Reaction of Biological Aldehydes with Proteins and Cellular Targets Jonathan A Doorn PhD Medicinal and Natural Products Chemistry College of Pharmacy The University of Iowa Overview Keywords ID: 791500
Download The PPT/PDF document "2010 Logan Workshop on Reactive Toxicity..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
2010 Logan Workshop on Reactive Toxicity:Reaction of Biological Aldehydes with Proteins and Cellular Targets
Jonathan A. Doorn, Ph.D.Medicinal and Natural Products ChemistryCollege of Pharmacy The University of Iowa
Slide2OverviewKeywords: reactive intermediates, protein modification, neurotoxicity/neurodegeneration, dopamine catabolism
Questions: How are reactive intermediates generated at aberrant levels?Is protein modification occurring? What are the targets? Can we predict targets?What is the consequence of protein modification? Disease?
Goals
Novel targets for therapeutic intervention.
Biomarkers for disease pathogenesis.
Slide3Background:Parkinson’s disease (PD)First described: J. Parkinson (1817); biochemistry (1950’s)Changes in biochemistry, biologyLoss/impairment of dopamine producing neurons – substantia
nigraProtein aggregation (Lewy bodies)Dopamine is a neurotransmitter involved in coordination of movement
What causes PD? Thought to involve oxidative stress.
BenMoyal-Segal & Soreq (2006)
J. Neurochem
. 97, 1740-1755.
Dinis-Oliveira et al (2006)
NeuroTox
27, 1110-1122..
Slide4Background:Oxidative stress: lipid peroxidationAldehydes formed via lipid peroxidation (from ROS)
Lipid aldehydes: 4-hydroxy-2-nonenal (4HNE) and malondialdehyde (MDA) at < 50 μM (Esterbauer et al., 1991).
“Gold standards”
Slide5Background:Why dopaminergic cells? How is oxidative stress involved?ENDOGENOUS NEUROTOXIN? Auto-oxidation of dopamine (DA) (Graham, 1978)DA uptake into vesicles (VMAT2)
Formation of reactive oxygen speciesReactive ortho-quinone addition to thiols
5
Slide6Background:ENDOGENOUS NEUROTOXIN?? Oxidative deamination of DA 3,4-dihydroxyphenylacetaldehyde (DOPAL) catalyzed by MAO
(Elsworth and Roth, 1997.)
Products of oxidative stress (4HNE and MDA) inhibit ALDH enzymes at low
μ
M
(Rees et al., 2009;
Yunden
et al., 2009).
Slide7Background:Is DOPAL an ENDOGENOUS NEUROTOXIN? DOPAL is far more toxic than DA (Burke
et al., 2004; Burke, 2003)Why is it harmful to cells? DOPAL is reactive toward tissue/proteins (Ungar et al., 1973; Mattamal
et al., 1993)
How does DOPAL react with proteins? What are the targets?
Slide8GoalsElucidate mechanisms for the generation of DOPAL at aberrant levelsDetermine protein reactivity of DOPALIdentify reactive sites on proteins (amino acids)Measure rate of reactivityIdentify protein targetsDetermine functional consequence of protein modification and role of protein modification in disease
Slide9Overview of ExperimentsHow do we obtain DOPAL? Biosynthesis, synthesis.Model systems for DA catabolism:MitochondriaSynaptosomes (isolated nerve terminals)
Cells: dopaminergic PC6-3 & N27
Mitochondria
PC6-3 Cells
Synaptosome
Slide10Overview of ExperimentsDA model systems: advantages and disadvantagesMitochondriaSynaptosomesCellsProtein reactivity experimentsModel nucleophilesN-acetylated
Cys, His, Lys, Arg; glutathione (GSH)ProteinsMitochondria and cell lysatesProtein reactivity kineticsMeasure rate constantsVary concentration of nucleophile
10
Slide11ResultsInhibition of DOPAL metabolism in dopaminergic PC6-3 cells by a product of oxidative stress (4HNE)105 cells/plate, treated with NGF 4-5 daysSupplemented: 100 μM DA (DA DOPAL in situ)
60 min time-course; aliquots removedDA, DOPAL, DOPAC and DOPET via HPLC
Slide12ResultsInhibition of DOPAL metabolism in dopaminergic PC6-3 cells by a product of oxidative stress (4HNE)105 cells/plate, treated with NGF 4-5 daysSupplemented with 100 μM DA (DA DOPAL in situ)
60 min time-course; aliquots removed, protein precipitatedDA, DOPAL, DOPAC and DOPET monitored via HPLCA = % Control ALDH activity (DOPAC production)B = % Control [DOPAL]
*
Cytotoxic! (MTT)
Slide13ResultsDoes increase in [DOPAL] yield increase in DOPAL-protein modification?0.5 mg/mL rat striatal synaptosomes + 100 μM DAAdd 0-100 μ
M 4HNE and incubate 2hrsControls with 100 μM pargyline (MAO inhibitor)SDS-PAGE; gel transfer to nitrocellulose membraneDetect catechol-modified proteins with nitroblue tetrazolium (Paz et al., 1991)
1 2 3 4 5 6 7
Ln
Sample
% Control
Control 100
5
μ
M 4HNE 227
10
μ
M 4HNE 243
50
μ
M 4HNE 238
100
μ
M 4HNE 213
6 MAOI 49.8
7 MAOI/50
μ
M 4HNE 32.6
Slide14ResultsHow does DOPAL react with proteins?Oxidation to quinone; quinone plus thiol (Cys)Aldehyde plus amine (Lys)How reactive is DOPAL?Protein cross-linking?
Slide15ResultsIs DOPAL reactive toward protein amines (i.e. Lys)? What is the adduct?Peptide = RKRSRAE; incubate 4 hrs, 37 ºC, pH 7.4(A) 10 μM peptide(B) 100
μM DOPAL + 10 μM peptide(C) 100 μM DA + 10 μM peptideMALDI-TOF-MS analysis
15
Slide16ResultsIs DOPAL reactive toward protein amines (i.e. Lys) or thiols (i.e. Cys)? 0 mM 1 mM 5 mM 10 mM
Slide17ResultsIs DOPAL reactive toward protein amines (i.e. Lys, His, Arg) or thiols (i.e. Cys)?No significant reactivity towards N-acetyl Cys (yet…)
HPLC analysis of reaction (10 mM N-acetyl Cys)Change in N-acetyl Cys as judge by DTNBNo significant auto-oxidation of DOPAL to quinone
No reactivity towards N-acetyl His or N-acetyl
Arg
λ
max
= 520 nm
λ
max
= 410 nm
tyrosinase
, sodium
metaperiodate
Slide18ResultsHow reactive is DOPAL toward protein amines?1-10 mM Ac-Lys + 0.1 mM DOPALk = 2.0 M
-1min-1Compare to 4HNE:k = 0.0798 M-1min-1
10 mM Ac-Lys
10 mM Ac-Lys
k
= 0.42 M
-1
min
-1
Unstable w/o reduction!!
Needs NaCNBH
3
Slide19ResultsHow reactive is DOPAL toward protein amines?
a Reducing agent (NaCNBH3) included for stability. Without reducing agent, reactivity was very low, << 0.40 M-1min-1b None Detected. No significant reaction detected during the time-course.c Very low reactivity, estimated to be < 0.2 M-1min
-1
Slide20ResultsHow reactive is DOPAL toward protein amines?Protein (BSA, GAPDH) + CatecholsStain with NBT
1 2 3 4
BSA + catechol
1 = DA
2 = DOPAL3 = DOPAC4 = L-DOPA
A
B
GAPDH + catechol
1 = DA
2 = DOPAL
3 = DOPAC
4 = L-DOPA
20
Slide21ResultsCan DOPAL cross-link proteins? Is it a bifunctional electrophile?GAPDH + DOPALProtein mixture + DOPAL
Ascorbate sensitive = quinone?
Lane
1 Control
2 5 µM DOPAL
50 µM DOPAL
100 µM DOPAL
Control
5 µM DOPAL
50 µM DOPAL
100 µM DOPAL
2 hrs
4 hrs
Slide22ResultsCan DOPAL cross-link proteins? Is it a bifunctional electrophile?GAPDH + DOPALProtein mixture + DOPAL
Lane1 Control DOPAL Control
DOPAL
Control
DOPAL
2 hrs
4 hrs
6 hrs
Initial = 10 µM DOPAL; spike 5 µM DOPAL/hr
Final [DOPAL] = 12 µM (HPLC)
Slide23Work in ProgressProtein modification: proteomics based approachWhat are target proteins? Functional consequence?Identify proteins: tyrosine hydroxylase, aldehyde
dehydrogenase (mito)
NBT Staining
Synaptosomes
NBT Staining
PC6-3 Cells
[4HNE] (
μ
M) = 0 5 10 50
[4HNE] (
μ
M) = 0 10 100
Slide24SummaryRole of DOPAL, toxic intermediate of DA catabolism, in PDProtein modificationIdentification of targetsBiological significance of protein modification: Lys adducts and cross-linking
Slide25AcknowledgementsLab (past and present):Graduate students: Jennifer Rees, David Anderson. Erin Gagan, Laurie Eckert and Lydia Mexas
Pharmacy students: Nicole Brogden, Caroline Onel, Kathryn Nelson, Michael Hirsch, Elizabeth Wittchow, Natalie SimmonsPostdoctoral fellow:
Jinsmaa Yunden, Ph.D.
Research assistant:
Virginia FlorangSummer students: Charlie Ellithorpe,
Alicia Williams
Collaborators/consultants:
Stefan Strack, Ph.D. (Pharmacology, Iowa)
Dan
Liebler
, Ph.D. (Proteomics, Vanderbilt)
Tom Hurley, Ph.D. (Biochemistry, Indiana University)
Larry Robertson, Ph.D. (Public Health, Iowa)
Annette Fleckenstein, Ph.D. (Pharmacology, University of Utah)
Richard Nass, Ph.D. (Toxicology, Indiana University)
Un Kang, M.D. (Neurology, University of Chicago)
Slide26AcknowledgementsFinancial supportNIH R01 ES15507NIH K22 ES12982 (Career Award)UI OVPR Biological Sciences Funding ProgramPilot Grants from NIH P30 ES05605 (EHSRC)
Pilot Grant: Center for Health Effect of Environmental ContaminationTraining grants: T32 GM008365 and T32 GM067795University of Iowa College of Pharmacy