TY  - THES
AB  - The main topic of this thesis is the tumor suppressor protein APC (Adenomatous Polyposis Coli) and its many different functions. Only little is known about the structural architecture of the 2843 amino acids APC protein. A stable and trypsin-resistent domain close to the N-terminal end, termed APC129-250, could be identified. The crystal structure of APC129-250 could be solved. The domain is monomeric and consists of three [alpha]-helices forming two separate anti-parallel coiled coils. APC129-250 includes the nuclear export signal NES(165-174) at the C-terminal end of the first helix. Interestingly, the conserved hydrophobic amino acids of NES(165-174), which are necessary for the nuclear export activity, are buried in one of the coiled coils. Therefore these residues are not freely accessible for interaction partners. Nevertheless, APC129-250 is able to directly interact with the nuclear export factor Crm1. This interaction is even enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC129-250, which deletes two amino acids forming two of the major hydrophobic interactions within the coiled coil. These results hint to a regulatory mechanism of the APC nuclear export activity by NES masking.
The most important interaction partner of the APC protein is the proto-oncoprotein [beta]-Catenin. Together with other proteins of the Wnt-signaling pathway, APC regulates the intracellular concentration and localization of the [beta]-Catenin protein. Several repetitive motifs within the APC sequence are necessary for this interaction, at least three homologous stretches of 15 amino acids and seven homologous stretches of 20 amino acids. Biophysical measurements show that the third recombinantly expressed non-phosphorylated 20-amino acid repeat of the APC protein is able to bind to [beta]-Catenin in vitro. Surprisingly, phosphorylation is not necessary for the binding itself. Nevertheless, phosphorylation of conserved serines within the repetitive motifs, which was simulated by site-directed mutagenesis of serines to aspartates, increases the affinity of the APC protein to [beta]-Catenin significantly. Though the functional interaction between APC and [beta]-Catenin has been characterized on the cellular level in detail, the stoichiometric relation of this binding is still not known. Biochemical data of this study indicate a 1:1 binding stoichiometry between [beta]-Catenin and the homologous motifs within the APC sequence. The 20-amino acid repeats have similar binding affinities and do not influence each other in their binding to [beta]-Catenin.
Altogether this study answers several important questions around the APC protein and its interaction partners by biophysical and biochemical experiments.
DA  - 2002
KW  - , APC (Adenomatöse Polyposis Coli) , [beta]-Catenin , NES (Nukleäres Export Signal) , Wnt-Signalweg ,
LA  - ger
PY  - 2002
TI  - Das Tumorsuppressor-Protein APC : strukturelle und biochemische Aspekte
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-2099
Y2  - 2024-11-22T03:37:05
ER  -