Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

Inter-annual variability of precipitation constrains the production response of boreal Pinus sylvestris to nitrogen fertilization

© 2015 Published by Elsevier B.V.Tree growth resources and the efficiency of resource-use for biomass production determine the productivity of forest ecosystems. In nutrient-limited forests, nitrogen (N)-fertilization increases foliage [N], which may increase photosynthetic rates, leaf area index (L), and thus light interception (IC). The product of such changes is a higher gross primary production and higher net primary production (NPP). However, fertilization may also alter carbohydrate partitioning from below- to aboveground, increasing aboveground NPP (ANPP). We analyzed effects of long-term N-fertilization on NPP, and that of long-term carbon storing organs (NPPS) in a Pinus sylvestris forest on sandy soil, a wide-ranging forest type in the boreal region. We based our analyses on a combination of destructive harvesting, consecutive mensuration, and optical measurements of canopy openness. After eight-year fertilization with a total of 70gNm^-2, ANPP was 27±7% higher in the fertilized (F) relative to the reference (R) stand, but although L increased relative to its pre-fertilization values, IC was not greater than in R. On the seventh year after the treatment initiation, the increase of ANPP was matched by the decrease of belowground NPP (78 vs. 92gCm^-2yr^-1; ~17% of NPP) and, given the similarity of IC, suggests that the main effect of N-fertilization was changed carbon partitioning rather than increased canopy photosynthesis. Annual NPPS increased linearly with growing season temperature (T) in both treatments, with an upward shift of 70.2gCm^-2yr^-1 by fertilization, which also caused greater amount of unexplained variation (r²=0.53 in R, 0.21 in F). Residuals of the NPPS-T relationship of F were related to growing season precipitation (P, r²=0.48), indicating that T constrains productivity at this site regardless of fertility, while P is important in determining productivity where N-limitation is alleviated. We estimated that, in a growing season average T (11.5±1.0°C; 33-year-mean), NPPS response to N-fertilization will be nullified with P 31mm less than the mean (325±85mm), and would double with P 109mm greater than the mean. These results suggest that inter-annual variation in climate, particularly in P, may help explaining the reported large variability in growth responses to fertilization of pine stands on sandy soils. Furthermore, forest management of long-rotation systems, such as those of boreal and northern temperate forests, must consider the efficiency of fertilization in terms of wood production in the context of changes in climate predicted for the region.

Big or fast: two strategies in the developmental control of body size.

Adult body size is controlled by the mechanisms that stop growth when a species-characteristic size has been reached. The mechanisms by which size is sensed and by which this information is transduced to the growth regulating system are beginning to be understood in a few species of insects. Two rather different strategies for control have been discovered; one favors large body size and the other favors rapid development.