Ceramide synthases at the centre of sphingolipid metabolism and biology

Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1-CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology.     

Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition

Systems biology approaches that combine experimental data and theoretical modelling to understand cellular signalling network dynamics offer a useful platform to investigate the mechanisms of resistance to drug interventions and to identify combination drug treatments. Extending our work on modelling the PI3K/PTEN/AKT signalling network (SN), we analyse the sensitivity of the SN output signal, phospho-AKT, to inhibition of HER2 receptor. We model typical aberrations in this SN identified in cancer development and drug resistance: loss of PTEN activity, PI3K and AKT mutations, HER2 overexpression, and overproduction of GSK3β and CK2 kinases controlling PTEN phosphorylation. We show that HER2 inhibition by the monoclonal antibody pertuzumab increases SN sensitivity, both to external signals and to changes in kinetic parameters of the proteins and their expression levels induced by mutations in the SN. This increase in sensitivity arises from the transition of SN functioning from saturation to non-saturation mode in response to HER2 inhibition. PTEN loss or PIK3CA mutation causes resistance to anti-HER2 inhibitor and leads to the restoration of saturation mode in SN functioning with a consequent decrease in SN sensitivity. We suggest that a drug-induced increase in SN sensitivity to internal perturbations, and specifically mutations, causes SN fragility. In particular, the SN is vulnerable to mutations that compensate for drug action and this may result in a sensitivity-to-resistance transition. The combination of HER2 and PI3K inhibition does not sensitise the SN to internal perturbations (mutations) in the PI3K/PTEN/AKT pathway: this combination treatment provides both synergetic inhibition and may prevent the SN from acquired mutations causing drug resistance. Through combination inhibition treatments, we studied the impact of upstream and downstream interventions to suppress resistance to the HER2 inhibitor in the SN with PTEN loss. Comparison of experimental results of PI3K inhibition in the PTEN upstream pathway with PDK1 inhibition in the PTEN downstream pathway shows that upstream inhibition abrogates resistance to pertuzumab more effectively than downstream inhibition. This difference in inhibition effect arises from the compensatory mechanism of an activation loop induced in the downstream pathway by PTEN loss. We highlight that drug target identification for combination anti-cancer therapy needs to account for the mutation effects on the upstream and downstream pathways.     

Plant peroxisomes: biogenesis and function

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.     

mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Ancient trans-Atlantic flight explains locust biogeography: molecular phylogenetics of Schistocerca

The desert locust (Schistocerca gregaria) has been an important agricultural pest at least since biblical times. Although the ecology, physiology and behaviour of this insect species have been well characterized, its biogeographical origins and evolutionary history are more obscure. Schistocerca gregaria occurs throughout Africa, the Middle East and Western Asia, but all other species in the genus Schistocerca are found in the New World. Because S. gregaria has the capacity for extreme long-distance movement associated with swarming behaviour, dispersal may have played an important role in determining current distribution patterns. Some authors have argued that S. gregaria is the product of an eastward trans-Atlantic dispersal from North America to Africa; others consider it more likely that the New World taxa are the product of westward dispersal from Africa. Here, we present a mitochondrial DNA phylogeny of Schistocerca species that supports the monophyly of New World species (including the Galapagos endemic Halmenus) relative to S. gregaria. In concert with observed patterns of molecular divergence, and in contrast to previous morphological studies, our analysis indicates a single trans-Atlantic flight from Africa to South America, followed by extensive speciation and ecological divergence in the New World.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.