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

OverviewKeywords: 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?

GoalsNovel 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

movementWhat 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..

Background:Oxidative stress: lipid peroxidation

Aldehydes formed via lipid peroxidation (from ROS)

Lipid aldehydes: 4-hydroxy-2-nonenal (4HNE) and malondialdehyde (MDA) at < 50 μM (Esterbauer et al., 1991).

O

OH

C5H11

4HNE

O

MDA

O O OH

“Gold standards”

(i.e., Arachidonate)LIPID

-CH=CH-CH2-CH=CH-

RH

(INITIATOR)(INITIATOR)

RR••

(INITIATOR)(INITIATOR)

RR••

(INITIATOR)(INITIATOR)

RR••

(INITIATOR)(INITIATOR)

RR•• O2O2O2

Free Radical Intermediate

-CH=CH-CH-CH=CH-

Free Radical Intermediate

-CH=CH-CH-CH=CH-

Conjugated DieneFormation

-CH=CH-CH=CH-CH-

Conjugated DieneFormation

-CH=CH-CH=CH-CH- -CH=CH-CH=CH-CH-

Peroxy RadicalOO

-CH=CH-CH=CH-CH-

Peroxy RadicalOO

OOH

LIPID

OOH

LIPID

OOH

LIPID

-CH=CH-CH=CH-CH-

Hydroperoxide

OOH

-CH=CH-CH=CH-CH-

Hydroperoxide

OOH HydrocarbonsRadical speciesAldehydes

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

NH2HO

HO

NH2O

O

[O] NH2HO

HO

SProtein

Protein-SH

NH2HO

HO

NH2O

HO

NH2O

O

O2 O2 O2 O2

NH2HO

HO

VMAT2

5

Background: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 μM (Rees et al., 2009; Yunden et al., 2009).

DA DOPAL

DOPAC

DOPETALRNADPH

ALDH2NAD

MAODA DOPAL

DOPAC

DOPETALRNADPH

ALDH2NAD

MAO

HO

HONH2

HO

HOO

HO

HOO

OH

MAO ALDH

NAD

H2O2 ALRNADPH

HO

HOOH

DA DOPAL DOPAC

DOPET

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

HO

O Protein-NH2HO

HO

N-Protein

OHO

HO

OO

O

[O] OHO

HO

Protein

SProtein

GoalsElucidate 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 ExperimentsHow do we obtain DOPAL? Biosynthesis, synthesis.

Model systems for DA catabolism:

MitochondriaSynaptosomes (isolated nerve terminals)Cells: dopaminergic PC6-3 & N27

HO

HONH2

HO

HOO

HO

HOOH

SO3DOPAL

MAO

DA

HSO3

Mitochondria

PC6-3 CellsTyr

DOPA

DA

DOPAL

DOPAC

DA

DAR

DA

DA

R

DA

DA

DA

DA

Tyr

DOPA

DA

DOPAL

DOPAC

DA

DAR

DAR

DA

DA

RD

AR

DA

DA

DA

DA

DA

DA

DADA

DA

Synaptosome

DA MAO ALDH2NADDOPAL DOPAC

Overview of ExperimentsDA model systems: advantages and disadvantages

MitochondriaSynaptosomesCells

Protein reactivity experimentsModel 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

ResultsInhibition 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: 100 μM DA (DA DOPAL in situ) 60 min time-course; aliquots removed DA, DOPAL, DOPAC and DOPET via HPLC

HO

HOO

DOPALHO

HOO

OH

DOPAC

DA DOPAL

DOPAC

DOPETALRNADPH

ALDH2NAD

MAODA DOPAL

DOPAC

DOPETALRNADPH

ALDH2NAD

MAO

0 10 20 30 40 50 600.0

0.5

1.0

1.5

2.0 Control

2 M10 M25M50 M100 M

Time (min)

[DO

PA

C]

(M

)

0 10 20 30 40 50 600.0

0.1

0.2

0.3

0.4

0.5

0.6Control

2 M10 M25 M50 M100 M

Time (min)[D

OP

AL

] (

M)

ResultsInhibition 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

A = % Control ALDH activity (DOPAC production)B = % Control [DOPAL]

0 2 10 25 50 1000

25

50

75

100

125 *

****

[4HNE] (M)

% C

on

tro

l (D

OP

AC

)

0 2 10 25 50 1000

100

200

300

400

500

600

[4HNE] (M)

% C

on

tro

l [D

OP

AL

]

*

Cytotoxic! (MTT)

ResultsDoes 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 7Ln 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

ResultsHow does DOPAL react with proteins?

Oxidation to quinone; quinone plus thiol (Cys)Aldehyde plus amine (Lys)

How reactive is DOPAL?Protein cross-linking?

OHO

HO

OHO

HO

SProtein

NHO

HO

Protein

PROTEINMODIFICATION

ProteinCrosslinking

Protein

-SH

Protein-NH2

Results 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 MALDI-TOF-MS analysis

900 950 1000 1050 1100 1150 12000

102030405060708090

100Peptide (A)

m/z

% In

ten

sit

y

900 950 1000 1050 1100 1150 12000

102030405060708090

100 Peptide

DOPAL-Peptide(B)

m/z

% In

ten

sit

y

900 950 1000 1050 1100 1150 12000

102030405060708090

100Peptide (C)

m/z

% In

ten

sit

y

900 950 1000 1050 1100 1150 12000

102030405060708090

100Peptide (A)

m/z

% In

ten

sit

y

900 950 1000 1050 1100 1150 12000

102030405060708090

100 Peptide

DOPAL-Peptide(B)

m/z

% In

ten

sit

y

900 950 1000 1050 1100 1150 12000

102030405060708090

100Peptide (C)

m/z

% In

ten

sit

y

OHO

HO

NHO

HO

PeptidePeptide

134 Da Adduct

15

Results Is DOPAL reactive toward protein amines (i.e. Lys) or thiols

(i.e. Cys)? 0 mM 1 mM 5 mM 10 mM

OH

O

O

NH

ProteinNo Reaction with DOPAL

SH

NH2

NH

ProteinNo Reaction with DOPAL

SProtein Reaction with DOPAL

NH2

O

Citraconic Anhydride (Modifies Lys)

Traut's Reagent (Converts Amines to Thiols)

Iodoacetic Acid (Modifies Cys)

ResultsIs 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

No reactivity towards N-acetyl His or N-acetyl Arg

OHO

HO

OO

O???

λmax = 520 nm λmax = 410 nm

tyrosinase, sodium metaperiodate

ResultsHow reactive is DOPAL toward protein amines?

1-10 mM Ac-Lys + 0.1 mM DOPAL

k = 2.0 M-1min-1

Compare to 4HNE:k = 0.0798 M-1min-1

0 20 40 60 80 100 120 140 160 1802

3

4

5

Time (min)

ln (

% C

on

tro

l DO

PA

L )

0 2 4 6 8 100.00

0.01

0.02

0.03

N-Ac-Lys (mM)

-k' (

min

-1)

0 20 40 60 80 100 120 140 160 1802

3

4

5

Time (min)

ln (

% C

on

tro

l DO

PA

L )

0 2 4 6 8 100.00

0.01

0.02

0.03

N-Ac-Lys (mM)-k

' (m

in-1

)

10 mM Ac-Lys

10 mM Ac-Lys

k = 0.42 M-1min-1

Unstable w/o reduction!!Needs NaCNBH3

OHO

HO

OHO

H3CO

ResultsHow reactive is DOPAL toward protein amines?

Structure Compound k (M-1min-1)

OHO

HO

DOPAL

2.0 ± 0.036

MOPAL

0.42 ± 0.042a

DMPAL

NDb

PAL

< 0.2c

OHO

H3CO

OH3CO

H3CO

O

a 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

ResultsHow reactive is DOPAL toward protein amines?

Protein (BSA, GAPDH) + Catechols Stain with NBT

1 2 3 4

BSA + catechol1 = DA2 = DOPAL3 = DOPAC4 = L-DOPA

OHO

HO

NH2HO

HO

OHO

HO OH

DA

DOPAL

DOPAC

L-DOPANH2

HO

HO COOH

A

B

GAPDH + catechol1 = DA2 = DOPAL3 = DOPAC4 = L-DOPA

20

ResultsCan DOPAL cross-link proteins? Is it a bifunctional

electrophile? GAPDH + DOPAL Protein mixture + DOPAL 1 2 3 4 5 6 7 8

45

66

116

200

97

DOPAL NaCNBH3 Ascorbate0

20

40

60

80

100 *

% C

on

tro

l

Ascorbate sensitive = quinone?

Lane1 Control2 5 µM DOPAL3 50 µM DOPAL4 100 µM DOPAL

5 Control6 5 µM DOPAL7 50 µM DOPAL8 100 µM DOPAL

2 hrs

4 hrs

NO

O

ProteinN

HO

HO

ProteinAscorbate

ResultsCan DOPAL cross-link proteins? Is it a bifunctional

electrophile? GAPDH + DOPAL Protein mixture + DOPAL

1 2 3 4 5 6 MW

(kDa)

45

66

97

116

200

Lane1 Control2 DOPAL

3 Control4 DOPAL

5 Control6 DOPAL

2 hrs

4 hrs

6 hrs

Initial = 10 µM DOPAL; spike 5 µM DOPAL/hrFinal [DOPAL] = 12 µM (HPLC)

Work in ProgressProtein modification: proteomics based approach

What are target proteins? Functional consequence? Identify proteins: tyrosine hydroxylase, aldehyde

dehydrogenase (mito)

Time (min)

Inte

nsi

ty

m/z

Inte

nsi

ty

+1

+2

Protease

LC separation

MS analysis

(MS/MS)

Database search(MASCOT)

PeptidesIdentified

ProteinIdentified

Time (min)

Inte

nsi

ty

Time (min)

Inte

nsi

ty

m/z

Inte

nsi

ty

+1

+2

m/z

Inte

nsi

ty

+1

+2

Protease

LC separation

MS analysis

(MS/MS)

Database search(MASCOT)

PeptidesIdentified

ProteinIdentified

NBT StainingSynaptosomes

NBT StainingPC6-3 Cells

[4HNE] (μM) = 0 5 10 50 [4HNE] (μM) = 0 10 100

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

OHO

HO

NH2HO

HO

OHO

HO OH

OHHO

HONADPH

MAOALDH2

NAD

AR

Protein Modif icationCellular ToxicityDisease

Increased CytosolicDopamine

Inhibition of AldehydeMetabolism

NO

O

ProteinProtein-SH N

HO

HO

SProtein

Protein ?

AcknowledgementsLab (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, ElizabethWittchow, 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)

AcknowledgementsFinancial support

NIH 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