Aging and Oxidative Damage to Mitochondrial Proteins

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Presentation 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 State University August 26, 2005 HHMI Summer 2005 Fellowship Program

Oxygen radicals and aging ROS (H2O2, ·O2-, ·OH) Oxidative damage Heart disease and aging Source: http://onlinetc.its.brooklyn.cuny.edu/ Core81/chap3.html Overall goal: Characterize oxidative damage to mitochondrial proteins

Oxidative Phosphorylation Cell cytosol Outer membrane Intermembrane space Inner membrane Matrix Oxidative Phosphorylation H+ H+ H+ e- III I IV V O2 + H+ e- 4 + 4 2 H2O ADP + Pi H2O2, ·O2-, •OH ATP NADH Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

Formation of LPO products Linoleic acid ROS (H2O2, ·O2-, ·OH) Aldehyde functionality group 4-hydroxy-2-nonenal (4-HNE) 4-oxo-2-nonenal (4-ONE)

Oxylipid adduction to proteins Cysteine electrophilic C-3 Histidine 4-HNE Lysine

Oxidative Phosphorylation Cell cytosol Outer membrane Intermembrane space Inner membrane Matrix Oxidative Phosphorylation H+ H+ H+ e- III I IV V O2 + H+ e- 4 + 4 2 H2O ADP + Pi H2O2, ·O2-, •OH ATP NADH Adapted from Cooper, G. and Hausman, R., Cell: A Molecular Approach, 2003

Probing for damaged proteins ARP (Aldehyde-reactive probe) hydroxylamine biotin 4-HNE-adducted protein Aldehyde functionality group

Thioredoxin as an in vitro model

Thioredoxin as an in vitro model: Experimental Approach + HNE & ARP Thioredoxin + Trypsin Thioredoxin peptides

MS of ARP-labeled thioredoxin peptides 734.4 HNE-ARP [IIHLTDDSFDTDVLK]3+ + HNE 630.3 HNE [IIHLTDDSFDTDVLK]3+ 577.9 [IIHLTDDSFDTDVLK]3+ m/z

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 % m/z I-I HNE-ARP H L T D S F T-D V b2 227.1 b3 833.5 b7 1278 b6 1163 b8 1365 b9 1512 b10 1627 b12 1843 b13 1942 b4 946.6 b5 1048

Affinity chromatography = ARP ARP (Aldehyde-reactive probe) hydroxylamine biotin Avidin bead column

Affinity chromatography (cont’d) = ARP Avidin bead column

MS of unlabeled peptides 100 MIAPILDEIADEYQGK 1805.9 % 799.0 1441.8 2084.6 2727.4 3370.2 4013.0 m/z

MS of labeled peptides IIHLTDDSFDTDVLK SDKIIHLTDDSFDTDVLK 2201.1 100 IIHLTDDSFDTDVLK HNE-ARP % SDKIIHLTDDSFDTDVLK HNE-ARP 2201.1 2531.3 799.0 1441.8 2084.6 2727.4 3370.2 4013.0 m/z

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

Affinity chromatography ARP-labeled Complex IV ARP LPO IV avidin biotin reversible bond avidin biotin irreversible bond