Dataset Information

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde.

ABSTRACT:

Background

The aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an endogenous neurotoxin implicated in Parkinson's Disease. Elucidating protein targets of DOPAL is essential in understanding it's pathology. The enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of DOPAL.

Methods

GAPDH activity was measured via reduction of NAD⁺ cofactor (340 nm). Protein aggregation was assessed with SDS-PAGE methods and specific modification via chemical probes.

Results

Low micromolar levels of DOPAL caused extensive GAPDH aggregation and irreversibly inhibited enzyme activity. The inactivation of GAPDH was dependent on both the catechol and aldehyde moieties of DOPAL. It is suggested that Cys are modified and oxidized by DOPAL.

Conclusions

The mechanism by which DOPAL modifies GAPDH can serve as a mechanistic explanation to the pathological events in Parkinson's Disease.

SUBMITTER: Vanle BC

PROVIDER: S-EPMC5746426 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Publications

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde.

Vanle Brigitte C BC Florang Virginia R VR Murry Daryl J DJ Aguirre Arturo L AL Doorn Jonathan A JA

Biochemical and biophysical research communications 20170819 2

<h4>Background</h4>The aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an endogenous neurotoxin implicated in Parkinson's Disease. Elucidating protein targets of DOPAL is essential in understanding it's pathology. The enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of DOPAL.<h4>Methods</h4>GAPDH activity was measured via reduction of NAD<sup>+</sup> cofactor (340 nm). Protein aggregation was assessed with SDS-PAGE methods and specific modification ...[more]

PMID: 28830811

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Project description:α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVK < acrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.

Dataset Information

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde.

Background

Methods

Results

Conclusions

Publications

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde.

Similar Datasets

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