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Attenuation of radiation toxicity by the phosphine resistance factor dihydrolipoamide dehydrogenase (DLD).

ABSTRACT: Phosphine gas is an excellent fumigant for disinfesting stored grain of insect pests, but heavy reliance on phosphine has led to resistance in grain pests that threatens its efficacy. Phosphine-resistance was previously reported to be mediated by the enzyme DLD. Here we explore the relationship between phosphine toxicity and genotoxic treatments with the goal of understanding how phosphine works. Specifically, we utilized mutant lines either sensitive or resistant to phosphine, gamma irradiation or UV exposure. The phosphine-resistance mutation in the enzyme of energy metabolism, dihydrolipoamide dehydrogenase exhibited cross-resistance to UV and ionizing radiation. Two radiation-sensitive mutants that are defective in DNA repair as well as a mutant that is defective in the activation of the DAF-16 stress response transcription factor all exhibit sensitivity to phosphine that exceeds the sensitivity of the wild type control. A radiation resistance mutation in cep-1, the p53 orthologue, that is deficient in double strand break repair of DNA and is also deficient in apoptosis causes radiation-resistance results but sensitivity toward phosphine.

SUBMITTER: Alzahrani SM

PROVIDER: S-EPMC6478721 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Attenuation of radiation toxicity by the phosphine resistance factor dihydrolipoamide dehydrogenase (DLD).

Alzahrani Saad M SM Ebert Paul R PR

Scientific reports 20190423 1

Phosphine gas is an excellent fumigant for disinfesting stored grain of insect pests, but heavy reliance on phosphine has led to resistance in grain pests that threatens its efficacy. Phosphine-resistance was previously reported to be mediated by the enzyme DLD. Here we explore the relationship between phosphine toxicity and genotoxic treatments with the goal of understanding how phosphine works. Specifically, we utilized mutant lines either sensitive or resistant to phosphine, gamma irradiation ...[more]

PMID: 31015501

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Project description:Dihydrolipoamide dehydrogenase (DLD; E3) oxidizes lipoic acid. Restoring the oxidized state allows lipoic acid to act as a necessary electron sink for the four mitochondrial keto-acid dehydrogenases: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain α-keto-acid dehydrogenase, and 2-oxoadipate dehydrogenase. DLD deficiency (DLDD) is caused by biallelic pathogenic variants in DLD. Three major forms have been described: encephalopathic, hepatic, and myopathic, although DLDD patients exhibit overlapping phenotypes. Hyperlactatemia, hyperexcretion of tricarboxylic acid cycle (TCA) metabolites and branched-chain keto acids, increased plasma branched-chain amino acids and allo-isoleucine are intermittent metabolic abnormalities reported in patients with DLDD. However, the diagnostic performance of these metabolites has never been studied. Therefore, we sought to systematically evaluate the diagnostic utility of these biomarkers for DLDD. We retrospectively analyzed the results of biochemical testing of six unrelated DLDD patients, including values obtained during both well visits and acute decompensation episodes. Elevation of branched-chain amino acid concentrations was not consistently observed. We found that five of six patients in our cohort had a maximum lifetime value of allo-isoleucine of 6 μmol/L, showing that alloisoleucine elevations even during illness may be subtle. Urine organic acid analysis (UOA) during acute decompensation episodes was abnormal in all cases; however, the pattern of abnormalities had high intersubject variability. No single biomarker was universally present, even in patients experiencing metabolic decompensation. We also observed novel biochemical associations: three patients had hyperexcretion of TCA cycle metabolites during crisis; in two patients, 2-ketoadipic and 2-hydroxyadipic acids, by products of lysine degradation, were detected. We propose that these result from 2-oxoadipate dehydrogenase deficiency, an underappreciated biochemical abnormality in DLD. Given the diversity of biochemical profiles among the patients with DLDD, we conclude that accurate biochemical diagnosis relies on a high index of suspicion and multipronged biochemical analysis, including both plasma amino acid and urine organic acid quantitation during decompensation. Biochemical diagnosis during the well state is challenging. We emphasize the critical importance of multiple simultaneous biochemical tests for diagnosis and monitoring of DLDD. We also highlight the under-recognized role of DLD in the lysine degradation pathway. Larger cohorts of patients are needed to establish a correlation between the biochemical pattern and clinical outcomes, as well as a genotype-phenotype correlation.

Project description:Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.

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Attenuation of radiation toxicity by the phosphine resistance factor dihydrolipoamide dehydrogenase (DLD).

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Attenuation of radiation toxicity by the phosphine resistance factor dihydrolipoamide dehydrogenase (DLD).

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