Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

doi:10.1016/j.abb.2005.04.015

Archives of Biochemistry and Biophysics

Volume 438, Issue 2, 15 June 2005, Pages 217-222

https://doi.org/10.1016/j.abb.2005.04.015 Get rights and content

Abstract

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 °C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

Section snippets

Chemicals

HNE and HHE were purchased from Cayman Chemical (Ann Arbor, MI). Human erythrocyte GAPDH (e-GAPDH) and glyceraldehyde-3-phosphate (GAP) were from Sigma–Aldrich (St. Louis, MO). β-NAD was from Oriental Yeast (Tokyo, Japan). An anti-human GAPDH monoclonal antibody was from Biogenesis (6G5, England, UK). An anti-human GAPDH polyclonal antibody was from Santa Cruz Biotechnology (FL-335, Delaware Avenue, CA). The fluorogenic peptides of l-alanyl-l-alanyl-l-phenylalanine-4-methyl-coumaryl-7-amide

Inhibition and degradation of GAPDH by incubation with lipid peroxidation products

First, we tested the effects of various aldehydes produced by lipid peroxidation on GAPDH activity. Either HNE or HHE strongly inhibited the GAPDH activity of the U937 cell extract and e-GAPDH activity in the IC₅₀ range of 21.0–50.0 μM. However, the other four aldehydes tested, crotonaldehyde, 2-hexenal, 2-nonenal, and glyoxal, did not (IC₅₀ > 1 mM) (Table 1). We then tested the effects of these aldehydes on triggering the degradation of GAPDH in the U937 cell extract (Fig. 1). The density of the

Discussion

Aldehydes produced by lipid peroxidation reactions are mainly classified into three families [3]. HHE and HNE belong to the family of 4-hydroxy-2-alkenals; 2-hexenal, 2-nonenal, and crotonaldehyde to the family of 2-alkenals; and glyoxal to the family of ketoaldehydes. In the present study, HHE and HNE inhibited GAPDH activity with low IC₅₀ values and triggered GAPDH degradation in the U937 cell extract. On the other hand, 2-hexenal, 2-nonenal, crotonaldehyde, and glyoxal did not decrease GAPDH

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At the same time, drugs based on deprenyl or its derivatives are employed in the medication of Parkinsons disease (Selegeline, Rasalgine) and may be used as recently reported for anticocaine therapy [31]. Another potent binder of GAPDH is hydroxynonenal that was shown to interact directly with Cys149 and taken in high concentration it was found to completely inactivate the enzyme [32–33]. The idea of using reactive cystein(-s) as a target for the chemical blockade of the enzyme led us to perform molecular docking studies which resulted in the creation of three chemicals that were shown to affect the dynamics of GAPDH molecule melting, as established by DSC [34].
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Given the importance of cysteine, lysine, and histidine (protein carbonylation targets) in catalysis, proteins modified by reactive aldehydes are typically inactivated and may be targeted for degradation. However, some signaling systems are activated upon carbonylation [13–17]. Protein carbonylation is believed to play a major role in altering mitochondrial respiration and metabolic capacity [4,18,19] and recently Curtis et al. [10] have identified the mitochondrial phosphate carrier and two subunits of Complex I (NDUFA2 and NDUFA3) as critical carbonylation targets using the 3T3-L1 model cell system.
Protein carbonylation is the covalent modification of proteins by α,β-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC–MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.
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Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.
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The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an “aging effector” via the formation of 4-HNE-protein adducts, and a resulting change in protein function.
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Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Abstract

Section snippets

Chemicals

Inhibition and degradation of GAPDH by incubation with lipid peroxidation products

Discussion

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Prog. Lipid Res.

Neurobiol. Aging

J. Lipid Res.

Brain Res.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Virology

Mol. Aspects Med.

Biochem. Biophys. Res. Commun.