1. Presented by Paul Barrett
Sanders Lab
Biochemistry Department
Dissertation Defense
September 5th, 2013

2. Cases of Dementia Are Increasing World Wide

3. The Jack Curve Displays Biomarker
Progression during Alzheimer's Disease
Jack et al, The Lancent Neurology, 12, 2013
~20-30
Years

4. APP Can Undergo Multiple Cleavage
Pathways
Alzheimer’s Disease Involves the
Amyloid Precursor Protein (APP)
α-Secretase
γ-Secretase
β-Secretase
γ-Secretase

5. NMR Spectroscopy Can Obtain Information
Regarding Protein Structure and Function
http://en.wikipedia.org/wiki/NMR_tube

6. Solution NMR Structural Studies Require
A Cell Membrane Mimetic
http://micro.magnet.fsu.edu/cells/plasmamembrane/plasmamembrane.html
Micelle
Bicelle

7. C99 Possesses a
Unique Membrane Topology
Beel, Biochemistry. 2008, 47, 9428-46
α-secretase
cleavage
N
C
Ligand Binding Pocket?

8. Solution NMR Determined Structures
Validate Membrane Topology
N-terminus
N-terminal
Helix
N-loop
Transmembrane Helix
C-loop
C-terminal
Helix
Barrett et al, Science. 2012, 336, 1168-
1171

9. Solution NMR Determined Structures
Validate Membrane Topology
Barrett et al, Science. 2012, 336, 1168-
1171
NMR Ensemble
Lowest Energy
Structure

10. EPR Studies can Examine C99 in
Lipid Bilayers

11. Power Saturation Studies Validate
Membrane Topology in a Lipid
Bilayer
N-
terminal
Helix
N-Loop
Transmembrane Helix
C-Loop
C-terminal
Helix
Barrett et al, Science. 2012, 336,
1168-1171

12. DEER Experiments Show C99 is Curved
in Both Micelles and a Lipid Bilayer
Barrett et al, Science. 2012,
336, 1168-1171
34.0 ± 1 Å LMPG Micelle
33.5 ± 1 Å Lipid Vesicle

13. Curved and Straight TMDs Would have
Different DEER Results
Barrett et al, Science. 2012, 336, 1168-
1171
34 Å 45 Å
DEER Results Modeled TMD Distances
Curved TMD Straight TMD34.0 ± 1 Å LMPG Micelle
33.5 ± 1 Å Lipid Vesicle
vs

14. Strategic Glycines and Abundance of β-
Branched Amino Acids May Allow Curvature
=Gly708/709
=β-branched AA
=Remaining AA

15. DEER Experiments Show Glycine Residues are
Responsible for Transmembrane Flexibility
Barrett et al, Science. 2012, 336, 1168-
1171

16. A Flexibly Curved TM Helix May Be
Important for γ-Secretase Cleavage
Osenkowski, P, JMB, 385, 642-652, (2009)

17. APP Can Undergo Multiple Cleavage
Pathways
α-Secretase
γ-Secretase
β-Secretase
γ-Secretase

18. Evidence Suggests A Link Between
Cholesterol and Alzheimer’s Disease

19. To Investigate Cholesterol Binding, an
Alternative Membrane Mimetic was Used
Micelle
Bicelle
Bicelles can incorporate
cholesterol
http://micro.magnet.fsu.edu/cells/plasmamembrane/plasmamembrane.html

20. NMR Data Shows C99 Can
Specifically Bind Cholesterol
Barrett et al, Science. 2012, 336, 1168-
1171

21. Cholesterol Binding is
Physiologically Relevant
Barrett et al, Science. 2012, 336, 1168-
1171

22. Scanning Alanine Mutagenesis Shows Which Residues
are Important For Cholesterol Binding
G

23. Key Residues for Binding Map to the
Transmembrane Helix and N-Terminal Helix
Mutation to Ala does not impact binding
Mutation to Ala attenuates binding
Mutation to Ala eliminates binding
α-Secretase
Cleavage Site

24. Binding of Cholesterol May Traffic C99 to
Cholesterol Rich Membrane Domains
(Lipid Rafts)
• Aβ oligomerization occurs in cholesterol rich domains
• Mature hippocampal neurons contain the most cholesterol
rich domains of any cell

25. To Investigate C99 Membrane Partitioning,
Giant Unilamellar Vesicles Were Used
Micelle
Bicelle
• Exact Ratio of Lipids in GUVs Can Be
Manipulated
• Fluorescent Probes Can be Added
• Imaged by Confocal Microscopy
http://micro.magnet.fsu.edu/cells/plasmamembrane/plasmamembrane.html
Giant Unilamellar Vesicles
(GUVs)

26. GUVs Can Form Cholesterol
Rich Membrane Domains
(Lipid Rafts)
Non-Raft Marker Raft Marker Merge
GUVs contain a lipid ratio of 2:1:1
POPC/Sphingomyelin(SM)/Cholesterol

27. C99 Partitions to “Raft” Membrane
Domains
Non-Raft
Marker
Raft Marker Merge
Non-Raft
Marker
C99 Merge
GUVs contain a lipid ratio of 2:1:1
POPC/Sphingomyelin(SM)/Cholesterol

28. Alanine Mutants Verify C99 Partitioning is
Cholesterol Specific
Mutation to Ala does not impact binding
Mutation to Ala attenuates binding
Mutation to Ala eliminates binding
G
G
E

29. Cholesterol Binding is Require For C99 Membrane “Raft” Partitioning
WT C99
G704A
“Binding KO”
E693A
“Binding KO”
G696A
“WT Binding”
Non-Raft C99 Merge

30. Cholesterol Binding May Initiate the Amyloidogenic Pathway

31. Cholesterol Analogues can Promote
or Inhibit “Raft” Formation
1=Raftophilic
0=Raftophobic
The goal is to find an analogue that binds to C99 more
tightly than native cholesterol and does not partition to
lipid rafts
J. J. Wenz, Predicting the effect of steroids on membrane biophysical properties based on the molecular structure. Biochim.Biophys.Acta 1818, 896
(3/2012, 2012)

32. Difference Between Cholesterol
and Coprostanol is One Double
Bond
Cholesterol Coprostanol
We anticipated that binding
between Coprostanol and C99 will
be similar to cholesterol binding

33. C99 Can Specifically Bind The
Cholesterol Analogue Coprostanol

34. Coprostanol Binding Reduces C99
Partitioning to “Raft” Domains
0% Coprostanol
25% Cholesterol
2.5% Coprostanol
22.5% Chol
5% Coprostanol
20% Chol
0% Coprostanol
20% Chol
Non-Raft C99
Partition Coefficients were determined by taking the
ratio of C99 in the raft phase to C99 out of the raft phase

35. Conclusions and Future Directions
C99 Possesses a
Flexibly Curved
Transmembrane Helix
C99 Can Specifically
Bind Cholesterol
C99 Partitioning to Cholesterol Rich
Membrane Domains can Be
Pharmacologically Regulated
How does this curvature and
flexibility regulate C99 cleavage?
0%
Coprostanol
5%
Coprostanol
How does cholesterol binding and
partitioning promote Alzheimer’s disease?

36. Acknowledgments
• Sanders Lab
– Current Members
• Dr. Charles Sanders
• Dr. Yuanli Song*
• Dr. Wade Van Horn*
• Arina Hadziselimovic
– Previous Members
• Andrew Beel
• Collaborators
– EPR Experiments
• Dr. Eric Hustedt
• Dr. Sunghoon Kim
– γ-Secretase Assay
• Dr. Bruce Carter
• Dr. Emily Stanley
– GUV studies
• Dr. Anne Kenworthy
• Charles Day
• Committee members
– Dr. Charles Sanders
– Dr. Walter Chazin
– Dr. Anne Kenworthy
– Dr. Bruce Carter
– Dr. Richard Armstrong
• NMR Facility
– Dr. Markus Voehler
• Funding
– MBTG
• Grant number: 4-04-250-1181
– NIH
• Grant number: F31NS077681

37. Acknowledgments

38. Questions???