Transcript Aging and Oxidative Damage to Mitochondrial Proteins Tony Tong Dr. Claudia Maier Dr. Fred Stevens Department of Chemistry Department of Pharmaceutical Sciences Department of Biochemistry and Biophysics Oregon.

Aging and Oxidative Damage to
Mitochondrial Proteins
Tony Tong
Dr. Claudia Maier
Dr. Fred Stevens
Department of Chemistry
Department of Pharmaceutical Sciences
Department of Biochemistry and Biophysics
Oregon State University
August 26, 2005
HHMI Summer 2005 Fellowship Program
Oxygen radicals and aging
ROS (H2O2, ·O2-, ·OH)
Oxidative damage
Source: http://onlinetc.its.brooklyn.cuny.edu/ Core81/chap3.html
Heart disease and aging
Overall goal: Characterize oxidative damage to
mitochondrial proteins
Oxidative Phosphorylation
Cell cytosol
Outer membrane
H+
H+
H+
Intermembrane
space
H+
H+
H+
H+
H+
e-
I
Inner membrane
e-
III
H+
H+
H+
H+
IV
V
2 H2O
H+
Matrix
eNADH
H+
H+
O2 + 4 H+ + 4 e-
H+
ADP
ATP
+
H2O2, ·O2-, •OH
Pi
Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003
Formation of LPO products
O
HO
Linoleic acid
ROS (H2O2, ·O2-, ·OH)
O
OH
O
O
4-hydroxy-2-nonenal (4-HNE)
Aldehyde functionality group
4-oxo-2-nonenal (4-ONE)
Oxylipid adduction to proteins
OH
SH
O
Protein
S
Cysteine
Protein
OH
H
N
N
C5H11
O
OH
C5H11
O
Protein
N
N
Histidine
4-HNE
OH
H2N
electrophilic C-3
O
C5H11
HN
Protein
Protein
Lysine
Protein
Oxidative Phosphorylation
Cell cytosol
Outer membrane
H+
H+
H+
Intermembrane
space
H+
H+
H+
H+
H+
e-
I
Inner membrane
e-
III
H+
H+
H+
H+
IV
V
2 H2O
H+
Matrix
eNADH
H+
H+
O2 + 4 H+ + 4 e-
H+
ADP
ATP
+
H2O2, ·O2-, •OH
Pi
Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003
Probing for damaged proteins
biotin
O
HN
Aldehyde functionality group
NH
O hydroxylamine
H
N
S
OH
O
O
N
H
NH2
O
ARP
(Aldehyde-reactive probe)
4-HNE-adducted protein
Thioredoxin as an in vitro model
O
HN
NH
O
H
N
S
N
H
O
OH
O
N
O
Thioredoxin as an in vitro model:
Experimental Approach
+ HNE & ARP
Thioredoxin
+ Trypsin
Thioredoxin peptides
MS of ARP-labeled thioredoxin peptides
[IIHLTDDSFDTDVLK]3+
HNE-ARP
HNE
630.3
734.4
[IIHLTDDSFDTDVLK]3+
577.9
+ HNE
+ ARP
m/z
[IIHLTDDSFDTDVLK]3+
MS/MS of thioredoxin peptides
b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14
I-I-H-L-T-D-D-S-F-D-T-D-V-L-K
N-terminus
C-terminus
y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
100
HNE-ARP
I-I
L
H
b2
227.1
T
D
D
S
F
D
T-D
b3
833.5
%
b4
946.6 b5
1048
b6
1163
b7
b8
1278
1365
b9 b10
1512 1627
0
200
V
400
600
800
1000
m/z
1200
1400
1600
b12
1843 b13
1942
1800
Affinity chromatography
= ARP
biotin
O
HN
NH
O
H
N
S
hydroxylamine
O
N
H
O
ARP
(Aldehyde-reactive probe)
Avidin bead column
NH2
Affinity chromatography (cont’d)
= ARP
Avidin bead column
MS of unlabeled peptides
100
MIAPILDEIADEYQGK
1805.9
%
0
799.0
1441.8
2084.6
m/z
2727.4
3370.2
4013.0
MS of labeled peptides
100
IIHLTDDSFDTDVLK
%
HNE-ARP
SDKIIHLTDDSFDTDVLK
2201.1
HNE-ARP
2531.3
0
799.0
1441.8
2084.6
m/z
2727.4
3370.2
4013.0
Future work
• Maximize recovery of material from
beads
• Repeat with labeled mitochondrial
samples
• Find which and where proteins have
been adducted
Acknowledgements
• The labs of:
– Dr. Claudia Maier
– Dr. Fred Stevens
– Dr. Mike Schimerlik
– Dr. Emily Ho
• EHSC Mass Spectrometry Core Facility
• Dr. Kevin Ahern
• Dr. Chris Mathews
• Howard Hughes Medical Institute
• Sacrificed rats
Questions?
Avidin-immobilized beads
monomeric avidin
acrylamide
azlactone linker
Affinity chromatography
avidin
biotin
ARP
LPO
IV
reversible bond
ARP-labeled Complex IV
avidin
avidin
avidin
avidin
biotin
irreversible bond