GAPDH Is Conformationally and Functionally Altered in Association with Oxidative Stress in Mouse Models of Amyotrophic Lateral Sclerosis

Anson Pierce, Hamid Mirzaei, Florian Muller, Eric De Waal, Alexander B. Taylor, Shanique Leonard, Holly Van Remmen, Fred Regnier, Arlan Richardson, Asish Chaudhuri

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.

Original languageEnglish (US)
Pages (from-to)1195-1210
Number of pages16
JournalJournal of Molecular Biology
Volume382
Issue number5
DOIs
StatePublished - Oct 24 2008
Externally publishedYes

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Glyceraldehyde-3-Phosphate Dehydrogenases
Amyotrophic Lateral Sclerosis
Oxidative Stress
Creatine Kinase
chloroamide S-330
Lysine
Protein Unfolding
Muscle Proteins
Enzymes
Threonine
Heat-Shock Proteins
Tandem Mass Spectrometry
Hydrophobic and Hydrophilic Interactions
Liquid Chromatography
Protein Kinases
Serine
Tyrosine
Skeletal Muscle
Proteins
Amino Acids

Keywords

  • amyotrophic lateral sclerosis
  • BisANS
  • GAPDH
  • protein oxidation
  • protein unfolding

ASJC Scopus subject areas

  • Virology

Cite this

GAPDH Is Conformationally and Functionally Altered in Association with Oxidative Stress in Mouse Models of Amyotrophic Lateral Sclerosis. / Pierce, Anson; Mirzaei, Hamid; Muller, Florian; De Waal, Eric; Taylor, Alexander B.; Leonard, Shanique; Van Remmen, Holly; Regnier, Fred; Richardson, Arlan; Chaudhuri, Asish.

In: Journal of Molecular Biology, Vol. 382, No. 5, 24.10.2008, p. 1195-1210.

Research output: Contribution to journalArticle

Pierce, A, Mirzaei, H, Muller, F, De Waal, E, Taylor, AB, Leonard, S, Van Remmen, H, Regnier, F, Richardson, A & Chaudhuri, A 2008, 'GAPDH Is Conformationally and Functionally Altered in Association with Oxidative Stress in Mouse Models of Amyotrophic Lateral Sclerosis', Journal of Molecular Biology, vol. 382, no. 5, pp. 1195-1210. https://doi.org/10.1016/j.jmb.2008.07.088
Pierce, Anson ; Mirzaei, Hamid ; Muller, Florian ; De Waal, Eric ; Taylor, Alexander B. ; Leonard, Shanique ; Van Remmen, Holly ; Regnier, Fred ; Richardson, Arlan ; Chaudhuri, Asish. / GAPDH Is Conformationally and Functionally Altered in Association with Oxidative Stress in Mouse Models of Amyotrophic Lateral Sclerosis. In: Journal of Molecular Biology. 2008 ; Vol. 382, No. 5. pp. 1195-1210.
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abstract = "It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43{\%} and 41{\%} reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22{\%} reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.",
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AU - Mirzaei, Hamid

AU - Muller, Florian

AU - De Waal, Eric

AU - Taylor, Alexander B.

AU - Leonard, Shanique

AU - Van Remmen, Holly

AU - Regnier, Fred

AU - Richardson, Arlan

AU - Chaudhuri, Asish

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N2 - It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.

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