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P1‐061: STUDIES OF STRUCTURAL CHANGES IN AB PEPTIDE DURING INTERACTION WITH LIPID MEMBRANES AND CHANNEL FORMATION
Author(s) -
Przygonska Kaja,
Kulma Magda,
Tarnowski Krzysztof
Publication year - 2014
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2014.05.297
Subject(s) - liposome , chemistry , membrane , peptide , biophysics , intracellular , biochemistry , neurodegeneration , lipid bilayer , vesicle , western blot , microbiology and biotechnology , biology , medicine , disease , gene
LRP1 as described for Fe65. Methods: Interaction between GULP1 molecules was analysed usingGULP1 constructs carrying two different epitope tags (HA-tagged or GFP-tagged). To confirm GULP1-dimerization we employed a protein fragment complementation assay (PCA) where GULP1 was fused to non-bioluminescent amino-or carboxy-terminal fragments of humanized Gaussia Luciferase (hGLuc) or non-fluorescence amino-or caboxy-terminal fragments of Venus-YFP. Specific GULP1-dimerization reconstitutes luciferase/venus and provides a measurable bioluminescent/ fluorescence signal. Results: Upon co-immunoprecipitation, we observed that the GFP-tagged GULP1 was precipitated with HA-tagged GULP1. To confirm our results we did a protein complementation assay. We observed GULP1 dimerization in living cells and found that co-expression of APP enhances dimerization of GULP1. Furthermore, we analysed localization of GULP1 dimers by confocal microscopy as well as nuclear extraction assays showing that GULP1 dimers can be found both in the cytosol and the nucleus. Complex formation of APP, GULP1 and LRP1 was analysed by triple-immunoprecipitation. We could show that GULP1 functions as a molecular bridge between APP and LRP1. Conclusions: Taken together, these data identify the dimerization properties of GULP1 in vivo. GULP1 forms dimers/oligomers both in the cytosol and the nucleus and the dimerization is further promoted by co-expression of APP. Additionally, GULP1 dimerization enables simultaneous binding of both APP and LRP1.