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.

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.

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.