Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Abstract

Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/ Hsp70 and CHIP-mediated CFTR degradation.Cysteine string protein (Csp, DnaJC5) 2 is a member of the DnaJ/Hsp40 protein chaperone family (1, 2). Csp contains a short N-terminal sequence, followed by the J-domain, a linker region, and the central cysteine-rich region, which gives these proteins their name. The cysteine residues in this region are post-translationally modified by the palmitate groups that serve for the membrane attachment of Csp (3). This structure is followed by a C-terminal domain, which is the most variable region among the three Csp paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic junctions of Drosophila neurons (4). Csps are expressed at high levels in neurons and in cell types involved in regulated exocytosis (5-7), where they have been shown to govern exocytic secretory functions. For example, depolarizationinduced synaptic vesicle exocytosis is impaired in neurons from Csp-deficient Drosophila (8), and Csp overexpression produced decreases in stimulated insulin release from ␤-cells and stimulated catecholamine release from chromaffin cells (9 -11). Deletion of the Csp gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder characterized by developing neurodegenerative changes (12, 13).As for other Hsp40 homologs, the J-domain is responsible for the ability of Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity (14). Similar to other chaperone proteins, Csp can be found in many different protein complexes in the...

CSPα: the neuroprotective J proteinThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…CSPa knock out mice show profound neurodegeneration, which is ameliorated by transgenic expression of a-synuclein. regulator) from the ER, increase CFTR ubiquitination, and target CFTR to the proteosome for degradation (Schmidt et al 2009;Zhang et al 2002a).…”

Chaperone-assisted degradation: multiple paths to destruction

“…In this way, the co-chaperone promotes the degradation of aggregation-prone forms of huntingtin in neuronal cells (Westhoff et al, 2005;Howarth et al, 2007). Another J-protein recently shown to facilitate CHIP-mediated degradation is the cysteine string protein (CSP), which is abundantly expressed in neurons, where it localizes to presynaptic junctions (Schmidt et al, 2009). Deletion of the CSP gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder (Zinsmaier et al, 1994;Fernandez-Chacon et al, 2004).…”

“…Deletion of the CSP gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder (Zinsmaier et al, 1994;Fernandez-Chacon et al, 2004). CSP interacts directly with CHIP and facilitates interactions of CHIP with chaperone clients (Schmidt et al, 2009).…”

Ubiquitination of Inositol-requiring Enzyme 1 (IRE1) by the E3 Ligase CHIP Mediates the IRE1/TRAF2/JNK Pathway

“…It is generally accepted that CHIP is not localized to a specific organelle, whereas it is found on the ER outer membrane (12). Many studies have shown that CHIP is an E3 ubiquitin ligase for endoplasmic reticulum-associated degradation (ERAD) (13,14), a process involved in cell adaptation to ER stress.…”