Multilayered regulation of TORC1-body formation in budding yeast

The target of rapamycin kinase complex 1 (TORC1) regulates cell growth and metabolism in eukaryotes. In Saccharomyces cerevisiae, TORC1 activity is known to be controlled by the conserved GTPases, Gtr1/2, and movement into and out of an inactive agglomerate/body. However, it is unclear whether/how these regulatory steps are coupled. Here we show that active Gtr1/2 is a potent inhibitor of TORC1-body formation, but cells missing Gtr1/2 still form TORC1-bodies in a glucose/nitrogen starvation-dependent manner. We also identify 13 new activators of TORC1-body formation and show that seven of these proteins regulate the Gtr1/2-dependent repression of TORC1-body formation, while the remaining proteins drive the subsequent steps in TORC1 agglomeration. Finally, we show that the conserved phosphatidylinositol-3-phosphate (PI(3)P) binding protein, Pib2, forms a complex with TORC1 and overrides the Gtr1/2-dependent repression of TORC1-body formation during starvation. These data provide a unified, systems-level model of TORC1 regulation in yeast.National Institutes of Health [R01GM097329, T32GM008659]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Overactivity, impulsivity and repetitive behaviour in males with fragile X syndrome:Contrasting developmental trajectories in those with and without autism

Background: Hyperactivity and repetitive behaviour are characteristic features of fragile X syndrome (FXS). However, little is known about the influence of autism symptomatology on how these characteristics develop over time. We investigate the profiles and developmental trajectories of overactivity, impulsivity, and repetitive behaviour, in males with FXS over three time points spanning eight years. Method: Participants formed two subgroups, those who displayed elevated symptoms of autism at Time 1 (n=37; Mage=16.32; age range=6.61-43.51) and those who did not (n=32; Mage= 8.43; age range=8.94-47.49). Results: Participants without elevated symptoms of autism showed a reduction in impulsivity and repetitive questioning over time, whereas those with elevated symptoms of autism did not. Differences between the two subgroups in several topographies of repetitive behaviour emerged at Time 3 only. Conclusions: These results further understanding of the relationship between autistic phenomenology and behavioural characteristics in FXS

Down syndrome is an oxidative phosphorylation disorder

Down syndrome is the most common genomic disorder of intellectual disability and is caused by trisomy of chromosome 21. Several genes in this chromosome repress mitochondrial biogenesis. The goal of this study was to evaluate whether early overexpression of these genes may cause a prenatal impairment of oxidative phosphorylation negatively affecting neurogenesis. Reduction in the mitochondrial energy production and a lower mitochondrial function have been reported in diverse tissues or cell types, and also at any age, including early fetuses, suggesting that a defect in oxidative phosphorylation is an early and general event in Down syndrome individuals. Moreover, many of the medical conditions associated with Down syndrome are also frequently found in patients with oxidative phosphorylation disease. Several drugs that enhance mitochondrial biogenesis are nowadays available and some of them have been already tested in mouse models of Down syndrome restoring neurogenesis and cognitive defects. Because neurogenesis relies on a correct mitochondrial function and critical periods of brain development occur mainly in the prenatal and early neonatal stages, therapeutic approaches intended to improve oxidative phosphorylation should be provided in these periods.Funding sources: This work was supported by grants from Instituto de Salud Carlos III [FIS-PI17/00021, FIS-PI17/00166]; Fundación Mutua Madrileña [MMA17/01]; Precipita-FECYT crowdfunding program [PR194]; Gobierno de Aragón [LMP135_18, Grupos Consolidados B33_17R] and FEDER 2014–2020 “Construyendo Europa desde Aragón”. CIBERER is an initiative of the ISCIII

Multilayered Regulation of TORC1 Signaling in Saccharomyces cerevisiae

The Target of Rapamycin Complex 1 (TORC1) is a master regulator of cellular growth in eukaryotes. Much insight has been gained into how amino acid and nitrogen levels regulate TORC1 through the escape from rapamycin-induced growth arrest complex (EGOC), and its regulators including the Seh1-associated complex (SEAC). However, other nutrient levels and environmental stresses also act on TORC1, and far less is known about how these signals are transmitted to the complex. In two projects presented here we investigate the osmotic stress signaling network acting on TORC1 as well as regulators of TORC1 agglomeration that act in glucose and nitrogen starvation conditions. In the first investigation, we introduce a novel and reproducible high-throughput assay to screen for genes that affect TORC1 activity in stress conditions. We then use these methods to measure the expression of a TORC1 dependent ribosome biogenesis gene, NSR1, in ~4700 strains from the yeast knock-out library during osmotic stress. We show that 440 of these strains are not able to properly repress NSR1 transcription. The genes identified in the screen form a highly-connected network including 17 proteins that directly interact with TORC1. Secondary rapamycin-based assays performed on these strains allowed us to further characterize the network and show that more than 50 of the proteins act downstream of TORC1. The data derived from this work serve as a resource for our lab and others studying TORC1, and the assay itself is customizable and can be used to characterize any gene regulatory network. In the second study, we sought to further our understanding of the movement of TORC1 from its position distributed across the surface of the vacuolar membrane to a single agglomerate (TORC1-body) in starvation conditions. Previous work suggested that the AMPK in yeast, Snf1, indirectly promoted the phosphorylation of the TORC1 component Kog1. This phosphorylation event sped up aggregation of the complex by ~20 fold. In order to identify other signaling proteins that regulate TORC1-body formation we performed a screen examining the impact that nearly all non-essential kinases and phosphatases in yeast, as well as selected proteins from the previous high-throughput network, have on TORC1 agglomeration. We identified 13 new regulators of TORC1 body formation, including the PI(3)P binding protein Pib2. We also examined the impact of EGOC deletions and mutants had on body formation and discovered that active EGOC was an inhibitor of TORC1 aggregation. Together, we show that seven of the new regulators likely act at or above the EGOC dependent inhibition of TORC1 body formation; while others act at a later step to assist in body formation.Release after 01/02/202

Genome-Wide Analysis of the TORC1 and Osmotic Stress Signaling Network in Saccharomyces cerevisiae

The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/Npr3 and the small GTPases Gtr1/Gtr2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1-dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay—including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks

Mechanical fuel load reduction in Australia: a potential tool for bushfire mitigation

Australia is the most fire-prone of all continents, with large areas of the country affected by bushfires each year. Bushfires can have profound impacts on communities and on the environment. Traditionally, prescribed burns have been used as a fuel reduction treatment. However, the proportion of land that is subjected to prescribed burns in Australia has decreased since 1990. This is partly due to the increasingly smaller windows of opportunity available to conduct burns safely, concerns about operational costs, health impacts from smoke, and the social acceptability of prescribed burning. These issues suggest a need for considering alternative fuel reduction approaches in select urban and rural areas, such as the potential for mechanical fuel load reduction (MFLR) treatments. The aim of MFLR is to reduce size, likelihood and severity of bushfires. In this paper we outline key considerations associated with the implementation of MFLR trials in Australia. We discuss issues such as assessing the effectiveness of MFLR in reducing fire risk, biomass change, potential harvest systems, cost-benefit analysis, social considerations and potential impacts on biodiversity. We conclude the paper with some discussion on policy considerations around the MFLR trials

Hybrid Aptamer-Molecularly Imprinted Polymer (AptaMIP) Nanoparticles Selective for the Antibiotic Moxifloxacin

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Modified thymine bases, each containing a polymerizable group (either carboxymethylvinyl or acrylamide) at the 5-position, have been incorporated multiple times into an aptamer sequence allowing the sequence to act as the key recognition element in an aptamer-molecularly imprinted polymer (MIP) hybrid nanoparticle (aptaMIP NP) system for the molecular recognition of the antibiotic moxifloxacin. These materials combine the recognition properties of an aptamer, with the robustness and stability of a MIP, offering a “best-of-both-worlds” approach. Both aptaMIP nanoparticles offer 10-fold superior binding affinity and selectivity over conventional MIP nanoparticles (nanoMIPs), with KD values of 3.65 × ±0.9 nM, 5.72 ± 0.6 nM and 48.60 ± 7.0 nM for the carboxy aptaMIP, acrylamide aptaMIP and nanoMIP, respectively; and 100-fold superior affinity compared to the unfunctionalized aptamer only (0.325 ± 0.16 μM), all with excellent selectivity for the template antibiotic. When applied to a sensor platform (Surface Plasmon Resonance), the limit of detection based on the aptaMIP nanoparticles was approximately three-fold lower (0.51 nM and 0.56 nM for the carboxy aptaMIP and acrylamide aptaMIP, respectively) compared to the nanoMIP (1.4 nM). The introduction of the aptamer as a “macro-monomer” into the imprinted polymer scaffold is a promising strategy for significantly improving the properties of both components of the hybrid material (aptamer and MIP). These hybrid polymers, bearing nucleic acid recognition materials offer a powerful tool for robust high affinity selective molecular recognition