title: Reconstitution of CHIP E3 ubiquitin ligase activity.
abstract: CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation. This chaperone activity can be provided by the Hsp70/Hsp40 molecular chaperone system as seen in the NBD1-R ubiquitination assay. Alternatively, heat treatment of CHIP can activate its own innate substrate-binding activity and allow for efficient ubiquitination of model substrates, such as denatured luciferase. Here, we describe methods for purifying the recombinant proteins necessary for in vitro reconstitution of CHIP ubiquitin ligase activity, as well as two methods used to monitor CHIP ligase activity. One method allows for the measurement of the Hsp70- and Hsp40-dependent CHIP activity while the other measures the Hsp40- and Hsp70-independent activity of heat-activated CHIP.
authors: Department of Cell and Developmental Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors: Hong Yu
authors: HY
authors: Ren
authors: Cam
authors: C
authors: Patterson
authors: Douglas M
authors: DM
authors: Cyr
authors: Meredith F N
authors: MF
authors: Rosser
mesh_terms: D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D044767:Ubiquitin-Protein Ligases