The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the antiinflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomaincontaining protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent antiinflammatory effects of depolymerizing microtubule-targeting agents

EcR recruits dMi-2 and increases efficiency of dMi-2-mediated remodelling to constrain transcription of hormone-regulated genes

Gene regulation by steroid hormones plays important roles in health and disease. In Drosophila, the hormone ecdysone governs transitions between key developmental stages. Ecdysone-regulated genes are bound by a heterodimer of ecdysone receptor (EcR) and Ultraspiracle. According to the bimodal switch model, steroid hormone receptors recruit corepressors in the absence of hormone and coactivators in its presence. Here we show that the nucleosome remodeller dMi-2 is recruited to ecdysone-regulated genes to limit transcription. Contrary to the prevalent model, recruitment of the dMi-2 corepressor increases upon hormone addition to constrain gene activation through chromatin remodelling. Furthermore, EcR and dMi-2 form a complex that is devoid of Ultraspiracle. Unexpectedly, EcR contacts the dMi-2 ATPase domain and increases the efficiency of dMi-2-mediated nucleosome remodelling. This study identifies a non-canonical EcR-corepressor complex with the potential for a direct regulation of ATP-dependent nucleosome remodelling by a nuclear hormone receptor

Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies

Detection of protein S-sulfhydration by a tag-switch technique

Protein S-sulfhydration (forming -S-SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H2 S-mediated signaling pathways. In this study we report the first selective, "tag-switch" method which can directly label protein S-sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H2 S alone cannot lead to S-sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers which would facilitate the oxidation of H2 S to HS(.)

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 46