complex cooperates with a ubiquitin ligase complex containing RMA1, UBE2J1, and derlin-1 to monitor the folding status of CFTR and delFl508 in the cytosol and target the mutant form (CFTR-DeltaF508) to the proteosome (Sha et al., 2009; Younger et al., 2006).

References:

Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ, Yin LY, Patterson C (1999) Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol Cell Biol 19, 4535-45.

Imai Y, Soda M, Hatakeyama S, Akagi T, Hashikawa T, Nakayama KI, Takahashi R (2002) CHIP is associated with Parkin, a gene responsible for familial Parkinson’s disease, and enhances its ubiquitin ligase activity. Mol Cell 10, 55-67.

Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, Hohfeld J, Patterson C (2001) CHIP is a U-box-dependent E3 ubiquitin ligase: Identification of Hsc70 as a target for ubiquitylation. J Biol Chem 276, 42938-44.

Sha Y, Pandit L, Zeng S, Eissa NT (2009) A critical role for CHIP in the aggresome pathway. Mol Cell Biol 29, 116-28. 

Shin Y, Klucken J, Patterson C, Hyman BT, McLean PJ (2005) The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways. J Biol Chem 280, 23727-34.

Windheim M, Peggie M, Cohen P (2008) Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem J 409, 723-9.

Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, Fan CY, Patterson C, Cyr DM (2006) Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 126, 571-82.