The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Stephanie E. Hood
Dec 1, 2020; 61:1720-1732
Research Articles

Melissa Gómez
Dec 1, 2020; 61:1658-1674
Research Articles

Martin Prescher
Dec 1, 2020; 61:1605-1616
Research Articles

Frank C. Nichols
Dec 1, 2020; 61:1645-1657
Research Articles

Hideaki Kuge
Dec 1, 2020; 61:1747-1763
Research Articles

Prabuddha Dey
Dec 1, 2020; 61:1556-1564
Reviews

Auguste Dargent
Dec 1, 2020; 61:1776-1783
Research Articles

Natalie Bruiners
Dec 1, 2020; 61:1617-1628
Research Articles

Junhwan Kim
Dec 1, 2020; 61:1707-1719
Research Articles

Thibaut Bourgeois
Dec 11, 2020; 0:jlr.RA120000737v1-jlr.RA120000737
Research Articles

Jenny E. Kanter
Dec 8, 2020; 0:jlr.ILR120001217v1-jlr.ILR120001217
Images in Lipid Research

Stacey N Keenan
Dec 17, 2020; 0:jlr.RA120001126v1-jlr.RA120001126
Research Articles

This manuscript has been withdrawn by the Author.

Photoreceptors have high energy-demands and a high density of mitochondria that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) of fuel substrates. Although glucose is the major fuel for central nervous system (CNS) brain neurons, in photoreceptors (also CNS), most glucose is not metabolized through OXPHOS but is instead metabolized into lactate by aerobic glycolysis. The major fuel sources for photoreceptor mitochondria remained unclear for almost six decades. Similar to other tissues (like heart and skeletal muscle) with high metabolic rates, photoreceptors were recently found to metabolize fatty acids (palmitate) through OXPHOS. Disruption of lipid entry into photoreceptors leads to extracellular lipid accumulation, suppressed glucose transporter expression, and a duel lipid/glucose fuel shortage. Modulation of lipid metabolism helps restore photoreceptor function. However, further elucidation of the types of lipids used as retinal energy sources, the metabolic interaction with other fuel pathways, as well as the crosstalk among retinal cells to provide energy to photoreceptors is not yet known. In this review, we will focus on the current understanding of photoreceptor energy demand and sources, and potential future investigations of photoreceptor metabolism.

Vitamin A aldehyde covalently bound to opsin protein is embedded in a phospholipid-rich membrane that supports photon absorption and phototransduction in photoreceptor cell outer segments. Following absorption of a photon, the 11-cis-retinal chromophore of visual pigment in photoreceptor cells isomerizes to all-trans-retinal. To maintain photosensitivity 11-cis-retinal must be replaced. At the same time, however, all-trans-retinal has to be handled so as to prevent nonspecific aldehyde activity. Some molecules of retinaldehyde upon release from opsin are efficiently reduced to retinol. Other molecules are released into the lipid phase of the disc membrane where they form a conjugate (N-retinylidene-PE, NRPE) through a Schiff base linkage with phosphatidylethanolamine (PE). The reversible formation of NRPE serves as a transient sink for retinaldehyde that is intended to return retinaldehyde to the visual cycle. However, if instead of hydrolyzing to PE and retinaldehyde, NRPE reacts with a second molecule of retinaldehyde a synthetic pathway is initiated that leads to the formation of multiple species of unwanted bisretinoid fluorophores. We report on recently identified members of the bisretinoid family some of which differ with respect to the acyl chains associated with the glycerol backbone. We discuss processing of the lipid moieties of these fluorophores in lysosomes of retinal pigment epithelial (RPE) cells, their fluorescence characters and new findings related to light and iron-associated oxidation of bisretinoids.

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is, therefore, crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) emerges as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide-1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.

The essential fatty acid DHA (22:6, omega-3 or n-3) is enriched in and required for the membrane biogenesis and function of photoreceptor cells (PRC), synapses, mitochondria, etc. of the CNS. PRC DHA becomes an acyl chain at the sn-2 of phosphatidylcholine (PC), amounting to more than 50% of the PRC outer segment phospholipids, where phototransduction takes place. Very long chain PUFAs (VLC-PUFAs,n-3, ≥ 28 carbons) are at the sn-1 of this PC molecular species and interact with rhodopsin. PRC shed their tips (DHA-rich membrane disks) daily, which in turn are phagocytized by the retinal pigment epithelium (RPE), where DHA is recycled back to PRC inner segments to be used for the biogenesis of new photoreceptor membranes. Here, we review the structures and stereochemistry of novel elovanoid (ELV)-N32 and ELV-N34 to be ELV-N32: (14Z,17Z,20R,21E,23E,25Z,27S,29Z)-20,27-dihydroxydo-triaconta-14,17,21,23,25,29-hexaenoic acid; ELV-N34: (16Z,19Z,22R,23E,25E,27Z,29S,31Z)-22,29-dihydroxytetra-triaconta-16,19,23,25,27,31-hexaenoic acid. ELVs are low-abundance, high-potency, protective mediators. Their bioactivity includes enhancing of anti-apoptotic and pro-survival protein expression with concomitant downregulation of pro-apoptotic proteins when RPE is confronted with uncompensated oxidative stress (UOS). ELVs also target PRC/RPE senescence gene programming, the senescence secretory phenotype in the interphotoreceptor matrix (IPM), as well as inflammaging (chronic, sterile, low-grade inflammation). An important lesson on neuroprotection is highlighted by the ELV mediators that target the terminally differentiated PRC and RPE, sustaining a beautifully synchronized renewal process. The role of ELVs in PRC and RPE viability and function uncovers insights on disease mechanisms and the development of therapeutics for age-related macular degeneration (AMD), Alzheimer’s disease (AD), and other pathologies.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3’s role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approx. 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

Perilipin (PLIN) 5 is a lipid droplet-associated protein that coordinates intracellular lipolysis in highly oxidative tissues and is thought to regulate lipid metabolism in response to phosphorylation by protein kinase A (PKA). We sought to identify PKA phosphorylation sites in PLIN5 and assess their functional relevance in cultured cells and the livers of mice. We detected phosphorylation on S155, S161 and S163 of recombinant PLIN5 by PKA in vitro and identified S155 as a functionally important site for lipid metabolism. Expression of phosphorylation-defective PLIN5 S155A in Plin5 null cells resulted in decreased rates of lipolysis and triglyceride-derived fatty acid oxidation compared with cells expressing wildtype PLIN5. These differences in lipid metabolism were not associated with differences in the cellular distribution of PLIN5. Rather, FLIM-FRET analysis of protein-protein interactions showed that PLIN5 S155 phosphorylation regulates PLIN5 interaction with adipose triglyceride lipase (ATGL) at the lipid droplet, but not with the co-activator of ATGL, α-β hydrolase domain-containing 5 (ABHD5). Re-expression of PLIN5 S155A in the liver of Plin5 liver-specific null mice reduced lipolysis when compared to mice with wildtype PLIN5 re-expression, but was not associated with other changes in hepatic lipid metabolism, such as fatty acid oxidation, de novo lipogenesis and triglyceride secretion. Furthermore, glycemic control was impaired in mice with expression of PLIN5 S155A compared with mice expressing PLIN5. Together, these studies demonstrate that PLIN5 S155 is required for PKA-mediated lipolysis and builds on the body of evidence demonstrating a critical role for PLIN5 in coordinating lipid and glucose metabolism

For three decades, the LPL–specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1^fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1^fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1^fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-B via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS³). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb₃), globotriaosylsphingosine (lyso-Gb₃), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb₃ isoforms (various fatty acids) and lyso-Gb₃ analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb₃ and lyso-Gb₃ levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb₃ and particular lyso-Gb₃ analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb₃ and lyso-Gb₃ isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb₃ massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb₃ accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb₃ or lyso-Gb₃ may play major roles in the pathogenesis of Fabry disease, and that Gb₃ and lyso-Gb₃ are not responsible for the pathology in all tissues or cell types.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

Lipins are eukaryotic proteins with functions in lipid synthesis and the homeostatic control of energy balance. They execute these functions by acting as phosphatidate phosphatase enzymes in the cytoplasm and by changing gene expression after translocation into the cell nucleus, in particular under fasting conditions. Here, we asked whether nuclear translocation and the enzymatic activity of Drosophila Lipin serve essential functions and how gene expression changes, under both fed and fasting conditions, when nuclear translocation is impaired. To address these questions, we created a Lipin null mutant, a mutant expressing Lipin lacking a nuclear localization signal (Lipin^NLS), and a mutant expressing enzymatically dead Lipin. Our data support the conclusion that the enzymatic but not nuclear gene regulatory activity of Lipin is essential for survival. Notably, adult Lipin^NLS flies were not only viable but also exhibited improved life expectancy. In contrast, they were highly susceptible to starvation. Both the improved life expectancy in the fed state and the decreased survival in the fasting state correlated with changes in metabolic gene expression. Moreover, increased life expectancy of fed flies was associated with a decreased metabolic rate. Interestingly, in addition to metabolic genes, genes involved in feeding behavior and the immune response were misregulated in Lipin^NLS flies. Altogether, our data suggest that the nuclear activity of Lipin influences the genomic response to nutrient availability with effects on life expectancy and starvation resistance. Thus, nutritional or therapeutic approaches that aim at lowering nuclear translocation of lipins in humans may be worth exploring.

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.

Phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or P_i. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and P_i, and discusses the advantages and disadvantages of each PAP assay.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1–CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage–activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P2 levels on the plasma membrane. In accord with their effects on PI(4,5)P2 levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P2 levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.