Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate. The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaL::ADP. DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins. It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription. Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR. In the presence of Dha, DhaL is dephosphorylated and DhaL::ADP displaces DhaK and stimulates DhaR activity. In the absence of Dha, DhaL::ADP is converted by the PTS to DhaL::ATP, which does not bind to DhaR.

The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signaling activity. Here, we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signaling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A–ABLIM-Actin-MKL1 sequence, we identify the MKL1-interactor Jumonji-domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo.

No abstract

Hypoxic-ischemic (HI) insult in the perinatal phase harbors a high risk of encephalopathy in the neonate. Brain cells undergo apoptosis, initiating neurodegeneration. So far, therapeutic approaches such as cooling remain limited. Transplantation of mesenchymal stem cells (MSCs) exhibits therapeutic success despite the short-time survival in the host brain, providing strong evidence that their beneficial effects are largely based on secreted factors, including extracellular vesicles (EVs). The aim of this study was to investigate the effects of human Wharton's jelly MSC (hWJ-MSC)-derived EVs on neuroprotection and neuroregeneration, using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) mimicking HI injury in the mouse neuroblastoma cell line neuro2a (N2a). hWJ-MSC-derived EVs were isolated from cell culture supernatants by multistep centrifugation and identified by endosomal marker expression and electron microscopy. OGD/R significantly increased DNA fragmentation and caspase 3 ( Casp3) transcription in N2a cells relative to undamaged cells. OGD/R-mediated DNA fragmentation and Casp3 expression could be prevented as well as resolved by the addition of hWJ-MSC-derived EV before and after OGD, respectively. hWJ-MSC-derived EV also tended to increase the phosphorylation of the B cell lymphoma 2 (Bcl2) family member Bcl-2-antagonist of cell death (BAD) in N2a cells, when added prior or post OGD, thereby inactivating the proapoptotic function of BAD. Fluorescence confocal microscopy revealed the close localization of hWJ-MSC-derived EVs to the nuclei of N2a cells. Furthermore, EVs released their RNA content into the cells. The expression levels of the microRNAs (miRs) let-7a and let-7e, known regulators of Casp3, were inversely correlated to Casp3. Our data suggest that hWJ-MSC-derived EVs have the potential to prevent and resolve HI-induced apoptosis in neuronal cells in the immature neonatal brain. Their antiapoptotic effect seems to be mediated by the transfer of EV-derived let-7-5p miR.