Glutaminolysis and autophagy in cancer

The remarkable metabolic differences between cancer cells and normal cells result in the potential for targeted cancer therapy. The upregulation of glutaminolysis provides energetic advantages to cancer cells. The recently described link between glutaminolysis and autophagy, mediated by MTORC1, may constitute an attractive target for therapeutic strategies. A combination of therapies targeting simultane-ously cell signaling, cancer metabolism, and autophagy can solve therapy resistance and tumor relapse problems, commonly observed in patients treated with most of the current targeted therapies. In this review we summarize the mechanistic link between glutaminolysis and autophagy, and discuss the impacts of these processes on cancer progression and the potential for therapeutic intervention.     

3H]-MRE 2029-F20, a selective antagonist radioligand for the human A2B adenosine receptors

MRE 2029-F20 [N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] is a selective antagonist ligand of A2B adenosine receptors. For use as a radioligand, 1,3-diallyl-xanthine, the precursor of [3H]-MRE 2029-F20, was synthesized, and tritiated on the allyl groups. [3H]-MRE 2029-F20 bound to human A2B receptors expressed in CHO cells showed a KD value of 1.65+/-0.10 nM and Bmax value of 36+/-4 fmol/mg protein. [3H]-MRE2029-F20 represents a useful tool for the pharmacological characterization of human A2B adenosine receptor subtype.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

Improving the accumulation of recombinant human serum albumin (HSA) in transgenic tobacco plants by fusion with the N-terminal proline-rich domain of γ-zein (Zera)

In Vitro Cellular & Developmental Biology - Plant - Plants have long played a major role in human health as a source of herbal remedies. Recently, transgenic plants have become convenient...     

DRhoGEF2 and diaphanous regulate contractile force during segmental groove morphogenesis in the Drosophila embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.     

Artificial ubiquitylation is sufficient for sorting of a plasma membrane ATPase to the vacuolar lumen of Arabidopsis cells

Sorting of transmembrane proteins into the inner vesicles of multivesicular bodies for subsequent delivery to the vacuole/lysosome can be induced by attachment of a single ubiquitin or K63-linked ubiquitin chains to the cytosolic portion of the cargo in yeast and mammals. In plants, large efforts have been undertaken to elucidate the mechanisms of vacuolar trafficking of soluble proteins. Sorting of transmembrane proteins, by contrast, is still largely unexplored. As a proof of principle, that ubiquitin is involved in vacuolar sorting in plants we show that a translational fusion of a single ubiquitin to the Arabidopsis plasma membrane ATPase PMA-EGFP is sufficient to induce its endocytosis and sorting into the vacuolar lumen. Sorting of the artificial reporter is not dependent on ubiquitin chain formation, but involves ubiquitin's hydrophobic patch and can be inhibited by coexpression of a dominant-negative version of the ESCRT (endosomal sorting complex required for transport) related protein AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1). Our results suggest that ubiquitin can in principle act as vacuolar sorting signal in plants.     

The eicosanoid response to high dose UVR exposure of individuals prone and resistant to sunburn

High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E(2) is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4 h, p < 0.001), neutrophil infiltration (24 h, p = 0.01), epidermal COX-2 (24 h, p < 0.05) and 12-LOX (24 h, p < 0.01), and dermal eNOS (24 h, p < 0.05) proteins, although CD3+ lymphocyte infiltration showed an earlier increase in phototype III/IV (24 h, p < 0.05). Although erythema was equivalent at 72 h in both groups, phototype I/II showed higher PGE(2) accompanied by elevated 15-HETE, and a strong positive correlation was seen between these mediators (n = 18, r = 0.805, p = 0.0001). Hence anti-inflammatory eicosanoid 15-HETE may temper the pro-inflammatory milieu in sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions.     

NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.