Dokument: Strukturelle und funktionelle Analyse des molekularen Chaperons Calnexin
| Titel: | Strukturelle und funktionelle Analyse des molekularen Chaperons Calnexin | |||||||
| Weiterer Titel: | Structural and Functional Analysis of the Molecular Chaperone Calnexin | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=15165 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20100519-094638-2 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dipl.-Biol. Brockmeier, Achim [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Schmitt, Lutz [Gutachter] PD Dr. Schulte, Ulrich [Gutachter] | |||||||
| Stichwörter: | Calnexin, Endoplasmic reticulum, Glycoproteins, Protein folding, Quality control | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site as well as through a polypeptide-binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated client proteins at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. This thesis focuses on the ability of calnexin to interact with client proteins in a lectin-independent manner under physiological conditions of the ER lumen and provides new mechanistic insights into its molecular chaperone function.
(1) The successful development of a new aggregation suppression assay permitted the demonstration that the soluble ER-luminal domain of calnexin (S-Cnx) can indeed suppress the aggregation of a non-glycosylated client protein at physiological temperature and at the resting ER Ca2+ concentration of 0.4 mM. However, gradual reduction of Ca2+ below the resting level resulted in an increased aggregation suppression potency but was accompanied by a progressive loss of native S-Cnx structure as assessed by biochemical and biophysical studies. These assays permitted the characterization of a single Ca2+-binding site on S-Cnx with a Kd of 0.15 mM. (2) Furthermore it was shown that the aggregation suppression potency of S-Cnx is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to S-Cnx in the presence of 0.4 mM Ca2+ is 0.7 mM. In contrast to Ca2+, ATP did not alter the overall stability of S-Cnx but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings exemplify that calnexin can act as a potent molecular chaperone that is capable of suppressing the aggregation of client proteins through polypeptide-based interactions under conditions that exist within the ER lumen. (3) Since the luminal portion of calnexin consists of two domains, a globular domain, which comprises the lectin site, and an extended arm domain, it was of great interest to understand the function of these domains during interactions of calnexin with non-native protein conformers. For this reason the aggregation suppression abilities of various deletion mutants of S-Cnx were tested in the newly established aggregation assay. The arm domain alone showed no capacity to suppress aggregation. However, stepwise truncation of the arm domain in S-Cnx resulted in a progressive reduction in aggregation suppression potency. Thus aggregation suppression appears to be primarily mediated by the globular domain, but the arm domain is required for maximal function. (4) To characterize the putative polypeptide-binding site, hydrophobic peptides were used that were effective competitors of the ability of S-Cnx to suppress client protein aggregation. Direct binding experiments revealed a single site of peptide binding in the globular domain (Kd = 0.9 μM) at a location distinct from the lectin site. (5) Progressive truncation of the arm domain in S-Cnx had no effect on the binding of small peptides but reduced the binding affinity of S-Cnx for large, non-native client proteins. Because client proteins/peptides exhibited no binding to the isolated arm domain, the findings support a model in which calnexin suppresses aggregation through a polypeptide-binding site in its globular domain, with the arm domain enhancing aggregation suppression by sterically constraining large client proteins. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie | |||||||
| Dokument erstellt am: | 19.05.2010 | |||||||
| Dateien geändert am: | 17.05.2010 | |||||||
| Promotionsantrag am: | 22.01.2010 | |||||||
| Datum der Promotion: | 04.05.2010 |
