Establishing a Causative Link between Lipid Peroxidation and MBP Misfolding in Multiple Sclerosis...
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Transcript of Establishing a Causative Link between Lipid Peroxidation and MBP Misfolding in Multiple Sclerosis...
Establishing a Causative Link between Lipid Peroxidation and
MBP Misfolding in Multiple Sclerosis
Natalie K. Cygan
Scripps-Oxford Laboratory
June 13, 2007
Multiple Sclerosis (MS)
◘ Inflammatory, demyelinating disease of the CNS◘ Autoimmune disease◘ Affects over 1 million people worldwide◘ Symptoms include:
◘ changes in sensation ◘ visual problems◘ muscle weakness ◘ difficulties with coordination and speech ◘ severe fatigue ◘ cognitive impairment ◘ problems with balance
Myelin Sheath
Myelin Membrane
◘ Lipids constitute 70% of myelin dry weight
◘ Lipids contribute significantly to myelin structure and adhesion ◘ 25% of total lipid content is cholesterol◘ Cholesterol influences:
◘ Membrane thickness◘ Membrane fluidity◘ Ion leakage
Myelin Basic Protein (MBP)
◘ 18.5 kDa protein◘ Intrinsically unstructured protein ◘ Extensive post-translational modifications◘ Primary role: stabilization of the myelin sheath◘ Contains an immunodominant epitope
◘ Exposure of epitope leads to breakdown of myelin
Cholesterol Oxidation
HO
OH
O
Cholesterol
Atheronal B
Atheronal A
ROS
Aldolization
HO
OH
H+
N
R
Schiff Base
NH2
R
OOH
4-hydroxy nonenal, 4-HNE (7)
O O
malondialdehyde, MDA (6)
OO
glyoxal, GLO (4)
O
acrolein, ACR (8)
MBP immunodominant epitope exposurein the presence of lipid derived aldehydes
DIRECT INDIRECT
Schiff base formation Membrane effects
1. Enzymatic assay2. Antibody assays
1. Vesicle aggregation 2. Calcein leakage
Cathepsin D digestion
Cathepsin D
PE 27%Chol 44%PC 11%PS 13%Sphingo 3%PI 2%
Buffer Extrusion
MBPEpitope
F42-F43 F86-F87169aa
1-169
1-42
43-169
87-169
43-86
[18.5kDa]
[14kDa]
[4.5kDa]
[5kDa]
[8.6kDa]
18.5kDa 18.5kDa
0 1 2 3 4 5 6 7 24 0 1 2 3 4 5 6 7 24 Time (h)
MBP digestion in liposomes by Cathepsin D
MBP digestion in liposomes containing 4.4% atheronal
by Cathepsin D
14kDa14kDa
4.5kDa5kDa
46kDa46kDa
◘ Atheronals appear to inhibit Cathepsin D digestion◘ Peptide aldehydes have been shown to inhibit Cathepsin D
Membrane Effects
◘ Schiff base formation between atheronals and phosphatidylethanolamine (PE) alters the biophysical membrane properties (E. Wachtel, D. Bach, R. F. Epand et al., Biochemistry 45 (4), 1345 (2006).
◘ Ongoing experiments:◘ Turbidity assay ◘ Calcein encapsulated liposomes
Turbidity Calcein encapsulation
Add MBP
Aggregation Fusion
PE 27%Chol 39.6%PC 11%PS 13%Sphingo 3%PI 2%Ath 4.4%
PE 27%Chol 39.6%PC 11%PS 13%Sphingo 3%PI 2%Ath 4.4%
+ Calcein
0% atheronal 4.4% atheronal 20% atheronal
Turbidity
0.9
0.95
1
1.05
1.1
1.15
1.2
0 50 100 150 200 250 300 350 400 450
Time (sec)
A40
0/A
400i
nt
Time (sec)
A4
00/A
400
int
Turbidity
◘ Atheronals present in liposomes decreases turbidity
◘ Less interaction between membranes when atheronals are present
0% atheronal 4.4% atheronal 20% atheronal
Calcein encapsulated liposomes
0
1
2
3
4
5
6
7
8
9
10
0 50 100 150 200 250 300 350 400
Time (sec)
% R
ele
as
e
Calcein Encapsulated Liposomes
Time (sec)
% C
alce
in R
elea
sed
◘ Atheronals present in liposomes decrease % Calcein released
◘ Less fusion of membranes when atheronals are present
Conclusions
◘ Cathepsin D digestion- atheronals appear to inhibit Cathepsin D digestion
◘ Turbidity- less interaction between membranes when atheronals are present
◘ Calcein leakage- less fusion between membranes when atheronals are present
Future Work
◘ Detect conformational change in MBP◘ Enzymatic assay
◘ Stromelysin-1 (MMP-3)
◘ Epitope antibody ◘ ELISA◘ BIACORE
◘ Determine atheronal concentration in NAWM from healthy patients and MS patients