Protein Modification by a Reactive Intermediate of Catecholamine Metabolism

1
Protein Modification by a Reactive Intermediate
of Catecholamine Metabolism

• Jonathan A. Doorn
• Assistant Professor
• Medicinal and Natural Products Chemistry
• College of Pharmacy
• The University of Iowa

2
Background

• Neurodegenerative diseases
• Parkinsons disease (PD)
• Loss/impairment of dopamine (DA) producing
  neurons substantia nigra
• DA is a neurotransmitter involved in coordination
  of movement
• Result/symptoms tremors, rigidity, bradykinesia,
  poor balance
• 2nd most common neurodegenerative disease
• 1.5 million cases in US 60,000 diagnosed/year
• What causes PD?
• Genetics (minority)
• Environment
• Genes/environment

http//www.ninds.nih.gov http//www.parkinson.or
g Chung, K.K., et al. (2003) J. Neurol. 250
Suppl. 3, III 15-24. Di Monte, D.A. (2003)
Lancet Neurol. 9, 531-538.
3
Background

• Mechanism (general)?
• Oxidative stress
• Reactive oxygen species
• Toxic aldehydes formed via lipid peroxidation

Jenner, P. (2003) Ann. Neurol. 53 Suppl. 3,
S26-36. Yoritaka, A., et al. (1996) Proc Natl
Acad Sci. 93, 2696-2701.
4
Background

• Mechanism (general)?
• Failure of cellular machinery involved in protein
  turnover
• Proteasome involved in cellular maintenance
  degrades proteins
• Impaired proteasomal activity ? protein
  aggregation

Protein
Protein
Proteasome
Protein
Protein
Protein
Protein
Dawson, T.M. and Dawson, V.L. (2003) Science 302,
819-822. McNaught, K.S., and Olanow, C.W. (2003)
Ann Neurol. 53 Suppl. 3, S73-84.
5
Background

• Role of a reactive intermediate?
• Reactive intermediate of DA metabolism,
  3,4-dihydroxyphenyl acetaldehyde (DOPAL).
• Metabolism of DOPAL ? 3,4-dihydroxyphenylacetic
  acid (DOPAC) or 3,4-dihydroxyphenylethanol
  (DOPET).

Enzymes monoamine oxidase (MAO) aldehyde
dehydrogenase 2 (ALDH2) aldehyde reductase
(ALR)
Elsworth, J.D. and Roth, R.H. (1997) Exp. Neurol.
144, 4-9.
6
Background

• Role of a reactive intermediate?
• Aldehyde electrophile, reactive toward cellular
  nucleophiles.
• DOPAL is cytotoxic. May play role in disease.
• DOPAL regulated by several enzymes. What
  inhibits metabolism?
• Why is DOPAL harmful? Specific mechanism?

Burke, W.J. et al. (2004) Neurotoxicology 25,
101-115. Blashko, H. (1952) Pharmacol. Rev.
4,415-453. Ungar, F. et al. (1973) Biochem.
Pharmacol. 22, 1905-1913.
7
Background

• Mechanism for generation of DOPAL at aberrant
  levels?
• Interference with DOPAL metabolism
• Consequence of ?DOPAL? Protein modification.
• Enzyme inactivation?
• Impairment of proteasome system?

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Hypothesis/Aims

• Hypothesis Impairment of DOPAL metabolism ?
  aberrant levels of the reactive aldehyde
  intermediate ? (deleterious) protein
  modification.
• Aims
• Elucidate mechanism(s) for inhibition of DOPAL
  metabolism
• Identify proteins modified by the reactive
  aldehyde intermediate
• Determine functional consequence of protein
  adduction
• Enzyme inactivation
• Inhibition of proteasome

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Approaches/Methods

• Synthesis of aldehydes (enzymatic chemical)
• DOPAL metabolism HPLC
• Protein modification proteomics based approach
• 2D gel electrophoresis

Separation pI and MW Detection antibodies
toward adducts radiolabeled reactive
intermediates generated in situ
Liebler, D.C. (2002) Introduction to Proteomics,
Humana Press, Totowa, NJ.
10
Approaches/Methods

• Protein modification proteomics based approach
• LC/MS/MS analysis
• Protein assays proteasome activity

Liebler, D.C. (2002) Introduction to Proteomics,
Humana Press, Totowa, NJ.
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Summary

• Reactive intermediate chemistry/chemical
  toxicology
• Elucidate mechanism(s) for aberrant
  concentrations of aldehyde intermediates.
• Identify protein targets.
• Determine functional consequence of protein
  modification by endogenous aldehydes.
• Do reactive (aldehyde) intermediates play a role
  in human disease (e.g. Parkinsons)?

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Future Directions

• Identify specific versus non-specific DOPAL
  binding.
• DOPAL-modification of proteins in a cell/animal.
• Other reactive aldehyde intermediates of
  neurotransmitters (e.g. norepinephrine,
  serotonin)?
• Therapeutic intervention design/use of
  antioxidants and/or carbonyl scavangers
  (nucleophiles).