Download DOPAL and PD

Jonathan A. Doorn, Ph.D.
Medicinal and Natural Products Chemistry
College of Pharmacy
The University of Iowa
Overview
 Keywords: 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.
Background:
 Parkinson’s disease (PD)
 First described: J. Parkinson (1817); biochemistry (1950’s)
 Changes in biochemistry, biology


Loss/impairment of dopamine producing neurons – substantia nigra
Protein aggregation (Lewy bodies)
 Dopamine is a neurotransmitter involved in coordination of movement
 What causes PD? Thought to involve oxidative stress.
Dinis-Oliveira et al (2006) NeuroTox 27, 1110-1122..
BenMoyal-Segal & Soreq (2006) J. Neurochem. 97, 1740-1755.
Background:
 Oxidative stress: lipid peroxidation
 Aldehydes formed via lipid peroxidation (from ROS)
LIPID
(i.e.,
Arachidonate)
R• RH
-CH=CH-CH2-CH=CH-
(INITIATOR)
O2
-CH=CH-CH-CH=CH-
Free Radical
Intermediate
-CH=CH-CH=CH-CH-
-CH=CH-CH=CH-CH-
Conjugated Diene
Formation
OO
Peroxy Radical
-CH=CH-CH=CH-CHOOH
Hydroperoxide
LIPID
OOH
Hydrocarbons
Radical species
Aldehydes
 Lipid aldehydes: 4-hydroxy-2-nonenal (4HNE) and malondialdehyde
(MDA) at < 50 μM (Esterbauer et al., 1991).
O
“Gold standards”
C 5H 11
O
O
O
OH
4HNE
MDA
OH
5
Background:
 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 species
Reactive ortho-quinone  addition to thiols

HO
NH2
O2
O2
HO
O
NH2
O2
O2
HO
O
NH2
O
S
HO
HO
NH2
[O]
O
O
NH2
Protein-SH
HO
HO
Protein
NH2
Background:
 ENDOGENOUS NEUROTOXIN??

Oxidative deamination of DA  3,4-dihydroxyphenylacetaldehyde
(DOPAL) catalyzed by MAO (Elsworth and Roth, 1997.)
HO
NH 2
HO
O
MAO
HO
DA
MAO
H 2 O2
DOPAL
ALDH2
NAD
ALR
NADPH
DOPAL
O
ALDH
NAD
HO
DA
HO
A
NA LR
D
PH
HO
OH
DOPAC
HO
OH
DOPET
HO
DOPET
DOPAC

Products of oxidative stress (4HNE and MDA) inhibit ALDH enzymes at
low μM (Rees et al., 2009; Yunden et al., 2009).
Background:
 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?
HO
O
HO
Protein-NH2
HO
N-Protein
HO
S
HO
HO
O
[O]
O
O
O
Protein
HO
HO
Protein
O
Goals
 Elucidate mechanisms for the generation of DOPAL at
aberrant levels
 Determine protein reactivity of DOPAL
 Identify reactive sites on proteins (amino acids)
 Measure rate of reactivity
 Identify protein targets
 Determine functional consequence of protein modification
and role of protein modification in disease
Overview of Experiments
 How do we obtain DOPAL? Biosynthesis, synthesis.
HO
NH2
HO
HO
O
MAO
HO
HO
HSO3
HO
DA
OH
SO3
DOPAL
 Model systems for DA catabolism:
DA
MAO
ALDH2
NAD
DOPAL
DOPAC
 Mitochondria
 Synaptosomes (isolated nerve terminals)
 Cells: dopaminergic PC6-3 & N27
Synaptosome
DA
R
Tyr
Mitochondria
R
DA
DOPA DOPAC
DA
DOPAL
DA
DA
DA
DA
DA
DA
PC6-3 Cells
Overview of Experiments
 DA model systems: advantages and disadvantages
 Mitochondria
 Synaptosomes
 Cells
 Protein reactivity experiments
 Model nucleophiles



N-acetylated Cys, His, Lys, Arg; glutathione (GSH)
Proteins
Mitochondria and cell lysates
 Protein reactivity kinetics


Measure rate constants
Vary concentration of nucleophile
10
Results
 Inhibition of DOPAL metabolism in dopaminergic PC6-3 cells by a
product of oxidative stress (4HNE)
 105 cells/plate, treated with NGF 4-5 days
DA
 Supplemented: 100 μM DA (DA  DOPAL in situ)
MAO
 DA, DOPAL, DOPAC and DOPET via HPLC
[DOPAC] (M)
1.5
1.0
0.5
0.0
Control
2 M
10 M
25 M
50 M
100 M
0.5
0.4
0.3
0.2
0.1
0.0
0
10
20
30
40
50
Time (min)
HO
10
20
30
40
HO
OH
DOPAC
0
50
Time (min)
O
HO
60
DOPET
DOPAC
0.6
[DOPAL] (M)
Control
2 M
10 M
25M
50 M
100 M
ALR
NADPH
ALDH2
NAD
 60 min time-course; aliquots removed
2.0
DOPAL
O
HO
DOPAL
60
Results
 Inhibition of DOPAL metabolism in dopaminergic PC6-3 cells by a
product of oxidative stress (4HNE)
 105 cells/plate, treated with NGF 4-5 days
 Supplemented with 100 μM DA (DA  DOPAL in situ)
 60 min time-course; aliquots removed, protein precipitated
 DA, DOPAL, DOPAC and DOPET monitored via HPLC
% Control (DOPAC)
100
Cytotoxic! (MTT)
75
50
25
**
**
50
100
0
% Control [DOPAL]
*
125
600
500
400
300
200
*
100
0
0
2
10
25
[4HNE] (M)
0
2
10
25
50
[4HNE] (M)
A = % Control ALDH activity (DOPAC production)
B = % Control [DOPAL]
100
Results
 Does increase in [DOPAL] yield increase in DOPAL-protein modification?
 0.5 mg/mL rat striatal synaptosomes + 100 μM DA
 Add 0-100 μM 4HNE and incubate 2hrs
 Controls with 100 μM pargyline (MAO inhibitor)
 SDS-PAGE; gel transfer to nitrocellulose membrane
 Detect catechol-modified proteins with nitroblue tetrazolium (Paz et al., 1991)
1
2
3
4
5
6
7
Ln Sample
% Control
1
Control
100
2
5 μM 4HNE
227
3
10 μM 4HNE
243
4
50 μM 4HNE
238
5
100 μM 4HNE
213
6
MAOI
49.8
7
MAOI/50 μM 4HNE
32.6
Results
 How does DOPAL react with proteins?
 Oxidation to quinone; quinone plus thiol (Cys)
 Aldehyde plus amine (Lys)
 How reactive is DOPAL?
 Protein cross-linking?
Results
15
 Is 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 peptide
(A)
Peptide
950
1000
1050
1100
1150
1200
100
90
80
70
60
50
40
30
20
10
0
900
Peptide
DOPAL-Peptide
950
1000
HO
1050
1100
1150
1200
100
90
80
70
60
50
40
30
20
10
0
900
O
Peptide
(C)
Peptide
950
1000
1050
m/z
m/z
m/z
HO
(B)
% Intensity
100
90
80
70
60
50
40
30
20
10
0
900
% Intensity
% Intensity
 MALDI-TOF-MS analysis
HO
N
Peptide
HO
134 Da Adduct
1100
1150
1200
Results
 Is DOPAL reactive toward protein amines (i.e. Lys) or thiols (i.e. Cys)?
0 mM 1 mM 5 mM 10 mM
Results
 Is 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 DTNB
No significant auto-oxidation of DOPAL to quinone
tyrosinase, sodium metaperiodate
HO
HO
O
O
O
O
λmax = 520 nm
???
λmax = 410 nm
 No reactivity towards N-acetyl His or N-acetyl Arg
Results
 How reactive is DOPAL toward protein amines?
0.03
10 mM Ac-Lys
5
-k' (min-1)
ln (% Control DOPAL )
 1-10 mM Ac-Lys + 0.1 mM DOPAL
4
3
0.02
k = 2.0 M-1min-1
0.01
0.00
2
0
0
20 40 60 80 100 120 140 160 180
2
4
6
8
10
N-Ac-Lys (mM)
10 mM Ac-Lys
Compare to 4HNE:
k = 0.0798 M-1min-1
0.03
5
-k' (min-1)
ln (% Control DOPAL )
Time (min)
4
3
2
0
20 40 60 80 100 120 140 160 180
Time (min)
0.02
k = 0.42 M-1min-1
0.01
0.00
0
2
4
6
N-Ac-Lys (mM)
8
10
Unstable w/o reduction!!
Needs NaCNBH3
Results
 How reactive is DOPAL toward protein amines?
Structure
Compound
k (M-1min-1)
DOPAL
2.0 ± 0.036
MOPAL
0.42 ± 0.042a
NDb
DMPAL
PAL
a
< 0.2c
Reducing agent (NaCNBH3) included for stability. Without reducing agent, reactivity was very low,
<< 0.40 M-1min-1
b None Detected. No significant reaction detected during the time-course.
c Very low reactivity, estimated to be < 0.2 M-1min-1
Results
20
 How reactive is DOPAL toward protein amines?
 Protein (BSA, GAPDH) + Catechols
 Stain with NBT
1
2
3
4
A
BSA + catechol
1 = DA
2 = DOPAL
3 = DOPAC
4 = L-DOPA
B
GAPDH + catechol
1 = DA
2 = DOPAL
3 = DOPAC
4 = L-DOPA
Results
 Can DOPAL cross-link proteins? Is it a bifunctional electrophile?
 GAPDH + DOPAL
 Protein mixture + DOPAL
1
2
3
4
5
6
7
200
116
97
66
45
100
*
8
Lane
1 Control
2 5 µM DOPAL
3 50 µM DOPAL
4 100 µM DOPAL
5
6
7
8
Control
5 µM DOPAL
50 µM DOPAL
100 µM DOPAL
2 hrs
4 hrs
% Control
80
Ascorbate sensitive = quinone?
60
40
20
0
DOPAL
NaCNBH3
Ascorbate
Results
 Can DOPAL cross-link proteins? Is it a bifunctional electrophile?
 GAPDH + DOPAL
 Protein mixture + DOPAL
MW
(kDa)
200
116
97
66
45
1
2
3
4
5
6
Lane
1 Control
2 DOPAL
2 hrs
3
4
Control
DOPAL
4 hrs
5
6
Control
DOPAL
6 hrs
Initial = 10 µM DOPAL; spike 5 µM DOPAL/hr
Final [DOPAL] = 12 µM (HPLC)
Work in Progress
 Protein modification: proteomics based approach
 What are target proteins? Functional consequence?
 Identify proteins: tyrosine hydroxylase, aldehyde dehydrogenase (mito)
[4HNE] (μM) = 0
5
10 50
NBT Staining
Synaptosomes
[4HNE] (μM) = 0 10 100
NBT Staining
PC6-3 Cells
Protease
Peptides
Identified
Protein
Identified
LC separation
Database search
(MASCOT)
MS analysis
(MS/MS)
Time (min)
Intensity
Intensity
+1
+2
m/z
Summary

Role of DOPAL, toxic intermediate of DA catabolism, in PD



Protein modification
Identification of targets
Biological significance of protein modification: Lys adducts and cross-linking
?
Acknowledgements
 Lab (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 Simmons
Postdoctoral fellow: Jinsmaa Yunden, Ph.D.
Research assistant: Virginia Florang
Summer 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)
Acknowledgements
 Financial support
 NIH R01 ES15507
 NIH K22 ES12982 (Career Award)
 UI OVPR Biological Sciences Funding Program
 Pilot Grants from NIH P30 ES05605 (EHSRC)
 Pilot Grant: Center for Health Effect of Environmental Contamination
 Training grants: T32 GM008365 and T32 GM067795
 University of Iowa College of Pharmacy