The natural history of dihydrolipoamide dehydrogenase deficiency in Israel.

Dihydrolipoamide dehydrogenase (DLD) deficiency is an ultra-rare autosomal-recessive inborn error of metabolism, affecting no less than five mitochondrial multienzyme complexes. With approximately 30 patients reported to date, DLD deficiency was associated with three major clinical presentations: an early-onset encephalopathic phenotype with metabolic acidosis, a predominantly hepatic presentation with liver failure, and a rare myopathic phenotype. To elucidate the demographic, phenotypic, and molecular characteristics of patients with DLD deficiency within the Israeli population, data were collected from metabolic disease specialists in four large tertiary medical centers in the center and south of Israel. Pediatric and adult patients with biallelic variants in DLD were included in the study. A total of 53 patients of 35 families were included in the cohort. Age at presentation ranged between birth and 10 years. Wide phenotypic variability was observed, from asymptomatic individuals in their sixth decade of life, to severe, neonatal-onset disease with devastating neurological sequelae. Six DLD variants were noted, the most common of which was the c.685G>T (p.G229C) variant in homozygous form (24/53 patients, 45.3%; 13/35 families), observed mostly among patients of Ashkenazi-Jewish descent, followed by the homozygous c.1436A>T (p.D479V) variant, found in 20 patients of Bedouin descent (37.7%; 16/35 families). Overall, patients did not necessarily present as one of the previously described distinct clinical phenotypes. DLD deficiency is a panethnic disorder, with significant phenotypic variability, and comprises a continuum, rather than three distinct clinical presentations.

The diagnostic challenge of mild citrulline elevation at newborn screening.

Citrulline is a target analyte measured at expanded newborn screening (NBS) and its elevation represents a biomarker for distal urea cycle disorders and citrin deficiency. Altered ratios of citrulline with other urea cycle-related amino acids are helpful for the differential diagnosis. However, the use of cut-off values in screening programmes has raised the issue about the interpretation of mild elevation of citrulline levels detected at NBS, below the usual range observed in the "classical/severe" forms of distal urea cycle disorders and in citrin deficiency. Herein, we report ten subjects with positive NBS for a mild elevation of citrulline (<100 µmol/L), in whom molecular investigations revealed carriers status for argininosuccinate synthase deficiency, a milder form of argininosuccinate lyase deficiency and two other diseases, lysinuric protein intolerance and dihydrolipoamide dehydrogenase deficiency, not primarily affecting the urea cycle. To guide the diagnostic process, we have designed an algorithm for mild citrulline elevation (<100 µmol/L) at NBS, which expands the list of disorders to be included in the differential diagnosis.

Dihydrolipoamide dehydrogenase, pyruvate oxidation, and acetylation-dependent mechanisms intersecting drug iatrogenesis.

In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete ß-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

BACKGROUND: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. METHODS: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. RESULTS: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. CONCLUSIONS: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

The moonlighting activities of dihydrolipoamide dehydrogenase: Biotechnological and biomedical applications.

Dihydrolipoamide dehydrogenase (DLDH) is a homodimeric flavin-dependent enzyme that catalyzes the NAD+ -dependent oxidation of dihydrolipoamide. The enzyme is part of several multi-enzyme complexes such as the Pyruvate Dehydrogenase system that transforms pyruvate into acetyl-co-A. Concomitantly with its redox activity, DLDH produces Reactive Oxygen Species (ROS), which are involved in cellular apoptotic processes. DLDH possesses several moonlighting functions. One of these is the capacity to adhere to metal-oxides surfaces. This was first exemplified by the presence of an exocellular form of the enzyme on the cell-wall surface of Rhodococcus ruber. This capability was evolutionarily conserved and identified in the human, mitochondrial, DLDH. The enzyme was modified with Arg-Gly-Asp (RGD) groups, which enabled its interaction with integrin-rich cancer cells followed by "integrin-assisted-endocytosis." This allowed harnessing the enzyme for cancer therapy. Combining the TiO2 -binding property with DLDH's ROS-production, enabled us to develop several medical applications including improving oesseointegration of TiO2 -based implants and photodynamic treatment for melanoma. The TiO2 -binding sites of both the bacterial and human DLDH's were identified on the proteins' molecules at regions that overlap with the binding site of E3-binding protein (E3BP). This protein is essential in forming the multiunit structure of PDC. Another moonlighting activity of DLDH, which is described in this Review, is its DNA-binding capacity that may affect DNA chelation and shredding leading to apoptotic processes in living cells. The typical ROS-generation by DLDH, which occurs in association with its enzymatic activity and its implications in cancer and apoptotic cell death are also discussed.

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent.

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO2 as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin-mediated drug-delivery of RGD-modified DLDH (DLDHRGD ), leading to apoptotic cell death. In these experiments, DLDHRGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA-binding capability of this enzyme. To explore this interaction under cell-free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel-shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA-binding. A Docking algorithm for prediction of protein-DNA complexes, Paradoc, identified a putative DNA binding site at the C-terminus of DLDH. Our finding that TiO2 -bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

An Updated View on the Molecular Pathomechanisms of Human Dihydrolipoamide Dehydrogenase Deficiency in Light of Novel Crystallographic Evidence.

Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.

Extracytoplasmic diaphorase activity of Streptomyces coelicolor A3(2).

Metabolism and utilization of plant-derived aromatic substances are fundamental to the saprophytic growth of Streptomyces. Here, we studied an enzyme activity reducing 2,6-dichlorophenolindophenol and nitroblue tetrazolium in the culture supernatant of Streptomyces coelicolor A3(2). N-terminal amino acid sequencing of a nitroblue tetrazolium-reducing enzyme revealed that the enzyme corresponds to the SCO2180 product. The protein exhibited a marked similarity with dihydrolipoamide dehydrogenase, the E3 subunit of 2-oxo-acid dehydrogenase complex. A recombinant SCO2180 protein formed a homodimer and exhibited a diaphorase activity catalyzing NADH-dependent reduction of various quinonic substrates. Similar nitroblue tetrazolium-reducing activities were observed for other Streptomyces strains isolated from soil, implying that the diaphorase-catalyzed reduction of quinonic substances widely occurs in the extracytoplasmic space of Streptomyces.

Dihydrolipoamide dehydrogenase suppression induces human tau phosphorylation by increasing whole body glucose levels in a C. elegans model of Alzheimer's Disease.

The microtubule associated tau protein becomes hyperphosphorylated in Alzheimer's disease (AD). While hyperphosphorylation promotes neurodegeneration, the cause and consequences of this abnormal modification are poorly understood. As impaired energy metabolism is an important hallmark of AD progression, we tested whether it could trigger phosphorylation of human tau protein in a transgenic Caenorhabditis elegans model of AD. We found that inhibition of a mitochondrial enzyme of energy metabolism, dihydrolipoamide dehydrogenase (DLD) results in elevated whole-body glucose levels as well as increased phosphorylation of tau. Hyperglycemia and tau phosphorylation were induced by either RNAi suppression of the dld-1 gene or by inhibition of the DLD enzyme by the inhibitor, 2-methoxyindole-2-carboxylic acid (MICA). Although the calcium ionophore A23187 could reduce tau phosphorylation induced by either chemical or genetic suppression of DLD, it was unable to reduce tau phosphorylation induced by hyperglycemia. While inhibition of the dld-1 gene or treatment with MICA partially reversed the inhibition of acetylcholine neurotransmission by tau, neither treatment affected tau inhibited mobility. Conclusively, any abnormalities in energy metabolism were found to significantly affect the AD disease pathology.

Structural alterations induced by ten disease-causing mutations of human dihydrolipoamide dehydrogenase analyzed by hydrogen/deuterium-exchange mass spectrometry: Implications for the structural basis of E3 deficiency.

Pathogenic amino acid substitutions of the common E3 component (hE3) of the human alpha-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complexes lead to severe metabolic diseases (E3 deficiency), which usually manifest themselves in cardiological and/or neurological symptoms and often cause premature death. To date, 14 disease-causing amino acid substitutions of the hE3 component have been reported in the clinical literature. None of the pathogenic protein variants has lent itself to high-resolution structure elucidation by X-ray or NMR. Hence, the structural alterations of the hE3 protein caused by the disease-causing mutations and leading to dysfunction, including the enhanced generation of reactive oxygen species by selected disease-causing variants, could only be speculated. Here we report results of an examination of the effects on the protein structure of ten pathogenic mutations of hE3 using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), a new and state-of-the-art approach of solution structure elucidation. On the basis of the results, putative structural and mechanistic conclusions were drawn regarding the molecular pathogenesis of each disease-causing hE3 mutation addressed in this study.

Identification of an extracellular bacterial flavoenzyme that can prevent re-polymerisation of lignin fragments.

A significant problem in the oxidative breakdown of lignin is the tendency of phenolic radical fragments to re-polymerise to form higher molecular weight species. In this paper we identify an extracellular flavin-dependent dehydrolipoamide dehydrogenase from Thermobifida fusca that prevents oxidative dimerization of a dimeric lignin model compound, which could be used as an accessory enzyme for lignin depolymerisation.

Genetic Conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.).

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam, and Australia. Each of 4 strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from 2 independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mg L(-1) for a 48-h exposure at 25 °C. Thus, the mechanism of high-level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

Intermittent hypoxia preconditioning (IHP) has been shown to protect neurons against ischemic stroke injury. Studying how proteins respond to IHP may identify targets that can help fight stroke. The objective of the present study was to investigate whether mitochondrial dihydrolipoamide dehydrogenase (DLDH) would respond to IHP and if so, whether such a response could be linked to neuroprotection in ischemic stroke injury. To do this, we subjected male rats to IHP for 20 days and measured the content and activity of DLDH as well as the three α-keto acid dehydrogenase complexes that contain DLDH. We also measured mitochondrial electron transport chain enzyme activities. Results show that DLDH content was indeed upregulated by IHP and this upregulation did not alter the activities of the three α-keto acid dehydrogenase complexes. Results also show that the activities of the five mitochondrial complexes (I-V) were not altered either by IHP. To investigate whether IHP-induced DLDH upregulation is linked to neuroprotection against ischemic stroke injury, we subjected both DLDH deficient mouse and DLDH transgenic mouse to stroke surgery followed by measurement of brain infarction volume. Results indicate that while mouse deficient in DLDH had exacerbated brain injury after stroke, mouse overexpressing human DLDH also showed increased brain injury after stroke. Therefore, the physiological significance of IHP-induced DLDH upregulation remains to be further investigated.

Eclipta prostrata improves alveolar development of bronchopulmonary dysplasia via suppressing the NLRP3 inflammasome in a DLD-dependent manner.

OBJECTIVES: Bronchopulmonary dysplasia (BPD), the most common late morbidity in preterm infants, is characterized by impaired alveolar development caused by persistent lung inflammation. Studies have shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome-mediated inflammation is critically involved in the development of BPD. As a traditional Chinese medicinal herb, Eclipta prostrata (EAP) exhibits potent anti-inflammatory properties. Our study aims to investigate whether EAP could improve the lung development of BPD by suppressing the lung inflammatory response. METHODS: The BPD rat model was established by intra-amniotic injection of lipopolysaccharide (LPS) and postnatal exposure to hyperoxia. Changes in the NLRP3 inflammasome and pyroptosis were assessed by treatment with EAP. The effect of EAP on the NLRP3 inflammasome was tested in vitro using the THP-1 cell line and primary alveolar macrophages. Proteomics analysis was used to elucidate the mechanism of action of EAP. RESULTS: Histopathological and immunofluorescence results of lung tissues revealed that LPS and hyperoxia induced lung injury and triggered NLRP3 inflammasome activation and pyroptosis in alveolar macrophages. EAP ameliorated BPD lung injury, inhibited NLRP3 inflammasome activation and reduced gasdermin D (GSDMD) expression in alveolar macrophages. EAP downregulated the expression of NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1ß) and GSDMD in LPS-stimulated THP-1 macrophages and primary alveolar macrophages. In addition, proteomics analysis identified that dihydrolipoamide dehydrogenase (DLD) interacted with EAP. Inhibition of DLD activity abolished the protective effects of EAP. CONCLUSIONS: Our study suggested that EAP could attenuate arrest of alveolar development via inhibiting NLRP3 inflammasome in a DLD-dependent way, and could be a potential therapeutic method for BPD.

Dehydrogenase (DLD) Deficiency in an Iranian Patient with Recurrent Intractable Vomiting: Successful Treatment with Thiamine Supplementation.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a rare disease of genetic origin due to the malfunctioning of a shared subunit of three mitochondrial multi-enzyme complexes. Phenotypes of this disease are a set of clinical manifestations ranging from neonatal disorders to myopathy or recurrent episodes of liver failures, and vomiting for which no adequate or definitive treatment is currently available. This study described a case involving a 16-year-old boy who had experienced recurrent vomiting of unknown cause from age two. Normal value ranges for the basic metabolic panel were reported in previous years. The patient was admitted with Wernicke's encephalopathy after the last vomiting attack, also indicating metabolites of organic acids compatible with DLD deficiency. Whole exome sequencing identified a known pathogenic mutation in the DLD gene, leading to a diagnosis of DLD deficiency. Our patient was treated with a high dose of thiamine supplementation and continued treatment, has not experienced any vomiting attacks or related problems in the last two years and has adequately responded to the treatment prescribed. Normal urine organic acid levels in patients with recurrent vomiting cannot roll out DLD deficiency. However, although thiamine deficiency typically induces Wernicke's encephalopathy, it can also be implicated in pyruvate dehydrogenase complex (PDHc) deficiency, and high-dose thiamine therapy (with doses up to 30 mg/kg) is recommended for deficient patients.

Mitochondrial Dihydrolipoamide Dehydrogenase (mtLPD1): Expression, Purification, Activity, and Redox Regulation.

Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) is a central enzyme in primary carbon metabolism, since its function is required to drive four multienzymes involved in photorespiration, the tricarboxylic acid (TCA) cycle, and the degradation of branched-chain amino acids. However, in illuminated, photosynthesizing tissue a vast amount of mtLPD1 is necessary for glycine decarboxylase (GDC), the key enzyme of photorespiration. In light of the shared role, the functional characterization of mtLPD1 is necessary to understand how the three pathways might interact under different environmental scenarios. This includes the determination of the biochemical properties and all potential regulatory mechanisms, respectively. With regards to the latter, regulation can occur through multiple levels including effector molecules, cofactor availability, or posttranslational modifications (PTM), which in turn decrease or increase the activity of each enzymatic reaction. Gaining a comprehensive overview on all these aspects would ultimately facilitate the interpretation of the metabolic interplay of the pathways within the whole subcellular network or even function as a proof of concept for genetic engineering approaches. Here, we describe the typical workflow how to clone, express, and purify plant mtLPD1 for biochemical characterization and how to analyze potential redox regulatory mechanisms in vitro and in planta.

Oncogenic role of copper­induced cell death­associated protein DLD in human cancer: A pan­cancer analysis and experimental verification.

Copper ions can bind directly to lipoylated components of the tricarboxylic acid (TCA) cycle, triggering the aggregation of mitochondrial lipoylated proteins and the destabilization of Fe-S cluster proteins, resulting in copper-dependent cell death. Dihydrolipoamide dehydrogenase (DLD) is a key protein of the TCA cycle and constitutes the E3 component of the α-ketoglutarate dehydrogenase complex, which is deeply interconnected with the mitochondrial electron transfer chain in the TCA cycle. Tumor cells demonstrate dependency on glutaminolysis fuelling to carry out the TCA cycle and essential biosynthetic processes supporting tumor growth. Therefore, DLD plays an important role in the tumor biological process. However, to the best of our knowledge, no pan-cancer analysis is currently available for DLD. Therefore, the present study first explored the DLD expression profile in 33 tumors in publicly available datasets, including TIMER2, GEPIA2, UALCAN, cBioPortal and STRING. TIMER2, GEPIA2 and UALCAN were used for exploring gene expression; survival prognosis was detected by GEPIA2; genetic alteration was analysed by cBioPortal; immune infiltration data was obtained from TIMER2; interacting proteins of DLD were detected by STRING. DLD was found to be highly expressed in colon, liver, lung, stomach, renal, corpus uteri endometrial and ovarian cancers compared with normal tissues, and its high expression was associated with poorer prognosis in ovarian cancer. To the best of our knowledge, the present study provided the first comprehensive pan-cancer analysis of the oncogenic role of DLD across different tumors types. As the expression of DLD in ovarian cancer was high, and high expression is associated with poor prognosis, experimental verification of DLD in ovarian cancer was conducted. In the present study, DLD expression was found to be high in the ovarian cancer OC3 cell line, compared with the normal ovarian epithelial IOSE80 cell line by reverse transcription-quantitative PCR analysis. After knockdown of DLD expression, it was found that DLD regulated metabolic pathways by suppressing the intracellular NAD+/NADH ratio, which then in turn suppressed tumor cell proliferation detected by MTT assay. In conclusion, the present pan-cancer analysis of DLD demonstrated that DLD expression was associated with the clinical prognosis, immune infiltration and tumor mutational burden in 33 tumor types, and experimental verification in ovarian cancer was conducted. These results may contribute to the understanding of the role of DLD in tumorigenesis.

Roles of Dihydrolipoamide Dehydrogenase in Health and Disease.

Significance: Dihydrolipoamide dehydrogenase (DLDH) is a flavin-dependent disulfide oxidoreductase. The active form of DLDH is a stable homodimer, and its deficiencies have been linked to numerous metabolic disorders. A better understanding of redox and nonredox features of DLDH may reveal druggable targets for disease interventions or preventions. Recent Advances: In this article, the authors review the different roles of DLDH in selected pathological conditions, including its deficiency in humans, its role in stroke and neuroprotection, skin photoaging, Alzheimer's disease, and DLDH as a nondehydrogenating protein, and construction of genetically modified DLDH animal models for further studying the role of DLDH in specific pathological conditions. DLDH is also vulnerable to oxidative modifications in pathological conditions. Critical Issues: Novel animal models need to be constructed using gene knockdown techniques to investigate the redox- and nonredox roles of DLDH in related metabolic diseases. Specific small-molecule DLDH inhibitors need to be discovered. The relationship between modifications of specific amino acid residues in DLDH and given pathological conditions is an interesting area that remains to be comprehensively evaluated. Future Directions: Cell-specific or tissue-specific knockdown of DLDH creating specific pathological conditions will provide more insights into the mechanisms, whereby DLDH may have therapeutic values under a variety of pathological conditions. Antioxid. Redox Signal. 39, 794-806.