Cytidinediphosphate‐diacylglycerol synthase 5 is required for phospholipid homeostasis and is negatively involved in hyperosmotic stress tolerance

Cytidinediphosphate diacylglycerol synthase (CDS) uses phosphatidic acid (PA) and cytidinetriphosphate to produce cytidinediphosphate‐diacylglycerol, an intermediate for phosphatidylglycerol (PG) and phosphatidylinositol (PI) synthesis. This study shows that CDS5, one of the five CDSs of the Oryza sativa (rice) genome, has multifaceted effects on plant growth and stress responses. The loss of CDS5 resulted in a decrease in PG and PI levels, defective thylakoid membranes, pale leaves in seedlings and growth retardation. In addition, the loss of CDS5 led to an elevated PA level and enhanced hyperosmotic tolerance. The inhibition of phospholipase D (PLD)‐derived PA formation in cds5 restored the hyperosmotic stress tolerance of the mutant phenotype to that of the wild type, suggesting that CDS5 functions as a suppressor in PLD‐derived PA signaling and negatively affects hyperosmotic stress tolerance.     

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.     

Lifespan regulation in α/β posterior neurons of the fly mushroom bodies by Rab27

Brain function has been implicated to control the aging process and modulate lifespan. However, continuous efforts remain for the identification of the minimal sufficient brain region and the underlying mechanism for neuronal regulation of longevity. Here, we show that the Drosophila lifespan is modulated by rab27 functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure that shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/βp neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade‐offs in major life functions. Within the α/βp neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant‐negative S6K in the α/βp neurons increases lifespan. Furthermore, the expression of phospho‐mimetic S6 in α/βp neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR‐mediated protein synthesis in α/βp neurons is sufficient to promote longevity.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.     

Diet restriction‐induced healthy aging is mediated through the immune signaling component ZIP‐2 in Caenorhabditis elegans

Dietary restriction (DR) robustly delays the aging process in all animals tested so far. DR slows aging by negatively regulating the target of rapamycin (TOR) and S6 kinase (S6K) signaling pathway and thus inhibiting translation. Translation inhibition in C. elegans is known to activate the innate immune signal ZIP‐2. Here, we show that ZIP‐2 is activated in response to DR and in feeding‐defective eat‐2 mutants. Importantly, ZIP‐2 contributes to the improvements in longevity and healthy aging, including mitochondrial integrity and physical ability, mediated by DR in C. elegans. We further show that ZIP‐2 is activated upon inhibition of TOR/S6K signaling. However, DR‐mediated activation of ZIP‐2 does not require the TOR/S6K effector PHA‐4/FOXA. Furthermore, zip‐2 was not activated or required for longevity in daf‐2 mutants, which mimic a low nutrition status. Thus, DR appears to activate ZIP‐2 independently of PHA‐4/FOXA and DAF‐2. The link between DR, aging, and immune activation provides practical insight into the DR‐induced benefits on health span and longevity.     

Phosphatidic Acid Sequesters Sec18p from cis‐SNARE Complexes to Inhibit Priming

Yeast vacuole fusion requires the activation of cis‐SNARE complexes through priming carried out by Sec18p/N‐ethylmaleimide sensitive factor and Sec17p/α‐SNAP. The association of Sec18p with vacuolar cis‐SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane‐associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA‐rich membrane microdomains before transferring to cis‐SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA‐binding domain blocked Sec18p binding to PA‐liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis‐SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis‐SNAREs and complementation with plasmid‐encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p.      

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P₂ ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P₂ , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity.     

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD⁺‐dependent protein deacetylase, which regulates the expression of the ATP‐dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho‐mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)‐like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2–Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.     

The role of MAP4K3 in lifespan regulation of Caenorhabditiselegans

The TOR pathway is a kinase signaling pathway that regulates cellular growth and proliferation in response to nutrients and growth factors. TOR signaling is also important in lifespan regulation - when this pathway is inhibited, either naturally, by genetic mutation, or by pharmacological means, lifespan is extended. MAP4K3 is a Ser/Thr kinase that has recently been found to be involved in TOR activation. Unexpectedly, the effect of this protein is not mediated via Rheb, the more widely known TOR activation pathway. Given the role of TOR in growth and lifespan control, we looked at how inhibiting MAP4K3 in Caenorhabditiselegans affects lifespan. We used both feeding RNAi and genetic mutants to look at the effect of MAP4K3 deficiency. Our results show a small but significant increase in mean lifespan in MAP4K3 deficient worms. MAP4K3 thus represents a new target in the TOR pathway that can be targeted for pharmacological intervention to control lifespan.     

Defining the role of DAG,mitochondrial function,and lipid deposition in palmitate-induced proinflammatory signaling and its counter-modulation by palmitoleate

Chronic exposure of skeletal muscle to saturated fatty acids, such as palmitate (C16:0), enhances proinflammatory IKK-NFκB signaling by a mechanism involving the MAP kinase (Raf-MEK-ERK) pathway. Raf activation can be induced by its dissociation from the Raf-kinase inhibitor protein (RKIP) by diacylglycerol (DAG)-sensitive protein kinase C (PKC). However, whether these molecules mediate the proinflammatory action of palmitate, an important precursor for DAG synthesis, is currently unknown. Here, involvement of DAG-sensitive PKCs, RKIP, and the structurally related monounsaturated fatty acid palmitoleate (C16:1) on proinflammatory signaling are investigated. Palmitate, but not palmitoleate, induced phosphorylation/activation of the MEK-ERK-IKK axis and proinflammatory cytokine (IL-6, CINC-1) expression. Palmitate increased intramyocellular DAG and invoked PKC-dependent RKIP^Ser153 phosphorylation, resulting in RKIP-Raf1 dissociation and MEK-ERK signaling. These responses were mimicked by PMA, a DAG mimetic and PKC activator. However, while pharmacological inhibition of PKC suppressed PMA-induced activation of MEK-ERK-IKK signaling, activation by palmitate was upheld, suggesting that DAG-sensitive PKC and RKIP were dispensable for palmitate''s proinflammatory action. Strikingly, the proinflammatory effect of palmitate was potently repressed by palmitoleate. This repression was not due to reduced palmitate uptake but linked to increased neutral lipid storage and enhanced cellular oxidative capacity brought about by palmitoleate''s ability to restrain palmitate-induced mitochondrial dysfunction.     

Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans

TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P‐S6) was defined as a surrogate marker for TOR‐dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P‐S6 levels. The P‐S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P‐S6 to physiologic levels, whereas all four tested components were required to downregulate P‐S6 in low glucose. TOR was epistatic to PKA components with respect to P‐S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR‐dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.     

Loss of NDG‐4 extends lifespan and stress resistance in Caenorhabditis elegans

NDG‐4 is a predicted transmembrane acyltransferase protein that acts in the distribution of lipophilic factors. Consequently, ndg‐4 mutants lay eggs with a pale appearance due to lack of yolk, and they are resistant to sterility caused by dietary supplementation with the long‐chain omega‐6 polyunsaturated fatty acid dihommogamma‐linolenic acid (DGLA). Two other proteins, NRF‐5 and NRF‐6, a homolog of a mammalian secreted lipid binding protein and a NDG‐4 homolog, respectively, have previously been shown to function in the same lipid transport pathway. Here, we report that mutation of the NDG‐4 protein results in increased organismal stress resistance and lifespan. When NDG‐4 function and insulin/IGF‐1 signaling are reduced simultaneously, maximum lifespan is increased almost fivefold. Thus, longevity conferred by mutation of ndg‐4 is partially overlapping with insulin signaling. The nuclear hormone receptor NHR‐80 (HNF4 homolog) is required for longevity in germline less animals. We find that NHR‐80 is also required for longevity of ndg‐4 mutants. Moreover, we find that nrf‐5 and nrf‐6 mutants also have extended lifespan and increased stress resistance, suggesting that altered lipid transport and metabolism play key roles in determining lifespan.     

Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate

Phosphatidic acid (PA) and its phosphorylated derivative diacylglycerol pyrophosphate (DGPP) are lipid molecules that have been implicated in plant cell signaling. In this study we report the rapid but transient accumulation of PA and DGPP in suspension-cultured tomato (Lycopersicon esculentum) cells treated with the general elicitors, N,N',N",N"'-tetraacetylchitotetraose, xylanase, and the flagellin-derived peptide flg22. To determine whether PA originated from the activation of phospholipase D or from the phosphorylation of diacylglycerol (DAG) by DAG kinase, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used. DAG kinase was found to be the dominant producer of PA that was subsequently metabolized to DGPP. A minor but significant role for phospholipase D could only be detected when xylanase was used as elicitor. Since PA formation was correlated with the high turnover of polyphosphoinositides, we hypothesize that elicitor treatment activates phospholipase C to produce DAG, which in turn acts as substrate for DAG kinase. The potential roles of PA and DGPP in plant defense signaling are discussed.