Dihydrolipoyl dehydrogenase promotes white adipocytes browning by activating the RAS/ERK pathway and undergoing crotonylation modification.

Acetylation modification has a wide range of functional roles in almost all physiological processes, such as transcription and energy metabolism. Crotonylation modification is mainly involved in RNA processing, nucleic acid metabolism, chromosome assembly and gene expression, and it's found that there is a competitive relationship between crotonylation modification and acetylation modification. Previous study found that dihydrolipoyl dehydrogenase (DLD) was highly expressed in brown adipose tissue (BAT) of white adipose tissue browning model mice, suggesting that DLD is closely related to white fat browning. This study was performed by quantitative real-time PCR (qPCR), Western blotting (WB), Enzyme-linked immunosorbent assay (ELISA), Immunofluorescence staining, JC-1 staining, Mito-Tracker Red CMXRos staining, Oil red O staining, Bodipy staining, HE staining, and Blood lipid quadruple test. The assay revealed that DLD promotes browning of white adipose tissue in mice. Cellularly, DLD was found to promote white adipocytes browning by activating mitochondrial function through the RAS/ERK pathway. Further studies revealed that the crotonylation modification and acetylation modification of DLD had mutual inhibitory effects. Meanwhile, DLD crotonylation promoted white adipocytes browning, while DLD acetylation did the opposite. Finally, protein interaction analysis and Co-immunoprecipitation (Co-IP) assays identified Sirtuin3 (SIRT3) as a decrotonylation and deacetylation modification enzyme of regulates DLD. In conclusion, DLD promotes browning of white adipocytes by activating mitochondrial function through crotonylation modification and the RAS/ERK pathway, providing a theoretical basis for the control and treatment of obesity, which is of great significance for the treatment of obesity and obesity-related diseases in the future.

Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.

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.

Lipoamide dehydrogenase (LADH) deficiency: medical perspectives of the structural and functional characterization of LADH and its pathogenic variants.

(Dihydro)lipoamide dehydrogenase (LADH) deficiency is an autosomal recessive genetic metabolic disorder. It generally presents with an onset in the neonatal age and premature death. The clinical picture usually involves metabolic decompensation and lactic acidosis that lead to neurological, cardiological, and/or hepatological outcomes. Severity of the disease is due to the fact that LADH is a common E3 subunit to the pyruvate, alpha-ketoglutarate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes and is also part of the glycine cleavage system; hence, a loss in LADH activity adversely affects several central metabolic pathways simultaneously. The severe clinical manifestations, however, often do not parallel the LADH activity loss, which implies the existence of auxiliary pathological pathways; stimulated reactive oxygen species (ROS) production as well as dissociation from the relevant multienzyme complexes proved to be auxiliary exacerbating pathomechanisms for selected disease-causing LADH mutations. This review provides an overview on the therapeutic challenges of inherited metabolic diseases, structural and functional characteristics of the mitochondrial alpha-keto acid dehydrogenase complexes, molecular pathogenesis and structural basis of LADH deficiency, and relevant potential future medical perspectives.

A novel synthetic sRNA promoting protein overexpression in cell-free systems.

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3' end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS.

Identification of Genes and Metabolic Pathways Involved in Resin Yield in Masson Pine by Integrative Analysis of Transcriptome, Proteome and Biochemical Characteristics.

Masson pine (Pinus massoniana L.) is one of the most important resin-producing tree species in southern China. However, the molecular regulatory mechanisms of resin yield are still unclear in masson pine. In this study, an integrated analysis of transcriptome, proteome, and biochemical characteristics from needles of masson pine with the high and common resin yield was investigated. The results showed that chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl C), carotenoids (Car), glucose (Glu), gibberellin A9 (GA9), gibberellin A15 (GA15), and gibberellin A53 (GA53) were significantly increased, whereas fructose (Fru), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-ILE), gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A19 (GA19), and gibberellin A24 (GA24) were significantly decreased in the high resin yield in comparison with those in the common one. The integrated analysis of transcriptome and proteome showed that chlorophyll synthase (chlG), hexokinase (HXK), sucrose synthase (SUS), phosphoglycerate kinase (PGK), dihydrolipoamide dehydrogenase (PDH), dihydrolipoamide succinyltransferase (DLST), 12-oxophytodienoic acid reductase (OPR), and jasmonate O-methyltransferases (JMT) were consistent at the transcriptomic, proteomic, and biochemical levels. The pathways of carbohydrate metabolism, terpenoid biosynthesis, photosynthesis, and hormone biosynthesis may play crucial roles in the regulation of resin yield, and some key genes involved in these pathways may be candidates that influence the resin yield. These results provide insights into the molecular regulatory mechanisms of resin yield and also provide candidate genes that can be applied for the molecular-assisted selection and breeding of high resin-yielding masson pine.

Dichloroacetate and thiamine improve survival and mitochondrial stress in a C. elegans model of dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Amplification refractory mutation system based real-time PCR (ARMS-qPCR) for rapid resistance characterization of Tribolium castaneum to phosphine.

Resistance of Tribolium castaneum to phosphine is related to point mutations in DNA code corresponding to amino acid changes associated with a core metabolic enzyme dihydrolipoamide dehydrogenase (DLD), but the mutation patterns vary among different resistant populations. Thus, there is a great need to develop a cost-effective method to detect core mutations in T. castaneum, which would be the key factor to understand the molecular basis of phosphine resistance. Amplification refractory mutation system-based quantitative Real-Time PCR (ARMS-qPCR) is an ideal method that can rapidly detect point mutations. Here, the P45S and G131D mutations existed in the DLD of T. castaneum selected from strong Chinese resistance phenotypes, and the DLD P45S mutation, which represents a strong phosphine resistance allele, was confirmed as the most abundant mutation to determine strong resistance genotypes. Our study found that 85 out of 120 beetles carried the P45S resistance allele, including 51 homozygous and 34 heterozygous individuals. Moreover, there was a strong linear relationship (R2 = 0.917) between the resistance ratio and the resistance allele frequency among the strongly resistant populations. Our data showed that the ARMS-qPCR method that we developed could rapidly determine strong resistance phenotypes of T. castaneum to phosphine by detecting the DLD P45S mutation. These results not only provide a detailed example for developing an ARMS-qPCR-based method to characterize pesticide resistance, but also support further elucidation of the molecular basis of phosphine resistance.

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.

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.

Characteristics of Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase of the parasitic nematode Haemonchus contortus and its modulatory functions on goat PBMCs in vitro.

Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase (BLAODA), as an essential excretion of Haemonchus contortus (HcESPs), was identified to have antigenic functions. T helper-9 (Th9) cells secrete interleukin (IL)-9, a signature cytokine associated with tumour immunology, allergy and autoimmunity. Nonetheless, the understanding of modulatory functions of BLAODA on Th9 and other immune cells is limited. In this study, the BLAODA gene was cloned, and the recombinant (r) protein of BLAODA (rHcBLAODA) was expressed and immunoblotting was performed. The results revealed that HcBLAODA gene was successfully cloned and rHcBLAODA protein was expressed. The localization of rHcBLAODA was confirmed on the surface of gut sections from adult H. contortus. The rHcBLAODA protein capability to react precisely with anti-H. contortus antibodies were confirmed by immunoblotting and immunofluorescence assay (IFA). Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced Th9 cells generation, IL-9 expression, nitric oxide production and cell apoptosis while suppressing the cells proliferation and cells migration depending on the concentration. Overall, these findings suggest that rHcBLAODA protein could modulate the host immune response by inducing Th9 cells to secrete IL-9 cytokine in vitro.

Minimal peptide length required to span the mitochondrial protein translocases in Trypanosoma brucei.

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.

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.

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.

Whole Cell Active Inhibitors of Mycobacterial Lipoamide Dehydrogenase Afford Selectivity over the Human Enzyme through Tight Binding Interactions.

Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Zinc Switch in Pig Heart Lipoamide Dehydrogenase: Steady-State and Transient Kinetic Studies of the Diaphorase Reaction.

Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M-1·s-1, (9 ± 2) × 104 M-1·s-1, and (1.6 ± 0.5) × 105 M-1·s-1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.

Dihydrolipoamide dehydrogenase regulates cystine deprivation-induced ferroptosis in head and neck cancer.

Ferroptosis is a new form of regulated cell death driven by iron-dependent lipid peroxidation. Glutaminolysis and tricarboxylic acid cycle are involved in ferroptosis, but the underlying metabolic process remains unclear. We examined the role of dihydrolipoamide dehydrogenase (DLD) in ferroptosis induction in head and neck cancer (HNC). The effects of cystine deprivation or sulfasalazine treatment and of DLD gene silencing/overexpression were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed with regard to cell death, lipid reactive oxygen species (ROS) and mitochondrial iron production, mitochondrial membrane potential, mRNA/protein expression, and α-ketoglutarate dehydrogenase (KGDH)/succinate/aconitase activities. Cystine deprivation induced ferroptosis via glutaminolysis. Cystine deprivation or import inhibition using sulfasalazine induced cancer cell death and increased lipid ROS and mitochondrial iron levels, which had been significantly decreased by short-interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting DLD (P < 0.01) but not by dihydrolipoyl succinyltransferase. The same results were noted in an in vivo mouse model transplanted with vector or shDLD-transduced HN9 cells. After cystine deprivation or sulfasalazine treatment, mitochondrial membrane potential, mitochondrial free iron level, KGDH activity, and succinate content significantly increased (P < 0.001), which had been blocked by DLD siRNA or shRNA and were consequently rescued by resistant DLD cDNA. Cystine deprivation caused iron starvation response and mitochondrial iron accumulation for Fenton reaction and ferroptosis. Our data suggest a close association of DLD with cystine deprivation- or import inhibition-induced ferroptosis.

Dihydrolipoamide dehydrogenase of Vibrio splendidus is involved in adhesion to Apostichopus japonicus.

Vibrio splendidus is one of the most opportunistic marine pathogens and infects many important marine animals, including the sea cucumber Apostichopus japonicus. In this study, two genes named DLD1 and DLD2, encoding dihydrolipoamide dehydrogenase (DLD) homologues in pathogenic V. splendidus, were cloned, and conditionally expressed in Escherichia coli BL21 (DE3). The enzymatic activities of DLD1 and DLD2 showed that they both belonged to the NADH oxidase family. Both DLD1 and DLD2 were located on the outer membrane of V. splendidus as detected by whole-cell ELISA. To study the adhesion function of DLD1 and DLD2, polyclonal antibodies were prepared, and antibody block assay was performed to detect the normal function of the two proteins. DLD1 and DLD2 were determined to play important roles in adhesion to different matrices and the adhesive ability of V. splendidus reduced more than 50% when DLD1 or DLD2 was defective.

Bio-Fenton reaction involved in the cleavage of the ethoxylate chain of nonionic surfactants by dihydrolipoamide dehydrogenase from Pseudomonas nitroreducens TX1.

Bacteria in the environment play a major role in the degradation of widely used man-made recalcitrant organic compounds. Pseudomonas nitroreducens TX1 is of special interest because of its high efficiency to remove nonionic ethoxylated surfactants. In this study, a novel approach was demonstrated by a bacterial enzyme involved in the formation of radicals to attack ethoxylated surfactants. The dihydrolipoamide dehydrogenase was purified from the crude extract of strain TX1 by using octylphenol polyethoxylate (OPEOn) as substrate. The extent of removal of OPEOs during the degradation process was conducted by purified recombinant enzyme from E. coli BL21 (DE3) in the presence of the excess of metal mixtures (Mn2+, Mg2+, Zn2+, and Cu2+). The metabolites and the degradation rates were analyzed and determined by liquid chromatography-mass spectrometry. The enzyme was demonstrated to form Fenton reagent in the presence of an excess of metals. Under this in vitro condition, it was shown to be able to shorten the ethoxylate chains of OPEOn. After 2 hours of reaction, the products obtained from the degradation experiment revealed a prominent ion peak at m/z = 493.3, namely the ethoxylate chain unit is 6 (OPEO6) compared to OPEO9 (m/z = 625.3), the main undegraded surfactant in the no enzyme control. It revealed that the concentration of OPEO15 and OPEO9 decreased by 90% and 40% after 4 hours, respectively. The disappearance rates for the OPEOn homologs correlated to the length of the exothylate chains, suggesting it is not a specific enzymatic reaction which cleaves one unit by unit from the end of the ethoxylate chain. The results indicate the diverse and novel strategy by bacteria to catabolize organic compounds by using existing housekeeping enzyme(s).

Subsequent cooling-circulation after radiofrequency and microwave ablation avoids secondary indirect damage induced by residual thermal energy.

PURPOSE: We aimed to investigate the exact role of residual thermal energy following microwave ablation (MWA) and radiofrequency ablation (RFA) at the final ablation and transition zones and determine whether residual thermal energy could be dissipated by subsequent cooling-circulation. METHODS: In an ex vivo study, MWA and RFA were performed on fresh porcine liver, and central and border temperatures were compared. In an in vivo study, MWA and RFA were performed to the livers of New Zealand white rabbits. Tissue samples were stained with α-NADH-diaphorase. The coagulation zones (NADH-negative) and transition zones (lightly NADH-stained) of different groups were compared at different time points. RESULTS: In the ex vivo model, the residual thermal energy after MWA and RFA could be dispersed by subsequent cooling-circulation due to the temperature decreasing rapidly. In the in vivo study, the coagulation volume in the ablation group was larger than that in the cooling-circulation group (P < 0.05) 2 days after ablation. In the ablation group, the damaged zone (the transition zone plus the coagulation zone) on α-NADH-diaphorase-stained images increased rapidly within 2 hours after ablation and slowly reached the maximum on day 2. However, the damaged zones did not change significantly at the three time points observed in the cooling-circulation group. CONCLUSION: The residual thermal energy in MWA and RFA induced secondary damage beyond the direct coagulation zone, and it could be dissipated by subsequent cooling-circulation, contributing to smaller ablation and transition zones.