The Amyloid Precursor Protein Has a Flexible Transmembrane Domain and Binds Cholesterol Science 336, 1171 (2013) Coach Prof. : Dr. Chung-I Chang Sit-in Prof.: Dr. Wei Yuan Yang Presenter: Han-Ying Wu Date: 11:00am to 12:00am, Oct. 9, 2013 1
Outlines What is Amyloid Precursor Protein? The relationship between Aβ and APP and AD The importance of studying the structure of ADD The method and instruments Nuclear Magnetic Resonance Spectroscopy (NMR) Electron paramagnetic resonance (EPR) The complex of C99 Results Discussion The importance of binding Cholesterol Conclusion 2
Amyloid Precursor Protein (APP) This region contians surfaced α-helix, random loops, and TMD Amyloid Precursor Protein (APP) is a single transmembrane protein that undergoes sequential proteolysis to generate multiple peptides, including the amyloid β peptide (Aβ) Hartmann T, et. Nat. Med. 1997,3 (9): 1016 20. 3
Amyloid-β Amyloid beta (Aβ) is a peptide of 36 43 amino acids that is processed from the Amyloid precursor protein (APP). It is also the notorious hall-mark of Alzheimer s disease(ad) Hartmann T, et. Nat. Med. 1997,3 (9): 1016 20..Lichtenthaler SF. Curr Alzheimer Res. 2011;9(2):165 177 4
Aβ related disease Extracellular free Aβ Aβ oligomers (soluble) Aβ fibrils (insoluble) Abnormal signal transductions, increased oxidative stress, disrupted Ca 2+ homeostasis Neuron dysfunction and degeneration Mattson, M. P. (2004) Pathways towards and away from Alzheimer s disease. Nature 430, 631 639. Weiner, H. L., and Frenkel, D. (2006) Immunology and immunotherapy of Alzheimer s disease. Nat. Rev. Immunol. 6, 404 416 5
Why to study the conformation of APP In previous studying, the main focus is the fragment containing TMD, others tried to employed by low-resolution methods In order to gain further understanding of Aβ aggregation, it is necessary for scientists to investigate the whole structure and to realize how the γ-secretase cleave C99 immediately and efficiently In earlier research, the dimerization of APP may result from the GXXXG motifs in APP-TMD Frieden, C. (2007) Protein Sci. 16, 2334 2344 F Munter, L. M., et al. (2007) EMBO J. 26, 1702 1712. 6
Other lecture A. Using a low-resolution method B. Focus on TMD-containing region 7
NMR Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a magnetic field absorb and re-emitelectromagnetic radiation. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and the magnetic properties of this nuclei. http://lucas.lakeheadu.ca/luil/nuclear-magnetic-resonance-nmr-facility/ 8
Paramagnetic Relaxation Enhancement(PRE) of NMR In order to gain higher resolution and significant NMR signals Bio-molecular (especially for protein ), there are a lot of PRE methods for obtain the reliable and clear NMR signals In this lecture, the author adopt the thiolreactive nitroxide free radical probe, 1-oxyl-2,2,5,5-tetramethylpyrroline-3- methylmethanethiosulfonate, to enhance the alignment of the NMR protein structure of C99 in LMPG micelles 9
Power saturation EPR EPR(electron paramagnetic resonance) is similar to NMR but the electron replace the nuclei. The method is that first couples an EPR spin label to selected positions on the protein of interest, and then measures the depth of the spin label in the membrane by monitoring its rate of collisions with extrinsic paramagnetic probes residing in the aqueous phase and in the lipid bilayer. Nathan J. Malmberg, et al. Annu. Rev. Biophys. Biomol. Struct. 2005. 34:71 90 10
The complex of C99 lyso-myristoylphosphatidylglycerol (LMPG) POPC The peptide sequence of APP C99 POPG A. J. Beel et al., Biochemistry 47, 9428 (2008). 11
The structure of C99 in LMPG micelles by NMR Two α-helix (N- and C- Helix both are surfaceembed ) 30 lowest-energy structures 12
The space-filling surface representation Representation structure form previous figure, which may explain the weakness of the dimerization by GXXXG motif 13
EPR study for relative depth of C99 TMD N-helix C-helix 14
The mutation of G708L, G709L The raw data of double electronelectron resonance EPR Shows the curvature of C99 15
The titration of cholesterol and C99 Black: E693, Blue: G700, Red: G704 {mole percent = [moles cholesterol/(moles DMPC + moles cholesterol)] 100}. DHPC/DMPC bicelles 16
The titration results Chemical shift change G704A mutation of the titration 17
The mutation result of binding cholesterol Ala-scanning mutagenesis result Red: eliminated binding, Yellow: significant binding, Green: partial binding 18
The importance of binding Cholesterol Associating with cholesterol, C99 may partition into the lipid rafts where β- and γ- secretase also associate with chlesterol Binding to the C99, Cholesterol may play as cofactor for γ-secretase to cleave C99 For C99, binding cholesterol reduces the cleavage by α-secretase For C99, binding cholesterol accelerates the process of Amyloid-β profibril 19
Conclusion Investigation of the C99 structure and the observation of the binding formation with cholesterol provide insight for amyoidogenesis. The elastically curved TMD of C99 presents the insight how the γ-secretase recognizes and proteolyzes this region. The GXXXG motif play crucial role in cholesterol binding The structure of C99 can assist the design of C99- selective AD therapeutics 20
Thanks for your attention 21
Ref. SM 11 22
The NMR data of C99 in micelles http://www.bmrb.wisc.edu/data_library/summary/showgeneralsf.php?accnum=15775 &Sf_framecode=assigned_chem_shift_list_1&data_type=assigned_chemical_shifts http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbid=15775 23
QuickChange Mutagenesis 24
Function of AB 25
DEER EPR 26
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Figure 1 28