Aging and Oxidative Damage to Mitochondrial Proteins Tony Tong Dr. Claudia Maier Dr. Fred Stevens...

Aging and Oxidative Damage to Mitochondrial Proteins

Tony TongDr. Claudia MaierDr. Fred Stevens

Department of ChemistryDepartment of Pharmaceutical Sciences

Department of Biochemistry and BiophysicsOregon State University

August 26, 2005HHMI Summer 2005 Fellowship Program

Oxygen radicals and aging

Oxidative damage

Overall goal: Characterize oxidative damage to mitochondrial proteins

ROS (H2O2, ·O2-, ·OH)

Source: http://onlinetc.its.brooklyn.cuny.edu/ Core81/chap3.html Heart disease and aging

Cell cytosol

Outer membrane

Intermembrane space

Inner membrane

Matrix

H+H+

H+

H+H+H+

H+

H+

V IV III I

H+

H+

H+ H+ H+

H+ H+ H+

NADH

O2 + H+ e-4 + 4

2 H2O

ADP

+

Pi

ATP

Oxidative Phosphorylation

e-

e-

e-

H2O2, ·O2-, •OH

Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

Formation of LPO products

Linoleic acid

4-hydroxy-2-nonenal (4-HNE) 4-oxo-2-nonenal (4-ONE)

ROS (H2O2, ·O2-, ·OH)

HO

O

Aldehyde functionality group

O

OH

O

O

Oxylipid adduction to proteins

4-HNE

C5H11

OH

O

S

Protein

C5H11

OH

O

HN

Protein

C5H11

OH

O

N

N

Protein

SH

Protein

Cysteine

N

HN

Protein

H2N

Protein

Histidine

Lysine

electrophilic C-3

O

OH

Cell cytosol

Outer membrane

Intermembrane space

Inner membrane

Matrix

H+H+

H+

H+H+H+

H+

H+

V IV III I

H+

H+

H+ H+ H+

H+ H+ H+

NADH

O2 + H+ e-4 + 4

2 H2O

ADP

+

Pi

ATP

Oxidative Phosphorylation

e-

e-

e-

H2O2, ·O2-, •OH

Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

Probing for damaged proteins

NHO

HN

S

O

HN

NH

O

O

NH2

ARP(Aldehyde-reactive probe)

hydroxylamine

biotin

4-HNE-adducted protein

Aldehyde functionality group

O

OH

Thioredoxin as an in vitro model

O

OH

NHO

HN

S

O

HN

NH

O

O

N

Thioredoxin as an in vitro model:Experimental Approach

+ HNE & ARPThioredoxin

+ Trypsin

Thioredoxin peptides

MS of ARP-labeled thioredoxin peptides

577.9

[IIHLTDDSFDTDVLK]3+

m/z

+ ARP

734.4

HNE-ARP

[IIHLTDDSFDTDVLK]3+

+ HNE

630.3

HNE

[IIHLTDDSFDTDVLK]3+

MS/MS of thioredoxin peptides

I-I-H-L-T-D-D-S-F-D-T-D-V-L-K

b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14

y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

N-terminus C-terminus

200 400 600 800 1000 1200 1400 1600 1800

100

0

%

m/z

I-I

HNE-ARP

H L T D D S F D T-D V

b2

227.1 b3

833.5

b7

1278b6

1163

b8

1365

b9

1512

b10

1627b12

1843 b13

1942

b4

946.6 b5

1048

Avidin bead column

Affinity chromatography

= ARP

NHO

HN

S

O

HN

NH

O

O

NH2

ARP(Aldehyde-reactive probe)

hydroxylamine

biotin

Affinity chromatography (cont’d)

= ARP

Avidin bead column

MS of unlabeled peptides

1805.9MIAPILDEIADEYQGK

799.0 1441.8 2084.6 2727.4 3370.2 4013.0m/z

100

0

%

MS of labeled peptides

2531.3

2201.1

799.0 1441.8 2084.6 2727.4 3370.2 4013.0m/z

100

0

%

IIHLTDDSFDTDVLK

HNE-ARP SDKIIHLTDDSFDTDVLK

HNE-ARP

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

acrylamide monomeric avidin

azlactone linker

biotin

Affinity chromatography

avidin

avidin

avidin avidin

avidin

ARP-labeled Complex IV

ARP LPO IV

biotin

irreversible bond

reversible bond