Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location.
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ABSTRACT:
SUBMITTER: Danson MJ
PROVIDER: S-EPMC1147910 | biostudies-other | 1987 May
REPOSITORIES: biostudies-other
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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.
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Project description:The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.
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Project description:The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456-E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.
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