Investigation of GABARAP complexes with apoptosis-related proteins and structural characterization of GABARAP lipidation
Investigation of GABARAP complexes with apoptosis-related proteins and structural characterization of GABARAP lipidation
Autophagy is a bulky protein degradation mechanism to maintain cellular homeostasis. The first decisive event in autophagy is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. The $\gamma$-aminobutyric acid type A receptor-associa...
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Personal Name(s): | Ma, peixiang (Corresponding author) |
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Contributing Institute: |
Strukturbiochemie; ICS-6 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2011
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Physical Description: |
VI, 139 S : Ill., graph. Darst |
Dissertation Note: |
Universität Düsseldorf, Diss., 2010 |
ISBN: |
978-3-89336-699-6 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
ohne Topic |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health
33 |
Subject (ZB): | |
Publikationsportal JuSER |
Autophagy is a bulky protein degradation mechanism to maintain cellular homeostasis. The first decisive event in autophagy is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. The $\gamma$-aminobutyric acid type A receptor-associated protein (GABARAP) is one of the key proteins in autophagosome formation. Its C-terminus can be conjugated with a lipid via a ubiquitin-like modification process. The lipidated-GABARAP is vital for understanding the mechanisms of protein selective recognition, cellular cargo engulfment and autophagosome formation. Due to the inefficiency of the enzyme catalyzed lipidation process $\textit{in vitro}$, a chemical conjugation method was developed in this work. The lipid conjugated protein was then structurally and functionally investigated in a suitable membrane environment for the first time. The nanodisc technology was employed to mimic the physicochemical environment and native state of the lipidated proteins. One nanodisc encompasses the lipid conjugated GABARAP and about 150 lipids, encircled by two copies of the apolipoprotein A-I derived membrane scaffold proteins (MSP). Using diffusion ordered NMR spectroscopy (DOSY), the lipidated-GABARAP tail was shown to be incorporated into the lipid bilayer. The $^{1}$H line widths of the [U-$^{15}$N]-labeled lipidated-GABARAP are in the range of 40 to 54 Hz. The narrow $^{1}$H line widths demonstrate the superiority of nanodiscs for the study of membrane proteins using solution NMR. The 2D $^{1}$H-$^{15}$N-HSQC spectra revealed that only the residues spatially close to the C-terminus are affected by the lipidation and membrane insertion, while the residues forming the hydrophobic pockets are not affected. Further titration experiments with a calreticulin derived peptide (178-188) indicated that lipidated-GABARAP is still able to interact with its interaction partners via the hydrophobic pockets, which is similar to the unlipidated-GABARAP. From these results, GABARAP is proposed to be able to act as a receptor on the autophagosomal membrane to recognize the interacting proteins and engulf them into autophagosomes. [...] |