Lipoamide Dehydrogenase Research Articles

Proteomics Approach of Rapamycin Anti-Tumoral Effect on Primary and Metastatic Canine Mammary Tumor Cells In Vitro.

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC50. Then, cell lines were treated with rapamycin IC50 dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC50 dose for all different tumor cells between 2 and 10 μM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC50 were higher when compared to other studies.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1β, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

TLR4 Agonist Combined with Trivalent Protein JointS of Streptococcus suis Provides Immunological Protection in Animals.

Streptococcus suis (S. suis) serotype 2 (SS2) is the causative agent of swine streptococcosis and can cause severe diseases in both pigs and humans. Although the traditional inactive vaccine can protect pigs from SS2 infection, novel vaccine candidates are needed to overcome its shortcomings. Three infection-associated proteins in S. suis—muramidase-released protein (MRP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and DLD, a novel putative dihydrolipoamide dehydrogenase—have been previously identified by immunoproteomic assays. In this study, the effective immune protection of the recombinant trivalent protein GAPDH-MRP-DLD (JointS) against SS2, SS7, and SS9 was determined in zebrafish. To improve the immune efficacy of JointS, monophosphoryl lipid A (MPLA) as a TLR4 agonist adjuvant, which induces a strong innate immune response in the immune cells of mice and pigs, was combined with JointS to immunize the mice. The results showed that immunized mice could induce the production of a high titer of anti-S. suis antibodies; as a result, 100% of mice survived after SS2 infection. Furthermore, JointS provides good protection against virulent SS2 strain infections in piglets. Given the above, there is potential to develop JointS as a novel subunit vaccine for piglets to prevent infection by SS2 and other S. suis serotypes.

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.

Detailed evaluation of pyruvate dehydrogenase complex inhibition in simulated exercise conditions

The mammalian pyruvate dehydrogenase complex (PDC) is a mitochondrial multienzyme complex that connects glycolysis to the tricarboxylic acid cycle by catalyzing pyruvate oxidation to produce acetyl-CoA, NADH, and CO2. This reaction is required to aerobically utilize glucose, a preferred metabolic fuel, and is composed of three core enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). The pyruvate-dehydrogenase-specific kinase (PDK) and pyruvate-dehydrogenase-specific phosphatase (PDP) are considered the main control mechanism of mammalian PDC activity. However, PDK and PDP activity are allosterically regulated by several effectors fully overlapping PDC substrates and products. This collection of positive and negative feedback mechanisms confounds simple predictions of relative PDC flux, especially when all effectors are dynamically modulated during metabolic states that exist in physiologically realistic conditions, such as exercise. Here, we provide, to our knowledge, the first globally fitted, pH-dependent kinetic model of the PDC accounting for the PDC core reaction because it is regulated by PDK, PDP, metal binding equilibria, and numerous allosteric effectors. The model was used to compute PDH regulatory complex flux as a function of previously determined metabolic conditions used to simulate exercise and demonstrates increased flux with exercise. Our model reveals that PDC flux in physiological conditions is primarily inhibited by product inhibition (∼60%), mostly NADH inhibition (∼30–50%), rather than phosphorylation cycle inhibition (∼40%), but the degree to which depends on the metabolic state and PDC tissue source.

CLTRN, Regulated by NRF1/RAN/DLD Protein Complex, Enhances Radiation Sensitivity of Hepatocellular Carcinoma Cells Through Ferroptosis Pathway

Radiation therapy is a viable treatment option for patients with unresectable hepatocellular carcinoma (HCC). However, radiation resistance and adverse effects are issues that needs to be addressed. Herein, for the first time, we investigated the ability of collectrin (CLTRN) to enhance radiosensitivity in patients with HCC. Transcriptome sequencing technology (RNA-seq technology) was used to analyze the transcription-level changes in the genes in HepG2 cells before and after x-ray irradiation. Combining the results with the HCC tissue RNA-seq data, we determined the ultimate target gene through bioinformatics analysis and cellular verification. A series of cellular and molecular biology techniques were applied invitro and invivo to confirm whether CLTRN can enhance radiosensitivity in HCC cells. Subsequently, the downstream action mechanism, the upstream transcription factor, and the interaction proteins of CLTRN were determined. First, we confirmed that CLTRN is the target gene for radiation therapy and verified the association between CLTRN and radiosensitivity. Invivo and invitro experiments were performed. Investigation of the gene regulatory mechanism revealed that the genes analyzed at the transcriptome level after CLTRN overexpression were mostly enriched in the glutathione metabolic pathway. As glutathione metabolism forms a vital link in ferroptosis, we surmised that CLTRN is associated with ferroptosis. This was confirmed through detection of cellular iron, determination of reactive oxygen species levels, use of transmission electron microscopy, and monitoring of ferroptosis-related protein indicators. Lastly, we investigated whether nuclear respiratory factor 1 is the upstream transcription factor of CLTRN and whether dihydrolipoamide dehydrogenase and members of the RAS oncogene family are its interacting proteins. CLTRN is a vital regulator of radiation sensitivity and could serve as a novel therapeutic target or prognostic marker in HCC treatment.

Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC (p < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively (p < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner (p < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.

A Novel Lipoate-Protein Ligase, Mhp-LplJ, Is Required for Lipoic Acid Metabolism in Mycoplasma hyopneumoniae.

Lipoic acid is a conserved cofactor necessary for the activation of several critical enzyme complexes in the aerobic metabolism of 2-oxoacids and one-carbon metabolism. Lipoate metabolism enzymes are key for lipoic acid biosynthesis and salvage. In this study, we found that Mycoplasma hyopneumoniae (M. hyopneumoniae) Mhp-Lpl, which had been previously shown to have lipoate-protein ligase activity against glycine cleavage system H protein (GcvH) in vitro, did not lipoylate the lipoate-dependent subunit of dihydrolipoamide dehydrogenase (PdhD). Further studies indicated that a new putative lipoate-protein ligase in M. hyopneumoniae, MHP_RS00640 (Mhp-LplJ), catalyzes free lipoic acid attachment to PdhD in vitro. In a model organism, Mhp-LplJ exhibited lipoate and octanoate ligase activities against PdhD. When the enzyme activity of Mhp-LplJ was disrupted by lipoic acid analogs, 8-bromooctanoic acid (8-BrO) and 6,8-dichlorooctanoate (6,8-diClO), M. hyopneumoniae growth was arrested in vitro. Taken together, these results indicate that Mhp-LplJ plays a vital role in lipoic acid metabolism of M. hyopneumoniae, which is of great significance to further understand the metabolism of M. hyopneumoniae and develop new antimicrobials against it.

Malonyl-proteome profiles of Staphylococcus aureus reveal lysine malonylation modification in enzymes involved in energy metabolism

BackgroundProtein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen.ResultsUsing anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position − 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes.ConclusionsData presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

Downregulation of dihydrolipoyl dehydrogenase by UVA suppresses melanoma progression via triggering oxidative stress and altering energy metabolism

Melanoma, the most severe form of skin cancer, has poor prognosis and is resistant to chemotherapy. Targeting cancer metabolism is a promising approach in cancer therapeutics. Dihydrolipoyl dehydrogenase (DLD) is a mitochondrial enzyme with diaphorase activity. Here we report a pivotal role of DLD in melanoma cell progression and proliferation. Suppression DLD expression by low intensity UVA (125 mJ/cm2) increased intracellular ROS production and decreased mitochondrial membrane potential thereby inducing autophagy cell death which were confirmed by increased LC3BII and decreased p62 expression in melanoma cells. Knockdown of DLD in melanoma cells also showed similar results. More so, suppression of DLD significantly inhibits in vivo melanoma growth and tumor proliferation. In addition, suppression of DLD increased the NAD+/NADH ratio in melanoma cells and also inhibits TCA cycle related metabolites. DLD downregulation markedly increased α-ketoglutarate and decreased succinic acid suggesting that DLD suppression may have decreased TCA cycle downstream metabolites, resulting in the alteration of mitochondrial energy metabolism Thus the downregulation of DLD induced autophagic cell death in melanoma cells and inhibits in vivo tumor growth and proliferation by increasing ROS production and altering energy metabolism. Our findings suggest that DLD plays a pivotal role in melanoma progression and proliferation.

Isolation and identification of three water-soluble selenoproteins in Se-enriched Agaricus blazei Murrill

Selenoproteins in selenium (Se)-enriched vegetables play an important role in human health. In this study, three water-soluble selenoproteins PR-Se-1, PR-Se-2 and PR-Se-3 in Agaricus blazei Murrill (ABM) were isolated by anion exchange chromatography, gel filtration chromatography and SDS-PAGE. Sequence analyses performed by HPLC–MS/MS showed that PR-Se-1, a 114024 Da selenoprotein with 1019 amino acids (AAs), is an isoenzyme of isocitrate dehydrogenase. PR-Se-2, a 53983 Da selenoprotein with 508 AAs, is a kind of dihydrolipoyl dehydrogenase. PR-Se-3, a 47179 Da selenoprotein with 415 AAs, is a kind d-proline reductase. Se content is high at 26.1 μg/g, and selenocystine is the predominant Se unit in the three selenoproteins. Se content of ABM is 9.15 μg/g, and the organic form of Se accounts for ~81% of total Se content. ABM could be a promising source of Se in Se-poor regions.

Differences in the antibody response to adult Fasciola hepatica excretory/secretory products in experimentally and naturally infected cattle and sheep

Fasciola hepatica (the liver fluke) is a common, global parasite of livestock. It can be highly pathogenic and has health and welfare implications for infected individuals. Typically, in ruminants, infections are sub-clinical, but if undiagnosed, they can lead to significant production losses. Accurate diagnosis is crucial to identify infection. Antibody detection ELISAs are commonly used to diagnose infection due to their high sensitivity and specificity and are typically based on native fluke excretory/secretory (ES) products or cathepsin L1 (CL1), the immunodominant antigen within ES products. These tests have been developed based on the antibody response of experimentally infected animals; however, this response has not been well characterised in naturally infected animals. We compared the antibody recognition of a recombinant CL1 (rCL1) antigen and native adult fluke ES products. Whilst samples from experimentally infected animals showed strong recognition of rCL1, serum antibodies from naturally infected animals did not. These results were confirmed by peptide array. Immunoblotting sera against ES products showed that experimentally infected animals had a strong, specific response to CL1/CL2 proteins whilst antibodies from naturally infected animals recognised multiple proteins and had a variable response to CL1/CL2. Mass spectrometry of proteins separated by 2D SDS PAGE, identified several antigens recognised by serum antibodies from a naturally infected cow, including cathepsins L1, L2 and L5, glutathione S-transferase and a dihydrolipoyl dehydrogenase. Overall, these results show that the antibody response in naturally infected animals to adult fluke ES products is qualitatively different to experimentally infected animals. This suggests that a diagnostic test based on CL1 alone may not be appropriate for diagnosis of natural F. hepatica infections in sheep and cattle.

Identification of secreted and cellular antigens of Staphylococcus aureus causing dairy sheep mastitis and their potential for vaccine development

Staphylococcus aureus is the leading cause of clinical mastitis and is associated with persistent subclinical infections in ewes, significantly compromising the quality and quantity of milk productions. To date, vaccines intended for use in sheep have been mainly focused on biofilm production traits, but many S. aureus pathogenic isolates do not produce biofilm, including those circulating in Sardinia, one of the leading sheep milk producers in Europe. The aim of this work was to identify suitable immunodominant, alternative candidates to biofilm components for vaccine and diagnostic development. An immunoproteomics study was carried out by testing sera from naturally infected sheep with a prevalent S. aureus lineage against cellular and secreted antigens, followed by tandem mass spectrometry identification of the most prominent immunogens. Four cellular and three secreted S. aureus antigens elicited a strong humoral host immune response. The four cellular antigens were the housekeeping proteins pyruvate kinase, elongation Factor Tu, dihydrolipoyl dehydrogenase, and alpha-keto acid dehydrogenase. The three secreted antigens were the bifunctional autolysin (Atl) and the two components of the Panton-Valentine leukocidin, lukF-PV/lukM, demonstrating the carriage of prophage phiPV83 in a sheep isolate and the strong response of the sheep host against them. In consideration of the key role played by these secreted proteins in S. aureus replication and immune evasion, these antigens may represent suitable candidates for developing vaccines eliciting a more successful immunological protection in areas where non-biofilm forming Staphylococcus spp. are the most widespread intramammary pathogens.

Pioglitazone corrects dysregulation of skeletal muscle mitochondrial proteins involved in ATP synthesis in type 2 diabetes

ContextIn this study, we aimed to identify the determinants of mitochondrial dysfunction in skeletal muscle (SKLM) of subjects with type 2 diabetes (T2DM), and to evaluate the effect of pioglitazone (PIO) on SKLM mitochondrial proteome. MethodsTwo different groups of adults were studied. Group I consisted of 8 individuals with normal glucose tolerance (NGT) and 8 with T2DM, subjected to SKLM mitochondrial proteome analysis by 2D-gel electrophoresis followed by mass spectrometry-based protein identification. Group II included 24 individuals with NGT and 24 with T2DM, whose SKLM biopsies were subjected to immunoblot analysis. Of the 24 subjects with T2DM, 20 were randomized to receive placebo or PIO (15 mg daily) for 6 months. After 6 months of treatment, SKLM biopsy was repeated. ResultsMitochondrial proteomic analysis on Group I revealed that several mitochondrial proteins involved in oxidative metabolism were differentially expressed between T2DM and NGT groups, with a downregulation of ATP synthase alpha chain (ATP5A), electron transfer flavoprotein alpha-subunit (ETFA), cytochrome c oxidase subunit VIb isoform 1 (CX6B1), pyruvate dehydrogenase protein X component (ODPX), dihydrolipoamide dehydrogenase (DLDH), dihydrolipoamide-S-succinyltransferase (DLST), and mitofilin, and an up-regulation of hydroxyacyl-CoA-dehydrogenase (HCDH), 3,2-trans-enoyl-CoA-isomerase (D3D2) and delta3,5-delta2,4-dienoyl-CoA-isomerase (ECH1) in T2DM as compared to NGT subjects. By immunoblot analysis on SKLM lysates obtained from Group II we confirmed that, in comparison to NGT subjects, those with T2DM exhibited lower protein levels of ATP5A (−30%, P = 0.006), ETFA (−50%, P = 0.02), CX6B1 (−30%, P = 0.03), key factors for ATP biosynthesis, and of the structural protein mitofilin (−30%, P = 0.01). T2DM was associated with a reduced abundance of the enzymes involved in the Krebs cycle DLST and ODPX (−20%, P ≤ 0.05) and increased levels of HCDH and ECH1, enzymes implicated in the fatty acid catabolism (+30%, P ≤ 0.05).In subjects with type 2 diabetes treated with PIO for 6 months we found a restored SKLM protein abundance of ATP5A, ETFA, CX6B1, and mitofilin. Moreover, protein levels of HCDH and ECH1 were reduced by −10% and − 15% respectively (P ≤ 0.05 for both) after PIO treatment. ConclusionType 2 diabetes is associated with reduced levels of mitochondrial proteins involved in oxidative phosphorylation and an increased abundance of enzymes implicated in fatty acid catabolism in SKLM. PIO treatment is able to improve SKLM mitochondrial proteomic profile in subjects with T2DM.

Succinic acid production from pulp and paper industry waste: A transcriptomic approach

The fermentative production of biobased chemicals and polymers using crude lignocellulose hydrolysates is challenging due to the presence of various inhibitory compounds and multiple sugars. This study evaluates the metabolic response of Actinobacillus succinogenes for the production of succinic acid using spent sulphite liquor (SSL) as feedstock derived from industrial acidic sulphite pulping of Eucalyptus globulus hardwood. A transcriptomic approach led to significant insights on gene regulation of the major metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle, pyruvate metabolism and oxidative phosphorylation) in batch cultures carried out on SSL and compared with glucose and xylose. Significantly overexpressed genes in SSL compared to glucose and xylose were fructose biphosphate aldolase (> 1.18-fold change) in the catabolism, phosphoenolpyruvate carboxykinase (> 1.59-fold change) and malate dehydrogenase (> 1.49-fold change) in the TCA cycle, citrate lyase (> 1.7-fold change), dihydrolipoamide dehydrogenase (> 0.88-fold change), pyruvate dehydrogenase E2 (> 1.63-fold change) and pyruvate formate lyase (> 0.61-fold change), involved in acetyl-CoA pathways. Finally, C4 tricarboxylic transporters were overexpressed (DCU (> 1.61-fold change) and 0079 (> 4.19-fold change). SSL was responsible for the upregulation of genes involved in the TCA cycle and oxidative phosphorylation, while xylose showed similar results with SSL in the oxidative phosphorylation.

Unique genetic variants in dihydrolipoamide dehydrogenase (dld) gene confer strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens)

The rusty grain beetle, Cryptolestes ferrugineus, a major pest of stored commodities, has developed very high levels (>1000×) of resistance to the fumigant phosphine. Resistance in this species is remarkably stronger than reported in any other stored product pests demanding the need to understand the molecular basis of this trait. Previous genetic studies in other grain insect pests identified specific variants in two major genes, rph1 and rph2 in conferring the strong resistance trait. However, in C. ferrugineus, although the gene, rph1 was identified as cytochrome-b5-fatty acid desaturase, the rph2 gene has not been reported so far. We tested the candidate gene for rph2, dihydrolipoamide dehydrogenase (dld) using the recently published transcriptome of C. ferrugineus and identified three variants, L73N and A355G + D360H, a haplotype, conferring resistance in this species. Our sequence analysis in resistant strain and phosphine selected resistant survivors indicates that these variants occur either alone as a homozygote or a mixture of heterozygotes (i.e complex heterozygotes) both conferring strong resistance. We also found that one of the three variants, possibly L73N expressing “dominant” trait at low frequency in resistant insects. Comparison of dld sequences between Australian and Chinese resistant strain of this species confirmed that the identified variants are highly conserved. Our fitness analysis indicated that resistant insects may not incur significant biological costs in the absence of phosphine selection for 19 generations. Thus, we propose that the observed high levels of resistance in C. ferrugineus could be primarily due to the characteristics of three unique variants, L73N and A355G + D360H within dld.

Bacillus cereus LS2 from Apostichopus japonicus antagonizes Vibrio splendidus growth

In this study, a bacterium identified as Bacillus cereus LS2 was isolated from healthy sea cucumbers and possessed strong antagonistic activity toward Vibrio splendidus, an important pathogen to Apostichopus japonicus. The antagonistic activity of strain LS2 to the growth of V. splendidus was 86.83% ± 1.47%. The antagonistic substance, with molecular weight less than 3 kDa, was sensitive to trypsin but stable at high temperatures. The antagonistic substances could be precipitated by acid and dissolved in anhydrous methanol. Further, the in-situ hydrolysis and ninhydrin staining assay showed that the antagonistic substance was a peptide. On the basis of these characteristics, the antagonistic substance was postulated to be a kind of lipopeptide. The analysis of differentially expressed proteins revealed that the expression of dihydrolipoamide dehydrogenase 1 (DLDH1) in V. splendidus could be upregulated in the presence of the LS2 supernatant. Furthermore, the overexpression of DLDH1 reduced the growth of V. splendidus by 12.5% compared with that of the wild-type strain. Our findings suggest that LS2 could be used as a potential bacterial antagonist to control V. splendidus infections by upregulating the DLDH1 expression in V. splendidus.

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.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

In vitro reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum

Pyruvate dehydrogenase (PDH) and 2‐oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate‐dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate‐dependent 2‐oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH‐ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty‐four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2‐E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2‐E3 subcomplex. Our in vitro reconstituted system showed CgE1p‐ and CgE1o‐dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2‐E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2‐E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact‐sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.