Please use this identifier to cite or link to this item: http://dx.doi.org/10.18419/opus-774
Authors: Gnann, Andreas
Title: Identifizierung neuer Faktoren, die am Abbau von humanem CFTR in Hefe beteiligt sind
Other Titles: Identification of new factors that are involved in the degradation of human CFTR in yeast
Issue Date: 2004
metadata.ubs.publikation.typ: Dissertation
URI: http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-19107
http://elib.uni-stuttgart.de/handle/11682/791
http://dx.doi.org/10.18419/opus-774
Abstract: Mukoviszidose oder Cystische Fibrose (CF) ist die häufigste Erbkrankheit der weißen Erdbevölkerung. Ursache von CF sind Mutationen des ABC-Transporters Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), der primär als Chloridkanal in apikalen Epithelien verschiedener Gewebe fungiert. Die mit 70% häufigste Mutation ist die Deletion eines Phenylalanins an Position 508 (?F508) in der Nukleotidbindedomäne 1. Diese führt zu einer Fehlfaltung des Proteins, die von der zellulären Protein-Qualitätskontrolle (QC) erkannt und über die ER-assoziierte Degradation (ERAD) dem Ubiquitin-Proteasom-System zum Abbau zuführt wird. Die genaue Aufklärung dieser Mechanismen ist ein wesentlicher Punkt in der Mukoviszidose-Grundlagenforschung. Denn ?F508-CFTR ist trotz Fehlfaltung grundsätzlich funktionsfähig und könnte durch gezielte Beeinflussung dieser Mechanismen vor dem Abbau geschützt und in der Folge austransportiert werden. Da die Prozesse der QC und der ERAD in Hefe wesentlich besser charakterisiert sind und nötige Mutanten vorhanden oder leichter zu generieren sind, wurde das humane Protein in der Bäckerhefe Saccharomyces cerevisiae exprimiert. Die Mechanismen von QC und ERAD sind von der Hefe bis zum Menschen hoch konserviert. Frühere Studien belegen, dass in der Hefe Saccharomyces cerevisiae exprimiertes CFTR von Hefe-QC und ERAD erkannt und auch hier über den Ubiquitin-Proteasom-Weg abgebaut wird. In dieser Arbeit wurden neue Faktoren identifiziert, die an Qualitätskontrolle und ERAD von CFTR beteiligt sind. Es konnte gezeigt werden, dass das Hefelektin Htm1p in den Abbauprozess von CFTR involviert ist. Die Komplementation einer Htm1p-Defizienz in Hefezellen durch die Expression des Säuger-Orthologs EDEM bestätigte nicht nur die Bedeutung dieser Lektine für den Abbau von CFTR, sondern belegte zudem die hohe Übereinstimmung von QC und ERAD zwischen Hefe und Säuger. Weiterhin wurde gezeigt, dass der Abbau von CFTR von den E3-Ligasen Der3p/Hrd1p und Doa10p abhängig ist. Der trimere Proteinkomplex Cdc48-Ufd1-Npl4 spielt eine entscheidende Rolle für den Abbau von CFTR ebenso, wie für einen verwandten ABC-Transporter der Hefe, Pdr5p.
The endoplasmic reticulum (ER) is responsible for folding, modification and delivery of secretory proteins to their site of action (Glick, 2002; Haigh and Johnson, 2002). It contains a highly active protein quality control system (QC) which scans the folding process of secretory proteins and retains those species which are unable to fold (Elgaard and Helenius, 2003). They are eliminated by a process called ER associated degradation (ERAD) via the ubiquitin proteasome system (Kostova and Wolf, 2003). Malfunction of these processes is the cause of many diseases (Kostova and Wolf, 2002; Rutishauser and Spiess, 2002). One of the most common hereditary diseases amongst the white population which is directly linked to QC and ERAD is cystic fibrosis (Kerem et al., 1989; Riordan et al., 1989). Here a mutated ABC transporter and chloride channel of the apical membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized by the QC as being malfolded and degraded via the ubiquitin-proteasome system. The most frequent mutation of CFTR which causes this effect is the deletion of a phenylalanine at position 508 in the first nucleotide binding domain (?F508). Interestingly even wild-type CFTR is very unstable and shows a degradation rate of about 70% (Jensen et al., 1995; Ward and Kopito, 1994). It is important to note that ?F508 CFTR can still function as a chloride channel (Dalemans et al., 1991) but due to QC and ERAD it fails to be transported to the apical plasma membrane. This could be overcome by lower temperature (Denning et al., 1992), high glycerol (Sato et al., 1996) or 4-phenylbutyrate (Rubenstein and Zeitlin, 2000). As none of these treatments is of therapeutic value other means have to be found to promote transport of mature ?F508 CFTR to the plasma membrane. Elucidation of the components of QC and ERAD which are responsible for the elimination of ?F508 CFTR is an important step in this direction. They may become targets for specific therapeutic manipulations to lead mutated but active CFTR to the cell surface. Previous studies have shown that the lectin calnexin interacts with CFTR in the ER lumen (Pind et al., 1994). Furthermore the bidirectional Sec61 translocon of the ER membrane was suggested to contribute to QC and ERAD (Bebök et al., 1998) as well as the cytosolic chaperones Hsp70/Hsc70, Hsp90 and the co-chaperone CHIP (Yang et al., 1993; Meacham et al.,1999; Loo et al., 1998; Meacham et al., 2001). Cellular QC and ERAD are highly conserved mechanisms from yeast to man (Ellgaard and Helenius, 2003; Kostova and Wolf, 2002, 2003). The ready availability of yeast mutants defective in QC and ERAD makes this organism a preferred model for investigation of these processes. Indeed the expression of CFTR in Saccharomyces cerevisiae has proven that the yeast components of QC and ERAD recognize this protein and degrade it via the proteasome in a ubiquitin dependent manner (Kiser et al., 2001; Zhang et al., 2001). These studies have uncovered the yeast ubiquitin conjugating enzymes Ubc6p and Ubc7p as well as the cytosolic Hsp70 as necessary components for the degradation of CFTR. Influence of the ubiquitin protein ligase Der3p/Hrd1p in CFTR degradation is somewhat controversial (Kiser et al., 2001; Zhang et al., 2001). Recently the mammalian counterparts of yeast Ubc6p, Ubc7p and Der3p/Hrd1p have been identified (Lenk et al., 2002; Fang et al., 2001; Tiwari and Weissman, 2001). Thus Saccharomyces cerevisiae proves to be an excellent model organism to further investigate the components of the QC and ERAD which are required for the degradation of CFTR. Mutants defective in newly discovered components of these processes have enabled testing of their involvement in CFTR degradation. These new experiments reveal that the ubiquitin protein ligases Der3p/Hrd1p and Doa10p seem to have a synergistic effect on the degradation of the CFTR protein. Furthermore the cytosolic trimeric Cdc48-Ufd1-Npl4 complex is found to be crucially required for proteasomal elimination of the protein. In addition the QC and degradation process of CFTR is considerably disturbed in a mutant defective in the ER lumenal lectin Htm1p. Most interestingly this defect can be complemented by the expression of the mammalian EDEM protein, showing that Htm1p and EDEM are functional homologues with respect to CFTR degradation.
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