A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease

Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation

Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Chemical Plausibility of Cu(lll) with Biological Ligation in pMMO

The mechanisms of dioxygen activation and methane C−H oxidation in particulate methane monooxygenase (pMMO) are currently unknown. Recent studies support a binuclear copper site as the catalytic center. We report the low-temperature assembly of a high-valent dicopper(III) bis(μ-oxide) complex bearing marked structural fidelity to the proposed active site of pMMO. This unprecedented dioxygen-bonded Cu(III) species with exclusive biological ligation directly informs on the chemical plausibility and thermodynamic stability of the bis(μ-oxide) structure in such dicopper sites and foretells unusual optical signatures of an oxygenation product in pMMO. Though the ultimate pMMO active oxidant is still debated, C–H oxidation of exogenous substrates is observed with the reported Cu(III) complexes. The assembly of a high valent species both narrows the search for relevant pMMO intermediates and provides evidence to substantiate the role of Cu(III) in biological redox processes

Chemical Plausibility of Cu(III) with Biological Ligation in pMMO

The mechanisms of dioxygen activation and methane C−H oxidation in particulate methane monooxygenase (pMMO) are currently unknown. Recent studies support a binuclear copper site as the catalytic center. We report the low-temperature assembly of a high-valent dicopper­(III) bis­(μ-oxide) complex bearing marked structural fidelity to the proposed active site of pMMO. This unprecedented dioxygen-bonded Cu­(III) species with exclusive biological ligation directly informs on the chemical plausibility and thermodynamic stability of the bis­(μ-oxide) structure in such dicopper sites and foretells unusual optical signatures of an oxygenation product in pMMO. Though the ultimate pMMO active oxidant is still debated, C–H oxidation of exogenous substrates is observed with the reported Cu­(III) complexes. The assembly of a high valent species both narrows the search for relevant pMMO intermediates and provides evidence to substantiate the role of Cu­(III) in biological redox processes

Direct Copper(III) Formation from O₂ and Copper(I) with Histamine Ligation

Histamine chelation of copper­(I) by a terminal histidine residue in copper hydroxylating enzymes activates dioxygen to form unknown oxidants, generally assumed as copper­(II) species. The direct formation of copper­(III)-containing products from the oxygenation of histamine-ligated copper­(I) complexes is demonstrated here, indicating that copper­(III) is a viable oxidation state in such products from both kinetic and thermodynamic perspectives. At low temperatures, both trinuclear Cu­(II)₂Cu­(III)­O₂ and dinuclear Cu­(III)₂O₂ predominate, with the distribution dependent on the histamine ligand structure and oxygenation conditions. Kinetics studies suggest the bifurcation point to these two products is an unobserved peroxide-level dimer intermediate. The hydrogen atom reactivity difference between the trinuclear and binuclear complexes at parity of histamine ligand is striking. This behavior is best attributed to the accessibility of the bridging oxide ligands to exogenous substrates rather than a difference in oxidizing abilities of the clusters