Oxidative stress via inhibition of the mitochondrial electron transport and Nrf-2-mediated anti-oxidative response regulate the cytotoxic activity of plumbagin.

Plumbagin, an anti-cancer agent, is toxic to cells of multiple species. We investigated if plumbagin targets conserved biochemical processes. Plumbagin induced DNA damage and apoptosis in cells of diverse mutational background with comparable potency. A 3-5 fold increase in intracellular oxygen radicals occurred in response to plumbagin. Neutralization of the reactive oxygen species by N-acetylcysteine blocked apoptosis, indicating a central role for oxidative stress in plumbagin-mediated cell death. Plumbagin docks in the ubiquinone binding sites (Q0 and Qi) of mitochondrial complexes I-III, the major sites for oxygen radicals. Plumbagin decreased oxygen consumption rate, ATP production and optical redox ratio (NAD(P)H/FAD) indicating interference with electron transport downstream of mitochondrial Complex II. Oxidative stress induced by plumbagin triggered an anti-oxidative response via activation of Nrf2. Plumbagin and the Nrf2 inhibitor, brusatol, synergized to inhibit cell proliferation. These data indicate that while inhibition of electron transport is the conserved mechanism responsible for plumbagin's chemotoxicity, activation of Nrf2 is the resulting anti-oxidative response that allows plumbagin to serve as a chemopreventive agent. This study provides the basis for designing potent and selective plumbagin analogs that can be coupled with suitable Nrf2 inhibitors for chemotherapy or administered as single agents to induce Nrf2-mediated chemoprevention.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man.

Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.

A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling.

Neural networks are balanced by inhibitory and excitatory neuronal activity. The formation of these networks is initially generated through neuronal subtype specification controlled by transcription factors. The basic helix-loop-helix (bHLH) transcription factor Ptf1a is essential for the generation of GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. The transcription factor Rbpj is a transducer of the Notch signaling pathway that functions to maintain neural progenitor cells. Here we demonstrate Ptf1a and Rbpj interact in a complex that is required in vivo for specification of the GABAergic neurons, a function that cannot be substituted by the classical form of the bHLH heterodimer with E-protein or Notch signaling through Rbpj. We show that a mutant form of Ptf1a without the ability to bind Rbpj, while retaining its ability to interact with E-protein, is incapable of inducing GABAergic (Pax2)- and suppressing glutamatergic (Tlx3)-expressing cells in the chick and mouse neural tube. Moreover, we use an Rbpj conditional mutation to demonstrate that Rbpj function is essential for GABAergic specification, and that this function is independent of the Notch signaling pathway. Together, these findings demonstrate the requirement for a Ptf1a-Rbpj complex in controlling the balanced formation of inhibitory and excitatory neurons in the developing spinal cord, and point to a novel Notch-independent function for Rbpj in nervous system development.

Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex.

PTF1a is an unusual basic helix-loop-helix (bHLH) transcription factor that is required for the development of the pancreas. We show that early in pancreatic development, active PTF1a requires interaction with RBPJ, the vertebrate Suppressor of Hairless, within a stable trimeric DNA-binding complex (PTF1). Later, as acinar cell development begins, RBPJ is swapped for RBPJL, the constitutively active, pancreas-restricted paralog of RBPJ. Moreover, the Rbpjl gene is a direct target of the PTF1 complex: At the onset of acinar cell development when the Rbpjl gene is first induced, a PTF1 complex containing RBPJ is bound to the Rbpjl promoter. As development proceeds, RBPJL gradually replaces RBPJ in the PTF1 complex bound to Rbpjl and appears on the binding sites for the complex in the promoters of other acinar-specific genes, including those for the secretory digestive enzymes. A single amino acid change in PTF1a that eliminates its ability to bind RBPJ (but does not affect its binding to RBPJL) causes pancreatic development to truncate at an immature stage, without the formation of acini or islets. These results indicate that the interaction between PTF1a and RBPJ is required for the early stage of pancreatic growth, morphogenesis, and lineage fate decisions. The defects in pancreatic development phenocopy those of Ptf1a-null embryos; thus, the first critical function of PTF1a is in the context of the PTF1 complex containing RBPJ. Action within an organ-specific transcription factor is a previously unknown function for RBPJ and is independent of its role in Notch signaling.

PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L.

PTF1 is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It comprises a dimer of P48/PTF1a (a pancreas and neural restricted basic helix-loop-helix [bHLH] protein) and a class A bHLH protein, together with a third protein that we show can be either the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L. In mature acinar cells, PTF1 exclusively contains the RBP-L isoform and is bound to the promoters of acinar specific genes. P48 interacts with the RBP subunit primarily through two short conserved tryptophan-containing motifs, similar to the motif of the Notch intracellular domain (NotchIC) that interacts with RBP-J. The transcriptional activities of the J and L forms of PTF1 are independent of Notch signaling, because P48 occupies the NotchIC docking site on RBP-J and RBP-L does not bind the NotchIC. Mutations that delete one or both of the RBP-interacting motifs of P48 eliminate RBP-binding and are associated with a human genetic disorder characterized by pancreatic and cerebellar agenesis, which indicates that the association of P48 and RBPs is required for proper embryonic development. The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H).