CHIP [6His-tagged]

Produktübersicht

• Artikelname: CHIP [6His-tagged]
• Artikelnummer: UBI-63-0001-025
• Hersteller Artikelnummer: 63-0001-025
• Alternativnummer: UBI-63-0001-025
• Hersteller: Ubiquigent
• Kategorie: Sonstiges
• Spezies Reaktivität: Human

Zusatzinformationen

• Species

  Human

• Source

  E. coli expression

• Quantity

  25 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

  ~38.3 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 ubiquitin conjugating activity of His-CHIP was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D3 (UbcH5c) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of His-CHIP for 30 minutes at 30°C in the presence of ubiquitin, His-UBE1, His-UBE2D3 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or His-CHIP (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of ATP (lanes 1 and 3).  Ubiquitin conjugates were detected in the absence of His-CHIP (lane 3) but they appeared to be more extensive in the presence of the E3 ligase.

 

Background:

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent 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 through 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 in the cytosol and target the mutant form (CFTR-DeltaF508) to the proteasome (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.

 

Produkteigenschaften

• Konzentration: 0.5 mg/ml
• Molekulargewicht: ~38.3 kDa
• NCBI: <a href=https://www.ncbi.nlm.nih.gov/gene/NP_005852 target=_blank rel=nofollow>NP_005852
• Quelle: E. coli expression
• Sequenz: Accession number: NP_005852. For full protein sequence information download the Certificate of Analysis pdf.