The neuronal mitochondrial metabolite
N
-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme
N
-acetyltransferase-8-like (NAT8L) and ...broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L
−/−
) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L
−/−
mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.
Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral ...membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.
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•Binding of phosphatidic acid (PA)-targets to PA is sensitive to curvature stress of the membrane.•Phosphatidylethanolamine (PE) affects PA-target binding to PA, directly and indirectly via changes in PA charge and negative curvature stress of the membrane respectively.•Lysophosphatidylcholine (LPC) abrogates PA target binding to PA by inducing positive curvature stress.•The PH2 domain of PDK1 shows different characteristics with respect to curvature compared to all other PA-binding domains tested.•Complex lipid mixtures and model lipid membranes are essential for lipid protein interaction studies – binding depends strongly on the physical chemistry of the membrane lipid composition.•Arabidopsis ECA1 binds PA in vitro and shows dependency on curvature stress similar to Opi1 and TGD2.
Metamorphic rutile from granulite facies metapelitic rocks of the Archean Pikwitonei Granulite Domain (PGD; Manitoba, Canada) provides constraints on the systematics of trace elements in rutile ...during high‐temperature conditions and subsequent slow cooling. Compositional profiles and maps of the Zr concentrations in rutile grains (120–600 μm) from three metapelitic gneisses were acquired by electron probe micro‐analysis, using a spatial resolution of down to 2 μm. Simultaneously, profiles were analysed for Nb, Cr and V, which have significantly different diffusion characteristics in rutile. The profiles of all elements show relatively homogeneous concentrations within most grains, but significant inter‐grain differences even within a single thin section. Some rutile grains display a slight concentration decrease from a neighbouring garnet towards the matrix for all measured elements. The lack of diffusion profiles for all analysed elements shows that these are highly immobile in rutile and that distributions of these elements are primary and preserve prograde information. The Nb and Cr concentrations overlap with ranges that are ascribed to different provenances indicating that source discrimination based on these elements is not possible in all cases. High retentiveness for Zr implies that the Zr‐in‐rutile geothermometer is highly robust to diffusive re‐equilibration, even during very slow cooling (<2 °C Ma−1) from granulite facies conditions. Most grains have high Zr contents (3000–4600 ppm). Differences between high Zr contents suggest that during growth under vapour‐absent conditions there may not be saturation of Zr in rutile, even if zircon is present. Therefore, several rutile grains need to be analysed in a sample to obtain a useful minimum peak temperature. The highest Zr concentrations correspond to ∼900 °C. This is significantly higher than previous peak temperature estimates of 820 °C based on two‐feldspar thermometry. On a regional scale this implies that part of the PGD was affected by ultra‐high temperature (UHT) metamorphism. It also implies that rutile is able to preserve primary compositions even to UHT conditions. This study shows that, if combined with textural information, Zr‐in‐rutile has the potential to be a very useful tool for estimating rutile crystallization temperatures and peak metamorphic conditions. For granulite facies rocks, Zr‐in‐rutile yields more reliable peak metamorphic temperatures than most other exchange geothermometers, which tend to partially re‐equilibrate by diffusion during cooling.
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•The four PA binding proteins SnRK2.10, TGD2C, PDK1-PH2 and ECA1/PICALM1A interact with DGPP in a manner that depends on lipid binding domains.•Epsin-like Clathrin Adaptor 1 (ECA1) − ...a plant salt stress signaling protein, binds most anionic lipids based on a combination of electrostatics and negative membrane curvature stress.•Efficient binding of PA by ECA1 requires a threshold level of negative curvature stress.•ECA1 binding to PA is strongly influenced by pH.
Epsin-like Clathrin Adaptor 1 (ECA1/ PICALM1A) is an A/ENTH domain protein that acts as an adaptor protein in clathrin-mediated endocytosis. ECA1 is recruited to the membrane during salt stress signaling in plants in a phosphatidic acid (PA)-dependent manner. PA is a lipid second messenger that rapidly and transiently increases in concentration under stress stimuli. Upon an increase in PA concentration another lipid, diacylglycerol pyrophosphate (DGPP), starts to accumulate. The accumulation of DGPP is suggested to be a cue for attenuating PA signaling during stress in plants. We showed in vitro that ECA1–PA binding is modulated as a function of membrane curvature stress and charge. In this work, we investigate ECA1 binding to DGPP in comparison with PA. We show that ECA1 has more affinity for the less charged PA, and this binding is pH dependent. Additionally, plant PA binding proteins SnRK2.10, TGD2C, and PDK1-PH2 were investigated for their interaction with DGPP, since no known DGPP binding proteins are available in the literature to date. Our results shed further light on DGPP and its interactions with membrane proteins which brings us closer toward understanding the complexity of protein interactions with anionic lipids, especially the enigmatic anionic lipid DGPP.
Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane ...trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning 31P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson−Hasselbalch-type formalism, yielding pK a 2 values of 6.96 ± 0.04 and 6.58 ± 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate PI(3,4)P2 as well as phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson−Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be −3.96 ± 0.10 for PI(3,4)P2 and −3.99 ± 0.10 for PI(4,5)P2. While for PI(3,5)P2 and PI(4,5)P2 the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P2. Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the 31P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P3 is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P3 of −5.05 ± 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P2 in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P2 is clustered even at 1 mol %.
The phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), is a key signaling lipid in the inner leaflet of the cell plasma membrane, regulating diverse signaling pathways ...including cell growth and migration. In this study we investigate the impact of the hydrogen-bond donor lipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the charge and phase behavior of PI(3,4,5)P3. PE and PI can interact with PI(3,4,5)P3 through hydrogen-bond formation, leading to altered ionization behavior and charge distribution within the PI(3,4,5)P3 headgroup. We quantify the altered PI(3,4,5)P3 ionization behavior using a multistate ionization model to obtain micro-pKa values for the ionization of each phosphate group. The presence of PE leads to a decrease in the pKa values for the initial deprotonation of PI(3,4,5)P3, which describes the removal of the first proton of the three protons remaining at the phosphomonoester groups at pH 4.0. The decrease in these micro-pKa values thus leads to a higher charge at low pH. Additionally, the charge distribution changes lead to increased charge on the 3- and 5-phosphates. In the presence of PI, the final deprotonation of PI(3,4,5)P3 is delayed, leading to a lower charge at high pH. This is due to a combination of hydrogen-bond formation between PI and PI(3,4,5)P3, and increased surface charge due to the addition of the negatively charged PI. The interaction between PI and PI(3,4,5)P3 leads to the formation of PI and PI(3,4,5)P3-enriched domains within the membrane. These domains may have a critical impact on PI(3,4,5)P3-signaling. We also reevaluate results for all phosphatidylinositol bisphosphates as well as for PI(4,5)P2 in complex lipid mixtures with the multistate ionization model.
Rationale
Strontium isotope ratios (87Sr/86Sr) in modern‐day marine environments are considered to be homogeneous (~0.7092). However, in the Baltic Sea, the Sr ratios are controlled by mixing ...seawater and continental drainage from major rivers discharging into the Baltic. This pilot study explores if variations in Sr can be detected in marine mammals from archaeological sites in the Baltic Sea.
Methods
87Sr/86Sr ratios were measured in tooth enamel from three seal species by laser ablation multi‐collector inductively coupled plasma mass spectrometry (LA‐MC‐ICP‐MS). The method enables micro‐sampling of solid materials. This is the first time that the method has been applied to marine samples from archaeological collections.
Results
The analyses showed inter‐tooth 87Sr/86Sr variation suggesting that different ratios can be detected in different regions of the Baltic Sea. Furthermore, the intra‐tooth variation suggests possible different geographic origin or seasonal movement of seals within different regions in the Baltic Sea through their lifetime.
Conclusions
The method was successfully applied to archaeological marine samples showing that: (1) the 87Sr/86Sr ratio in marine environments is not uniform, (2) 87Sr/86Sr differences might reflect differences in ecology and life history of different seal species, and (3) archaeological mobility studies based on 87Sr/86Sr ratios in humans should therefore be evaluated together with diet reconstruction.
The burial and exhumation of continental crust during collisional orogeny exert a strong control on the dynamics of mountain belts and plateaus. Constraining the rates and style of exhumation of ...deeply buried crust has proven difficult due to complexities in the local geology and thermochronometric methods typically used. To advance this field, we applied trace‐element and U‐Pb laser ablation inductively coupled plasma mass spectrometry analyses to rutile from eclogite and amphibolite samples from the Western Gneiss Complex of Norway—an archetypal continental (ultra)high‐pressure (UHP) terrane. Peak temperature and timing of midcrustal cooling were constrained for samples collected along a subduction‐ and exhumation‐parallel transect, using Zr‐in‐rutile thermometry and U‐Pb rutile geochronology, respectively. Peak temperatures decrease from 830 °C in the UHP domain to 730 °C at the UHP‐HP transition, remain constant at 730 °C across most of the terrane, and decrease to 620 °C at the eclogite‐out boundary. U‐Pb results show that most of the terrane cooled through 500 °C at 380–375 Ma except for the lowest grade region, where cooling occurred approximately 20 million years earlier. The results indicate that exhumation was a two stage process, involving (1) flexural rebound and slab flattening at depth combined with foreland‐directed extrusion, followed by (2) synchronous cooling below 500 °C across the, by then, largely flat‐lying Western Gneiss Complex. The latter implies and requires relatively homogeneous mass removal across a large area, consistent with erosion of an overlying orogenic plateau. The Caledonides were at near‐equatorial latitudes at the time. A Caledonian paleo‐plateau thus may represent a so far unrecognized factor in Devonian and Carboniferous atmospheric circulation and climate forcing.
Key Points
First application of microanalytical U‐Pb rutile thermochronology to Western Gneiss Complex rocks indicates exhumation in two stages
The WGC was exhumed by flexural bending and foreland‐directed thrusting, followed by flat‐slab exhumation in the middle crust
The latter is consistent with erosion of an overlying orogenic plateau, which would have been a major control of local and global climate
The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the ...underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R 0p) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R 0p values of PA and LPA were −46 Å and +20 Å, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca2+ did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R 0p of −1.3 × 102 Å). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.
Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure ...of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.
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