Dihydrolipoamide dehydrogenase (DLD) is a novel molecular target of bortezomib.

Proteasome inhibitors (PIs), such as bortezomib and calfizomib, were backbone agents in the treatment of multiple myeloma (MM). In this study, we investigated bortezomib interactors in MM cells and identified dihydrolipoamide dehydrogenase (DLD) as a molecular target of bortezomib. DLD catalyzes the oxidation of dihydrolipoamide to form lipoamide, a reaction that also generates NADH. Our data showed that bortezomib bound to DLD and inhibited DLD's enzymatic function in MM cells. DLD knocked down MM cells (DLD-KD) had decreased levels of NADH. Reduced NADH suppressed assembly of proteasome complex in cells. As a result, DLD-KD MM cells had decreased basal-level proteasome activity and were more sensitive to bortezomib. Since PIs were used in many anti-MM regimens in clinics, we found that high expression of DLD correlated with inferior prognosis of MM. Considering the regulatory role of DLD in proteasome assembly, we evaluated DLD targeting therapy in MM cells. DLD inhibitor CPI-613 showed a synergistic anti-MM effect with bortezomib in vitro and in vivo. Overall, our findings elucidated DLD as an alternative molecular target of bortezomib in MM. DLD-targeting might increase MM sensitivity to PIs.

dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Stoichiometry and architecture of the human pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.

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.

The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

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.

Thioredoxins o1 and h2 jointly adjust mitochondrial dihydrolipoamide dehydrogenase-dependent pathways towards changing environments.

Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX-based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild-type and the glycine decarboxylase T-protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)-dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C-labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine-tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions.

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.

Cuproptosis Related Gene DLD Associated with Poor Prognosis and Malignant Biological Characteristics in Lung Adenocarcinoma.

PURPOSE: Cuproptosis plays a crucial role in the biological function of cells. The subject of this work was to analyze the effects of cuproptosis-related genes (CRGs) on the prognosis and biological function in lung adenocarcinoma (LUAD). METHODS: In this study, RNA sequencing and clinical data of LUAD samples were screened from public databases and our institution. A CRG signature was identified by least absolute shrinkage and selection operator and Cox regression. In addition, this study analyzed the correlation between prognostic CRGs and clinicopathological features. Finally, this study studied the effect of inhibiting dihydrolipoamide dehydrogenase (DLD) expression on cell biological function. RESULTS: There were 10 CRGs that showed differential expression between LUAD and normal tissues (p<0.05). A prognostic signature (DLD and lipoyltransferase 1 [LIPT1]) was constructed. Survival analysis suggested that patients with LUAD in the high-risk group had shorter overall survival (OS) (p<0.05). High expression of DLD and low expression of LIPT1 were significantly associated with shorter OS (p<0.05). Immunohistochemical analysis revealed that, in LUAD tissues, DLD was highly expressed, whereas LIPT1 was not detected. Finally, inhibition of DLD expression could significantly restrain cell proliferation, invasion and migration. CONCLUSION: Overall, this prognostic CRG signature may play a pivotal role in LUAD outcome, while oncogene DLD may be a future therapeutic candidate for LUAD.

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.

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.

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.

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.

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.

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.