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.

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.

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.

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.

Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Pan-Cancer Analysis of the Cuproptosis-Related Gene DLD.

Background: Cancer affects millions of people each year and imposes a huge economic and social burden worldwide. Cuproptosis is a recently discovered novel mode of cell death. The exact function of the cuproptosis-related gene dihydrolipoamide dehydrogenase (DLD) and its role in pan-cancer is unknown. Methods: Data were retrieved from the GTEx, TCGA, and multiple online websites. These data were used to assess the expression, prognosis, and diagnostic value of DLD in various tumors. The relationship of DLD with immune microenvironment immunomodulators, immune checkpoints, tumor mutational load (TMB), microsatellite instability (MSI), and oncology drug sensitivity was explored by correlation analysis. Results: The mRNA and protein expression of DLD differs in most cancers. Survival analysis showed that DLD was associated with prognosis with KIRC, KIRP, KICH, and UCS. DLD had a strong diagnostic value in KIRC, GBM, PAAD, and LGG (AUC > 0.9). DLD promoter methylation affects the aberrant expression of LIHC, LUSC, PAAD, READ, and THCA. DLD was negatively correlated with stromal score, immune score, and ESTIMATE score in UCEC, TGCT, LUSC, and SARC. In UCS, resting memory CD4 T cells and activated NK cells were significantly correlated with DLD expression. Significant correlations were also observed between DLD expression and immunomodulators, immune checkpoints, TMB, and MSI in various cancers. Importantly, we also identified a number of potential drugs that may target DLD. Conclusion: DLD expression is associated with a variety of tumor prognoses and plays an integral role in tumorigenesis, tumor metabolism, and immunity.

Insight into the molecular mechanism of phosphine toxicity provided by functional analysis of cytochrome b5 fatty acid desaturase and dihydrolipoamide dehydrogenase in the red flour beetle, Tribolium castaneum.

Phosphine is the dominant chemical used in postharvest pest control. Widespread and highly frequent use of phosphine has been selected for pest insects, including Tribolium castaneum, which is highly resistant. Lipid peroxidation and reactive oxygen species (ROS) are two major factors determining phosphine toxicity; however, the mechanisms of production of these two factors in phosphine toxicity are still unknown. Here, we first determined the time course of phosphine-induced lipid peroxidation and ROS production in T. castaneum. Our results showed that lipid peroxidation occurs before ROS in the process of phosphine toxicity, and fumigated beetles with higher resistance levels were associated with weaker activity on lipid peroxidation and ROS. A significant decline in lipid peroxidation was observed in fumigated individuals after knockdown of cytochrome b5 fatty acid desaturase (Cyt-b5-r) via RNA interference (RNAi), indicating that Cyt-b5-r is critical for triggering phosphine-induced lipid peroxidation. Moreover, significant decreases in both ROS and mortality were detected in fumigated T. castaneum adults fed melatonin for 7 days, an inhibitor of lipid peroxidation. Cyt-b5-r RNAi also inhibited ROS production and mortality in phosphine-treated beetles. Meanwhile, a significant decrease in ROS production (68.4%) was detected in dihydrolipoamide dehydrogenase (DLD) knockdown individuals with phenotypes susceptible to phosphine, suggesting that lipid peroxidation initiates ROS with the expression of DLD. However, a significant increase in ROS (122.1%) was detected in the DLD knockdown beetles with strongly resistant phenotypes, indicating that the DLD-involved pathway may not be the only mechanism of ROS generation in phosphine toxicity and the existence of a moonlighting role in downregulating ROS in strongly resistant T. castaneum.

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase.

Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data.

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.

Enzyme histochemical characterization of orbital glands in fetuses of Indian buffalo (Bubalus bubalis).

Background: The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.

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.

Rg3 regulates myocardial pyruvate metabolism via P300-mediated dihydrolipoamide dehydrogenase 2-hydroxyisobutyrylation in TAC-induced cardiac hypertrophy.

The failing heart is characterized by an increase in glucose uptake and glycolytic rates that is not accompanied by a concomitant increase in glucose oxidation. Lower coupling of glucose oxidation to glycolysis possibly owes to unchanged or reduced pyruvate oxidation in mitochondria. Therefore, increasing pyruvate oxidation may lead to new therapies for heart disease. Dihydrolipoamide dehydrogenase (DLD) is a component of the pyruvate dehydrogenase complex (PDH). DLD mutations or defects are closely associated with metabolic diseases. However, few studies explore the effects of DLD mutants or acylation status on PDH activity and pyruvate metabolism. P300 is protein 2-hydroxyisobutyryltransferases in cells, and P300-dependent lysine 2-hydroxyisobutyrylation of glycolytic enzymes affects glucose metabolism. However, there are no relevant reports on the effect of 2-hydroxyisobutyrylation on the energy metabolism of heart failure, and it is worth further in-depth study. In this study, we showed that 2-hydroxyisobutyrylation is an essential protein translational modification (PTM) that regulates the activity of pyruvate dehydrogenase complex (PDHc). In a mouse model of transverse aortic constriction (TAC)-induced cardiac hypertrophy, the 2-hydroxyisobutylation of DLD was significantly increased, related to the decrease in PDH activity. In addition, our data provide clear evidence that DLD is a direct substrate of P300. As one of the main active ingredients of ginseng, ginsenoside Rg3 (Rg3) can reduce the 2-hydroxyisobutylation levels of DLD and restore the PDH activity by inhibiting the acyltransferase activity of P300, thereby producing beneficial effects whenever the heart is injured. Therefore, this study suggests a novel strategy for reversing myocardial hypertrophy.

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

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

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.