Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

The need to gain insights into the molecular details of peripheral membrane proteins’ specificity towards phosphatidic acid (PA) is undeniable. The variety of PA species classified in terms of acyl chain length and saturation translates into a complicated, enigmatic network of functional effects that exert a critical influence on cell physiology. As a consequence, numerous studies on the importance of phosphatidic acid in human diseases have been conducted in recent years. One of the key proteins in this context is mTOR, considered to be the most important cellular sensor of essential nutrients while regulating cell proliferation, and which also appears to require PA to build stable and active complexes. Here, we investigated the specific recognition of three physiologically important PA species by the mTOR FRB domain in the presence or absence of cholesterol in targeted membranes. Using a broad range of methods based on model lipid membrane systems, we elucidated how the length and saturation of PA acyl chains influence specific binding of the mTOR FRB domain to the membrane. We also discovered that cholesterol exerts a strong modulatory effect on PA-FRB recognition. Our data provide insight into the molecular details of some physiological effects reported previously and reveal novel mechanisms of fine-tuning the signaling cascades dependent on PA.

Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

Effects of Sex, Age, and Apolipoprotein E Genotype on Brain Ceramides and Sphingosine-1-Phosphate in Alzheimer’s Disease and Control Mice

Apolipoprotein ε4 (APOE)4 is a strong risk factor for the development of Alzheimer’s disease (AD) and aberrant sphingolipid levels have been implicated in AD. We tested the hypothesis that the APOE4 genotype affects brain sphingolipid levels in AD. Seven ceramides and sphingosine-1-phosphate (S1P) were quantified by LC-MSMS in hippocampus, cortex, cerebellum, and plasma of <3 months and >5 months old human APOE3 and APOE4-targeted replacement mice with or without the familial AD (FAD) background of both sexes (145 animals). APOE4 mice had higher Cer(d18:1/24:0) levels in the cortex (1.7-fold, p = 0.002) than APOE3 mice. Mice with AD background showed higher levels of Cer(d18:1/24:1) in the cortex than mice without (1.4-fold, p = 0.003). S1P levels were higher in all three brain regions of older mice than of young mice (1.7-1.8-fold, all p ≤ 0.001). In female mice, S1P levels in hippocampus (r = −0.54 [−0.70, −0.35], p < 0.001) and in cortex correlated with those in plasma (r = −0.53 [−0.71, −0.32], p < 0.001). Ceramide levels were lower in the hippocampus (3.7–10.7-fold, all p < 0.001), but higher in the cortex (2.3–12.8-fold, p < 0.001) of female than male mice. In cerebellum and plasma, sex effects on individual ceramides depended on acyl chain length (9.5-fold lower to 11.5-fold higher, p ≤ 0.001). In conclusion, sex is a stronger determinant of brain ceramide levels in mice than APOE genotype, AD background, or age. Whether these differences impact AD neuropathology in men and women remains to be investigated.

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S−1 mM−1), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.

Sphingolipids mediate polar sorting of PIN2 through phosphoinositide consumption at the trans-Golgi network

The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting.

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered domains (rafts)

ABSTRACTUsing efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of insulin receptor (IR) in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length. Increasing acyl chain length increases both bilayer width and the propensity to form ordered domains. To distinguish the effects of membrane width and domain formation, we incorporated purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths. IR activity increases with increased chain length, but more modestly than by increasing lipid acyl chain length in cells. This suggests that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases with sodium orthovanadate showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. The results are consistent with a model in which a combination of bilayer width and ordered domain formation modulate IR activity via effects upon IR conformation and accessibility to phosphatases.SignificanceThis study shows how methyl-α-cyclodextrin mediated lipid exchange can be used to probe the influence of lipid structure upon the functioning of a transmembrane receptor. Plasma membranes having a propensity to form Lo domains are required to support a high level of IR activity. The studies indicate this may reflect an effect of lipid environment upon IR domain localization, which in turn alters its conformation and vulnerability to phosphatases. Alterations in lipid composition could conceivably regulate IR activity in vivo.

Lipidomics Reveals Glycerophospholipid Metabolism Dysregulation in the Corpus Striatum of Mice Treated with Cefepime

Cefepime exhibits a broad spectrum of antimicrobial activity and thus is widely used for severe bacterial infection. Adverse effects on the central nervous system (CNS) have been reported in the patients treated with cefepime. Current explanation for the neurobehavioral effect of cefepime mainly attributes to its ability to across blood-brain barrier and to competitively bind to GABAergic receptor; however, the underlying mechanism is largely unknown. In this study, mice were intraperitoneally administered 80 mg/kg cefepime for different time period, including 1 day, 3 days, 5 days, 7 days and 10 days, and then LC/MS-MS-based metabolomics was used to investigate the effect of cefepime on the brain lipidomic profiling and metabolic pathway. Repeated cefepime treatment time-dependently caused anxiety-like behavior accompanied with the reduced locomotor activity in the open field test. Cefepime profoundly altered the lipid profile in the corpus striatum, and glycerophospholipid contributed to a large proportion among those significantly modified lipids. Cefepime also significantly modified acyl chain length and unsaturation of fatty acids. In addition, cefepime obviously altered the morphology of neurite, mitochondria and synaptic vesicles of striatal neuron in vitro. Collectively, our results show that cefepime reprograms glycerophospholipid metabolism of corpus striatum, which may underly its neurobehavioral effect.

Membrane Hydrophobicity Determines the Activation Free Energy of Passive Lipid Transport

ABSTRACTThe collective behavior of lipids with diverse chemical and physical features determines a membrane’s thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophospholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport, lipid desorption, which is rate-limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid’s local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane’s interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.SIGNIFICANCECell homeostasis requires spatiotemporal regulation of heterogeneous membrane compositions, in part, through non-vesicular transport of individual lipids between membranes. By systematically investigating how the chemical diversity present in glycerophospholipidomes and variations in membrane order influence the free energy barriers for passive lipid transport, we discover a correlation between the activation free energy and membrane hydrophobicity. By demonstrating how membrane hydrophobicity is modulated by local changes in membrane composition and order, we solidify the link between membrane physicochemical properties and lipid transport rates. Our results suggest that variations in cell membrane hydrophobicity may be exploited to direct non-vesicular lipid traffic.

Aging selectively dampens oscillation of lipid abundance in white and brown adipose tissue

Lipid metabolism is under the control of the circadian system and circadian dysregulation has been linked to obesity and dyslipidemia. These factors and outcomes have also been associated to, or affected by, the process of aging. Here, we investigated whether murine white (WAT) and brown (BAT) adipose tissue lipids exhibit rhythmicity and if this is affected by aging. To this end, we have measured the 24 h lipid profiles of WAT and BAT using a global lipidomics analysis of > 1100 lipids. We observed rhythmicity in nearly all lipid classes including glycerolipids, glycerophospholipids, sterol lipids and sphingolipids. Overall, ~ 22% of the analyzed lipids were considered rhythmic in WAT and BAT. Despite a general accumulation of lipids upon aging the fraction of oscillating lipids decreased in both tissues to 14% and 18%, respectively. Diurnal profiles of lipids in BAT appeared to depend on the lipid acyl chain length and this specific regulation was lost in aged mice. Our study revealed how aging affects the rhythmicity of lipid metabolism and could contribute to the quest for targets that improve diurnal lipid homeostasis to maintain cardiometabolic health during aging.

Deep-sea microbes as tools to refine the rules of innate immune pattern recognition

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus Moritella was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally.