Meredith F.N. Rosser

Sequential Quality-Control Checkpoints Triage Misfolded Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis arises from the misfolding and premature degradation of CFTR Delta F508, a Cl- ion channel with a single amino acid deletion. Yet, the quality-control machinery that selects CFTR Delta F508 for degradation and the mechanism for its misfolding are not well defined. We identified an ER membrane-associated ubiquitin ligase complex containing the E3 RMA1, the E2 Ubc6e, and Derlin-1 that cooperates with the cytosolic Hsc70/CHIP E3 complex to triage CFTR and CFTR Delta F508. Derlin-1 serves to retain CFTR in the ER membrane and interacts with RMA1 and Ubc6e to promote CFTRs proteasomal degradation. RMA1 is capable of recognizing folding defects in CFTR Delta F508 coincident with translation, whereas the CHIP E3 appears to act posttranslationally. A folding defect in CFTR Delta F508 detected by RMA1 involves the inability of CFTRs second membrane-spanning domain to productively interact with amino-terminal domains. Thus, the RMA1 and CHIP E3 ubiquitin ligases act sequentially in ER membrane and cytosol to monitor the folding status of CFTR and CFTR Delta F508.

Chaperone functions of the E3 ubiquitin ligase CHIP.

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination.

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508s correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTRs membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the ΔF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of ‘corrector’ drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients.Publication history: Republished from Current BioDatas Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Cystic Fibrosis Transmembrane Conductance Regulator as a Model Substrate to Study Endoplasmic Reticulum Protein Quality Control in Mammalian Cells

Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum (ER) to select misfolded proteins for degradation. Herein we describe methods that allow for the study of the pathway for proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). The experimental system described employs transiently transfected HEK-293 cells and is utilized to monitor the biogenesis of CFTR by Western blot and pulse-chase analysis.

Analysis of CFTR Folding and Degradation in Transiently Transfected Cells

Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.

The Use of Small Molecules to Correct Defects in CFTR Folding, Maturation, and Channel Activity

Cystic Fibrosis, one of the most common inherited lethal disease among Caucasians, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR protein acts as a gated Cl - channel at the apical membrane of epithelial cells, thereby facilitating proper hydration of mucosal linings. Disease causing muta- tions in the CFTR protein can affect a variety of steps in the biogenesis of a functional protein including the folding and trafficking of CFTR as well as the channel activity of plasma membrane-localized protein. Therefore, current research is focused on the use of small molecules to not only correct folding defects but also to enhance channel activity of mutant CFTR proteins. This review discusses the current knowledge of the folding, trafficking, and gating defects caused by CFTR mutations, the manner by which these defects are monitored by the cell, as well as the strategies which are cur- rently being utilized to develop and screen for small molecule therapeutics.

Do Hsp40s Act as Chaperones or Co-Chaperones?

The Hsp70 family plays an essential role in cellular protein metabolism by acting as a polypeptide binding and release factor that interacts with nonnative regions of proteins at different stages of their life cycles.1 Hsp40 proteins not only act as co-chaperones to facilitate complex formation between Hsp70 and client proteins, but it has also been proposed that Hsp40s use an intrinsic chaperone activity to bind and deliver the nonnative substrates to Hsp70. Herein, we review genetic, biochemical and structural data that describes the mechanisms by which Type I and Type II Hsp40 proteins act to bind substrates. The manner by which the functions of the Type I and Type II proteins are specified is also discussed.

Reconstitution of CHIP E3 Ubiquitin Ligase Activity

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.