Aging and Oxidative Damage to Mitochondrial Protein Subunits of Complex IV

Aging and Oxidative Damage to Mitochondrial Protein Subunits of Complex IV

Tony TongDr. Fred Stevens

Dr. Claudia MaierDuane Mooney

Department of ChemistryDepartment of Biochemistry and Biophysics

Oregon State UniversitySeptember 1, 2004

Tony TongDr. Fred Stevens

Dr. Claudia MaierDuane Mooney

Department of ChemistryDepartment of Biochemistry and Biophysics

Oregon State UniversitySeptember 1, 2004HHMI Summer 2004 Fellowship ProgramHHMI Summer 2004 Fellowship Program

Background:Aging and Oxygen RadicalsBackground:Aging and Oxygen RadicalsHeart disease is the #1 cause of death over age

65Large amounts of oxygen radicals are thought to

contribute to:protein oxidationmitochondrial dysfunction

Molecular mechanisms largely unknownFinding which proteins are damaged can provide

an insight into cardiac aging on the molecular level

Heart disease is the #1 cause of death over age 65

Large amounts of oxygen radicals are thought to contribute to:protein oxidationmitochondrial dysfunction

Molecular mechanisms largely unknownFinding which proteins are damaged can provide

an insight into cardiac aging on the molecular level

oxidative stressmyocardial aging

Background:Oxidative PhosphorylationBackground:Oxidative Phosphorylation

Background:Oxygen Radicals and Complex IV

Background:Oxygen Radicals and Complex IV Oxygen radicals can cause

peroxidation of nearby lipids These LPO products can then

adduct to proteins, ie Complex IV

Studies have shown that as adduction of 4-HNE (a LPO product) increases, Complex IV activity decreases

Oxygen radicals can cause peroxidation of nearby lipids

These LPO products can then adduct to proteins, ie Complex IV

Studies have shown that as adduction of 4-HNE (a LPO product) increases, Complex IV activity decreases

Source: Suh, J. H., Heath, S. H., Hagen, T. M. (2003). Two subpopulations of mitochondria in the aging rat heart display heterogeneous levels of oxidative stress. Free Radical Biology & Medicine 35 (9), 1064-1072

O

OH

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

= Young Rats

= Old Rats

Background:Adduction to Complex IVBackground:Adduction to Complex IVAdduction assumed

to be mostly on Cys, Lys, His (most nucleophilic)

Adduction assumed to be mostly on Cys, Lys, His (most nucleophilic)

O

OH

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

Aldehyde functionality group

O

OH

4-HNE

C5H11

OH

O

S

Prote in

C5H11

OH

O

HN

Prote in

C5H11

OH

O

SH

Prote in

H2N

Prote in

N

HN

Prote in

N

N

Prote in

CysteineLysine

Histidine

Background:Complex IVBackground:Complex IVaka cytochrome c oxidaseComprised of 13 polypeptide subunits

aka cytochrome c oxidaseComprised of 13 polypeptide subunits

Experimental:Labeling adducted Complex IV

Experimental:Labeling adducted Complex IVUse rat and mouse heart mitochondriaHICAT label addedLabel is aldehyde-specific (hydrazide binds

to lipid adducts)

Use rat and mouse heart mitochondriaHICAT label addedLabel is aldehyde-specific (hydrazide binds

to lipid adducts)

HICAT

HICATHICAT

+ HICATIV IV

HICAT(Hydrazide-functionalized Isotope-Coded Affinity Tag)

Biotin polyethyleneoxide 13C label

hydrazide

HN NH

S

HN

O

O

O

O

HN

O

CH2 CH2

O

NH NH2

O

OH

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

Aldehyde functionality group

Experimental:Isolating adducted Complex IV

Experimental:Isolating adducted Complex IVMembrane proteins separated by Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE)

Western blot with avidin-horseradish peroxidase (binds with biotin)

Adding H2O2 fluoresces Hrp

Membrane proteins separated by Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE)

Western blot with avidin-horseradish peroxidase (binds with biotin)

Adding H2O2 fluoresces Hrp

HICAT

HICATHICAT

IVBN-PAGE

Western blot

w/ Hrp

4˚C

Develop

film

Experimental:Isolating adducted Complex IV

Experimental:Isolating adducted Complex IVFluorescent spots overlaid with an identical

gelMatching spots are HICAT-reactiveAll gel spots excised and digested with

trypsinProteins extracted

Fluorescent spots overlaid with an identical gel

Matching spots are HICAT-reactiveAll gel spots excised and digested with

trypsinProteins extracted

Overlay film & 2nd gel

Gel pieces cut,digested, extracted

Experimental:ID of adducted Complex IV proteins

Experimental:ID of adducted Complex IV proteinsDigested proteins analyzed by LC/Q-ToF*

mass spectrometryDigested proteins analyzed by LC/Q-ToF*

mass spectrometry

*Liquid Chromatography/Quadrupole-Time of Flight

Brief Background:ProteomicsBrief Background:ProteomicsStudy of proteinsA certain sequence of amino acids defines a

proteinHighly unlikely that 2 different protein

fragments will have the same mass ID of a sequence ID of a protein

Study of proteinsA certain sequence of amino acids defines a

proteinHighly unlikely that 2 different protein

fragments will have the same mass ID of a sequence ID of a protein

Experimental:ID of proteinsExperimental:ID of proteinsQ-ToF-MS/MS sequentially breaks up protein

fragments further into amino acid residuesAnalysis by MASCOT search engine identifies

proteins

Q-ToF-MS/MS sequentially breaks up protein fragments further into amino acid residues

Analysis by MASCOT search engine identifies proteins

Results:Blue Native-PAGE GelResults:Blue Native-PAGE Gel

Mouse heart mitochondria

Results:Blue Native-PAGE BlotResults:Blue Native-PAGE Blot

Results:Blue Native-PAGE OverlayResults:Blue Native-PAGE Overlay

HICAT-reactive spots

Results:Mass spectrometry of peptides

Results:Mass spectrometry of peptides15 fragment ions correspond to peptide

ILYMMDEINNPVLTVK

15 fragment ions correspond to peptide ILYMMDEINNPVLTVK

Q-ToF-MS/MS Spectrum of a Complex IV Subunit II peptide

Results:Complex IV MASCOT Search Results

Results:Complex IV MASCOT Search ResultsSubunits I, II, III, IV, Va, Vb, VIIa

7 of 13 subunits detectedSubunit VIc detected in rat heart

mitochondria

Subunits I, II, III, IV, Va, Vb, VIIa

7 of 13 subunits detectedSubunit VIc detected in rat heart

mitochondria

Results:ETC Proteins in Blue Native-PAGE

Results:ETC Proteins in Blue Native-PAGE

HICAT-reactive spots

Complex IComplex VComplex III

Complex IV

Complex II

Results:Detecting HICAT adductionResults:Detecting HICAT adductionNone found in MASCOT searchingConcentration problem?

None found in MASCOT searchingConcentration problem?

Experimental:Future approachesExperimental:Future approaches2-D PAGE (2nd dimension w/SDS) for

increased resolutionAffinity chromatography coupled with MS

Only adducted peptide fragments will be detected

Allows ID of adducted residues

2-D PAGE (2nd dimension w/SDS) for increased resolution

Affinity chromatography coupled with MSOnly adducted peptide fragments will be

detectedAllows ID of adducted residues

Many thanks to…Many thanks to… The labs of:

Dr. Fred Stevens Dr. Claudia Maier Dr. Tory Hagen Dr. Emily Ho

Duane Mooney Andy Larkin Gretchen Clark-Scannell Brian Arbogast Dr. John Sowell Dr. Kevin Ahern Howard Hughes Medical Institute OSU Undergraduate Research, Innovation, Scholarship,

Creativity (URISC) Program You for listening!

The labs of: Dr. Fred Stevens Dr. Claudia Maier Dr. Tory Hagen Dr. Emily Ho

Duane Mooney Andy Larkin Gretchen Clark-Scannell Brian Arbogast Dr. John Sowell Dr. Kevin Ahern Howard Hughes Medical Institute OSU Undergraduate Research, Innovation, Scholarship,

Creativity (URISC) Program You for listening!

Questions?Questions?

Experimental:Affinity chromatographyExperimental:Affinity chromatographyBead-immobilized avidin (commercially

prepared)Monomeric & multimeric forms (mono is

weaker, biotin bond is reversible) Irreversible sites blocked w/biotinGlycine used to remove biotin from reversible

sitesProteins added, unbound protein washed awayBound protein eluted with formic acid

Bead-immobilized avidin (commercially prepared)

Monomeric & multimeric forms (mono is weaker, biotin bond is reversible)

Irreversible sites blocked w/biotinGlycine used to remove biotin from reversible

sitesProteins added, unbound protein washed awayBound protein eluted with formic acid

Results:Affinity chromatographyResults:Affinity chromatographyEarly work w/biotinylated insulin shows

presence of protein in elution samples and none in nonbiotinylated protein washes

However, no labeled peptides found in Q-ToF

In MALDI, peptides were detected, but not enough to do MS/MS

Early work w/biotinylated insulin shows presence of protein in elution samples and none in nonbiotinylated protein washes

However, no labeled peptides found in Q-ToF

In MALDI, peptides were detected, but not enough to do MS/MS

Results:Affinity Chromatography Test Run

Results:Affinity Chromatography Test Run

MALDI-ToF MS Spectrum of Insulin-Biotin

Results:HICAT Breakdown DetectedResults:HICAT Breakdown DetectedSmall peaks at 375 and 457 detectedSmall peaks at 375 and 457 detected

HN

NH

O H

HN

HS

O O O

NH

OHN

O

N

O

HN

OH

O

S

HN

H2N

OHO

O

m/z 375

m/z 489

m/z 457

m/z 855

m/z 801

m/z 270

100 200 300 400 500 600 700 800 900m/z

100

75

50

25

0

Re

lativ

e I

nte

nsi

ty (

%)

270

375

457

489 623.4 801

930MH+

855

MS/MS of 12C-HICAT-DDE-Glutathione

Results:The other bands?Results:The other bands?Lower bands: Cytochrome c (ETC

protein), actin, hemoglobin

Everywhere: Complex V subunits, citric acid cycle and ß-oxidation related proteins, myosin, albumin

Lower bands: Cytochrome c (ETC protein), actin, hemoglobin

Everywhere: Complex V subunits, citric acid cycle and ß-oxidation related proteins, myosin, albumin

Background:LPO Product FormationBackground:LPO Product Formation

COOH

COOH COOH

HOO OOH

COOH

OH

COOH

O

COOH

OH

COOH

O

COOH

OOH

HOO

COOH

O

OOH

O

O

OOHO

OH

O

O

COOH

O

COOH

HOO

OOH

Linoleic Acid

13-HPODE 9-HPODE

4-HPNE

4-HNE4-ONE

13-HODE13-KODE

2,4-DDE

9-KODE 9-HODE

O

COOH

O

O

Acrolein

Experimental:Biocytin-Hydrazide LabelExperimental:Biocytin-Hydrazide LabelShorter chain length vs. HICATCommercially available

Shorter chain length vs. HICATCommercially available

HN NH

S

HN

O

O

NHNH2

O

NH2

Biocytin-Hydrazide