Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)

Carotenoid biosynthesis was examined in a phylloplane yeast identified by ITS, 18S and 28S rDNA analysis as a Dioszegia sp. close to D. takashimae. In well-aerated flask or fermentor cultures, this strain produced essentially a single pigment confirmed as the xanthophyll plectaniaxanthin by NMR analysis, at concentrations of 103-175 microgg(-1) biomass dry weight. Detailed studies showed increases in plectaniaxanthin concentrations in the presence of 5 mM hydrogen peroxide (1.8-fold), 50 and 100 microM duroquinone (3.1- and 3.7-fold, respectively), and 2% ethanol (4.9-fold). Whereas oxidative stress is known to enhance the biosynthesis of torularhodin or astaxanthin in other red yeasts where they are associated with an antioxidant function, this is the first report implicating plectaniaxanthin in a similar role. At reduced aeration, biosynthesis of plectaniaxanthin was suppressed and its putative precursor gamma-carotene accumulated. The carotenoid cyclase inhibitor nicotine (5-20 mM) inhibited plectaniaxanthin formation, with lycopene accumulating stoichiometrically. Hydroxy groups at C-1' and C-2' therefore seem to be introduced late in plectaniaxanthin biosynthesis, following cyclization of the beta-ionone ring.     

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.     

GAPDH mediates nitrosylation of nuclear proteins

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.     

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

SIRT1 is involved in adrenocortical cancer growth and motility

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF-II (insulin-like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up-regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.     

Dalmatian: spotting the difference in cohesin protectors

The cohesin complex prevents separation of chromosomes following their duplication until the appropriate time during cell division. In vertebrates, establishment and maintenance of cohesin‐dependent linkages depend on two distinct proteins, sororin and shugoshin. New findings published in The EMBO Journal show that in Drosophila, the function of both of these cohesin regulators is carried out by a single hybrid protein, Dalmatian.