Necrosis and ethylene-inducing peptide 1–like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate ...microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.
Glycerophospholipids, the structural components of cell membranes, have not been considered to be spatial cues for intercellular signaling because of their ubiquitous distribution. We identified ...lyso-phosphatidyl-β-D-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, and demonstrated its role in modality-specific repulsive guidance of spinal cord sensory axons. LysoPtdGlc is locally synthesized and released by radial glia in a patterned spatial distribution to regulate the targeting of nociceptive but not proprioceptive central axon projections. Library screening identified the G protein–coupled receptor GPR55 as a high-affinity receptor for LysoPtdGlc, and GPR55 deletion or LysoPtdGlc loss of function in vivo caused the misallocation of nociceptive axons into proprioceptive zones. These findings show that LysoPtdGlc/GPR55 is a lipid-based signaling system in glia-neuron communication for neural development.
Phototherapy converts lipophilic unconjugated bilirubin to hydrophilic bilirubin photoisomers, such as lumirubin. We comparatively used a blue light-emitting diode (LED) and a green fluorescent lamp ...(FL) as light sources for phototherapy of hyperbilirubinemic preterm neonates with the aim of examining potential differences in urinary lumirubin excretion between these two wavelengths. Urinary lumirubin levels were measured using a fluorescence assay with blue light exposure in the presence of the unconjugated bilirubin-inducible fluorescent protein UnaG, and denoted as urinary UnaG-bound bilirubin (UUB)/creatinine (Cr) (μg/mg Cr). Preterm neonates born at ≤ 33 weeks gestational age and treated with phototherapy were subjected to this study. The maximum UUB/Cr level during phototherapy per device intensity was compared between neonates treated with the blue LED and the green FL. A total of 61 neonates were examined to determine the maximum UUB/Cr levels. The median of maximum UUB/Cr excretion per light intensity of each device (μg/mg Cr/μW/cm2/nm) was 0.83 for the blue LED and 1.29 for the green FL (p = 0.01). Green light was found to be more effective than blue one for bilirubin excretion via urinary lumirubin excretion. This is the first spectroscopic study to compare the efficacy of phototherapy at different wavelengths using fluorescence assay.
Abstract
Lumirubin is the most prevalently excreted hydrophilic bilirubin photoisomer in phototherapy for neonatal jaundice caused by excess hydrophobic unconjugated bilirubin (ZZ-bilirubin). We ...developed a simple method to estimate the amount of lumirubin by monitoring the reverse photoisomerization of lumirubin to ZZ-bilirubin. Although lumirubin formation was long considered irreversible, exposure to blue light in the presence of the fluorescent protein UnaG, which binds specifically and tightly to ZZ-bilirubin, enables the reverse photoisomerization of lumirubin. This reaction was first detected using a fluorescence assay of neonatal urine sampled during phototherapy and purified lumirubin. The phenomenon of reverse photoisomerization of lumirubin was validated using liquid chromatography–mass spectrometry, which confirmed that lumirubin is reconverted to ZZ-bilirubin in the presence of UnaG. Analyses of 20 urine samples from 17 neonates revealed a significant correlation (correlation coefficient
r
= 0.978; 95% confidence interval 0.867–0.979;
P
< .001) between lumirubin and ZZ-bilirubin concentration before and after reverse photoisomerization. In general, the rate of photo-reconversion of lumirubin to ZZ-bilirubin is approximately 40%. In conclusion, we demonstrate here that lumirubin can be photo-reconverted to ZZ-bilirubin via exposure to blue light in the presence of UnaG. Utilizing this approach, urinary lumirubin levels can be estimated using an easy-to-perform fluorescence assay.
Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally ...misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.
Pore-forming toxins (PFTs) are soluble proteins that can oligomerize on the cell membrane and induce cell death by membrane insertion. PFT oligomers sometimes form hexagonal close-packed (hcp) ...structures on the membrane. Here, we show the assembling of the sphingomyelin (SM)-binding PFT, lysenin, into an hcp structure after oligomerization on SM/cholesterol membrane. This process was monitored by high-speed atomic force microscopy. Hcp assembly was driven by reorganization of lysenin oligomers such as association/dissociation and rapid diffusion along the membrane. Besides rapid association/dissociation of oligomers, the height change for some oligomers, possibly resulting from conformational changes in lysenin, could also be visualized. After the entire membrane surface was covered with a well-ordered oligomer lattice, the lysenin molecules were firmly bound on the membrane and the oligomers neither dissociated nor diffused. Our results reveal the dynamic nature of the oligomers of a lipid-binding toxin during the formation of an hcp structure. Visualization of this dynamic process is essential for the elucidation of the assembling mechanism of some PFTs that can form ordered structures on the membrane.
Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation ...studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
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•Pore-forming toxins (PFTs) represent highly specific lipid-binding proteins.•Non-toxic PFT derivatives are used to visualize lipids in live and fixed cells.•Specific PFTs are applied to visualize sphingomyelin and ceramide phosphoethanolamine.•PFTs revealed different pools of sphingomyelin in biological membranes.
GPRC5B recruitment of Src family kinases has been implicated in diet-induced insulin resistance. However, the mechanism of this action is not fully understood. Here, we report that GPRC5B-mediated ...phosphorylation of sphingomyelin synthase 2 (SMS2) by Fyn is a crucial step in the development of insulin resistance. Lipid-induced metabolic stress augments SMS2 phosphorylation by facilitating the interaction of GPRC5B and SMS2. SMS2 phosphorylation reduces its ubiquitination, and consequently increases SMS2 protein abundance. Although ceramide and diacylglycerol (DAG) have been known to be central mediators of lipid-induced insulin resistance, the accumulation of these lipids fails to impair insulin signaling in SMS2 knockout mouse embryonic fibroblasts (MEFs). Conversely, exogenous expression of a phosphomimetic SMS2 impairs insulin action in SMS2 knockout MEFs under metabolic stress conditions. We demonstrate that SMS2-generated DAG in sphingomyelin synthesis inhibits insulin signaling through JNK activation. Thus, GPRC5B links sphingolipid metabolism to diet-induced insulin resistance via SMS2-dependent DAG production.
•ZIC-HILIC method was developed for isometric separation of mono-glucosylated lipids.•ZIC-HILIC preferentially retained glucose- followed by galactose-featuring lipids.•ZIC-HILIC reliably resolved ...lipids based on their carbohydrate and lipid moieties.•GlcCer in mouse brain samples was analyzed in the presence of excess GalCer.•GlcCer in skin was quantified based on a difference of an extra hydroxyl group.
Mono-glycosylated sphingolipids and glycerophospholipids play important roles in diverse biological processes and are linked to a variety of pathologies, such as Parkinson disease. The precise identification of the carbohydrate head group of these lipids is complicated by their isobaric nature and by substantial differences in concentration in different biological samples. To overcome these obstacles, we developed a zwitterionic (ZIC)-hydrophilic interaction chromatography (HILIC) electrospray ionization tandem mass spectrometry method. ZIC-HILIC preferentially retains inositol, followed by glucose- and galactose-featuring lipids. Comparison with unmodified silica gel HILIC stationary phase revealed different retention specificity. To evaluate the precision of ZIC-HILIC, we quantified glucosyl- (GlcCer) and galactosylceramides (GalCer) in seven different regions of the mouse brain and discovered that GlcCer and GalCer concentrations are inversely related. The highest GalCer (lowest GlcCer) content was found in the medulla oblongata and hippocampus, whereas the highest GlcCer (lowest GalCer) content was found in other regions. With a neutral loss scan, ZIC-HILIC resolved glucosylceramide species featuring non-hydroxylated fatty acid, hydroxylated fatty acid, and trihydroxy sphingoid bases in mouse epidermis samples. This demonstrates that our ZIC-HILIC-based approach is a valuable tool for characterizing the structural diversity of mono-glucosylated lipids in biological material and for quantifying these important lipids.
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