Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity

A novel mechanism potentially underlying fatty acid induced insulin resistance

Jingyue Yang, Beverly Gibson, Jacqueline Snider, Christopher M. Jenkins, Xianlin Han, Richard W. Gross

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 μM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [ 14C]-palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at ∼35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide 232VPTPNVSVVDLTRC*R245 was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.

Original languageEnglish (US)
Pages (from-to)11903-11912
Number of pages10
JournalBiochemistry
Volume44
Issue number35
DOIs
StatePublished - Sep 6 2005
Externally publishedYes

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Palmitoyl Coenzyme A
Glyceraldehyde-3-Phosphate Dehydrogenases
Enzyme activity
Cysteine
Insulin Resistance
Fatty Acids
Insulin
Lipoylation
Enzymes
Palmitates
Metabolites
Ions
Enzyme inhibition
Glucose
Acyl Coenzyme A
Autoradiography
Stoichiometry
Polyacrylamide Gel Electrophoresis
Proteins
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity : A novel mechanism potentially underlying fatty acid induced insulin resistance. / Yang, Jingyue; Gibson, Beverly; Snider, Jacqueline; Jenkins, Christopher M.; Han, Xianlin; Gross, Richard W.

In: Biochemistry, Vol. 44, No. 35, 06.09.2005, p. 11903-11912.

Research output: Contribution to journalArticle

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abstract = "The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 μM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [ 14C]-palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at ∼35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide 232VPTPNVSVVDLTRC*R245 was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.",
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