Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

Roles for PI(3,5)P2 in nutrient sensing through TORC1

TORC1, a conserved protein kinase, regulates cell growth in response to nutrients. Localization of mammalian TORC1 to lysosomes is essential for TORC1 activation. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)), an endosomal signaling lipid, is implicated in insulin-dependent stimulation of TORC1 activity in adipocytes. This raises the question of whether PI(3,5)P(2) is an essential general regulator of TORC1. Moreover, the subcellular location where PI(3,5)P(2) regulates TORC1 was not known. Here we report that PI(3,5)P(2) is required for TORC1 activity in yeast and regulates TORC1 on the vacuole (lysosome). Furthermore, we show that the TORC1 substrate, Sch9 (a homologue of mammalian S6K), is recruited to the vacuole by direct interaction with PI(3,5)P(2), where it is phosphorylated by TORC1. Of importance, we find that PI(3,5)P(2) is required for multiple downstream pathways via TORC1-dependent phosphorylation of additional targets, including Atg13, the modification of which inhibits autophagy, and phosphorylation of Npr1, which releases its inhibitory function and allows nutrient-dependent endocytosis. These findings reveal PI(3,5)P(2) as a general regulator of TORC1 and suggest that PI(3,5)P(2) provides a platform for TORC1 signaling from lysosomes

A pathway of targeted autophagy is induced by DNA damage in budding yeast

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response

A Signaling Lipid Associated with Alzheimer's Disease Promotes Mitochondrial Dysfunction

Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer's disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although "candidate gene" approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD

A Neurotoxic Glycerophosphocholine Impacts PtdIns-4, 5-Bisphosphate and TORC2 Signaling by Altering Ceramide Biosynthesis in Yeast

<div><p>Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(<i>O</i>-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(<i>O</i>-16:0/2:0) is also toxic to <i>Saccharomyces cerevisiae</i>, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂)-binding protein, was relocalized in response to PC(<i>O</i>-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(<i>O</i>-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P₂ leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(<i>O</i>-16:0/2:0) on the distribution of PM PtdIns(4,5)P₂ pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(<i>O</i>-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.</p></div

PC(<i>O</i>-16:0/2:0) inhibits TORC2 signaling.

<p>(<b>A</b>) <b>Phosphorylation of the TORC2 substrate Ypk1 is reduced following treatment.</b> TORC2-dependent Ypk1 (T662) phosphorylation status was assessed in whole cell extracts from vehicle (ethanol, EtOH), PC(<i>O</i>-16:0/2:0) (20 µM) or rapamycin (Rap, 200 ng/ml) treated wild type (YPH500) and <i>spo14</i>Δ (YKB2076) cells. Immunoblots were also probed with anti-sera for total Ypk1 to ensure equal loading. (<b>B</b>) <b><i>tor2-21</i></b><b> mutants display increased sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Strains expressing plasmid borne wild type <i>TOR2</i> or the temperature sensitive (ts) alleles <i>tor2-21</i> or <i>tor2-30</i> in a <i>tor1</i>Δ, <i>tor2</i>Δ or a combined <i>tor1</i>Δ <i>tor2</i>Δ background were plated in 10-fold serial dilutions on YPD plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml). Plates were incubated for 2 days at a permissive (25 C) or semi-permissive temperature (33 C). (<b>C</b>) <b>Overexpression of hyperactive Ypk2 suppresses sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Ypk2 wild type (Ypk2), hyperactive (D239A), kinase dead (K373A) and the double mutant (D239A and K373A) were transformed into wild type (SH100) and <i>tor2-21</i> (SH121) expressing cells. Growth was assessed following 2 days at permissive (25 C) and semi-permissive temperature (33 C) on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml).</p