Plant D-2-Hydroxyglutarate Dehydrogenase Participates in the Catabolism of Lysine Especially during Senescence

D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH(d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism

Particle-in-Cell algorithms on DEEP: The iPiC3D case study

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A novel cell-based screening assay for small-molecule MYB inhibitors identifies podophyllotoxins teniposide and etoposide as inhibitors of MYB activity

The transcription factor MYB plays key roles in hematopoietic cells and has been implicated the development of leukemia. MYB has therefore emerged as an attractive target for drug development. Recent work has suggested that targeting MYB by small-molecule inhibitors is feasible and that inhibition of MYB has potential as a therapeutic approach against acute myeloid leukemia. To facilitate the identification of small-molecule MYB inhibitors we have re-designed and improved a previously established cell-based screening assay and have employed it to screen a natural product library for potential inhibitors. Our work shows that teniposide and etoposide, chemotherapeutic agents causing DNA-damage by inhibiting topoisomerase II, potently inhibit MYB activity and induce degradation of MYB in AML cell lines. MYB inhibition is suppressed by caffeine, suggesting that MYB is inhibited indirectly via DNA-damage signalling. Importantly, ectopic expression of an activated version of MYB in pro-myelocytic NB4 cells diminished the anti-proliferative effects of teniposide, suggesting that podophyllotoxins disrupt the proliferation of leukemia cells not simply by inducing general DNA-damage but that their anti-proliferative effects are boosted by inhibition of MYB. Teniposide and etoposide therefore act like double-edged swords that might be particularly effective to inhibit tumor cells with deregulated MYB

The natural anti-tumor compound Celastrol targets a Myb-C/EBPβ-p300 transcriptional module implicated in myeloid gene expression.

Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBPβ, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBPβ by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPβ by disrupting the interaction of both transcription factors with p300. Myb, C/EBPβ and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBPβ-p300 module might explain its promising anti-leukemic activity

The natural anti-tumor compound Celastrol targets a Myb-C/EBPβ-p300 transcriptional module implicated in myeloid gene expression

Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBPβ, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBPβ by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPβ by disrupting the interaction of both transcription factors with p300. Myb, C/EBPβ and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBPβ-p300 module might explain its promising anti-leukemic activity

Celastrol disrupts the C/EBPβ-p300 interaction.

<p><b>A,B.</b> QT6 fibroblasts transfected with the indicated expression vectors were subjected to GFP-trap experiments. Total cell extracts (TCE) and GFP-trap samples (trap) were analyzed by western blotting with antibodies against GFP and p300. In panel B, the cells were also treated with Celastrol. <b>C.</b> QT6 fibroblasts were transfected with the Gal4-dependent reporter gene pG5E4-38luc, the β-galactosidase plasmid pCMVβ and expression vectors for Gal4-C/EBPβ and p300-VP19, as indicated. The transfected cells were incubated for 12 hours with or without Celastrol followed by analysis of luciferase activities. The luciferase activity was first normalized to the β-galactosidase activity. The normalized luciferase activity of the Gal4-C/EBPβ plus p300-VP16 transfected cells or the Gal4-VP16 transfected cells in the absence of Celastrol was then set to 100%. Asterisks indicate statistical significance (*** p < 0.001, Student’s t-test).</p

Celastrol does not affect DNA-binding of C/EBPβ but disrupts its cooperation with p300.

<p><b>A.</b> A radiolabeled double-stranded oligonucleotide with a consensus C/EBP binding site was incubated without nuclear extract, with nuclear extract from cells transfected with C/EBPβ expression vector and treated with Celastrol as indicated, or with nuclear extract from untransfected cells. Protein-DNA complexes were analyzed by native polyacrylamide gel electrophoresis. The top panel shows a western blot analysis of aliquots of the nuclear extracts stained with antibodies against C/EBPβ. <b>B.</b> QT6 cells transfected with expression vectors for C/EBPβ or a Jun-C/EBPβ hybrid protein and incubated with Celastrol were analyzed by northern blotting for expression of <i>MRP126</i> and <i>S17</i> mRNAs. <b>C.</b> QT6 cells transfected with expression vectors for C/EBPβ and p300 were treated with the indicated concentrations of Celastrol, harvested after 16 hours and analyzed by northern blotting for expression of <i>MRP126</i> and <i>S17</i> mRNAs. Numbers below the lanes indicate the amounts of <i>MRP126</i> mRNA relative to the S17 mRNA as control determined by quantification with a phosphor image analyzer. The signals for <i>MRP126</i> and <i>S17</i> mRNAs were obtained by sequential hybridization of the same blot with specific radiolabeled probes. <b>D.</b> QT6 cells transfected with expression vectors for C/EBPβ and p300/1751-2379 and cultivated for 24 hours with or without Celastrol. Aliquots of total cell extracts were then analyzed with antibodies against p300, pp300(Ser2280) and C/EBPβ. Black and white arrows mark the un-phosphorylated and highly phosphorylated p300.</p

Disruption of the C/EBPβ-p300 interaction is dependent on cysteine residues in the Taz2 domain of p300.

<p><b>A.</b> Sequence comparison of the p300 Taz2 domain from human, mouse and chicken. Cysteine and histidine residues involved in coordination of zinc ions and Cys-1789 and Cys-1790 are highlighted. <b>B-D.</b> QT6 fibroblasts transfected with the indicated expression vectors were subjected to GFP-trap experiments and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190934#pone.0190934.g003" target="_blank">Fig 3</a>. <b>E.</b> Amino acid sequences of different C/EBP family members implicated in Taz2-binding. Box A and Box B refer to α-helical regions assumed to directly interact with the Taz2 domain. <b>F.</b> Partial view of the Taz2 domain and its interaction with Box A and Box B sequences of C/EBPε. The peptide backbones of the Taz2 domain and of CEBPε are shown in brown and turquois, respectively. The position of Cys-1790 is shown. The figure was created from PDB entry 3t92.</p