Project description:Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.
Project description:Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.
Project description:Pleckstrin homology (PH) domains are membrane-binding lipid recognition proteins that interact with phosphatidylinositol phosphate (PIP) molecules in eukaryotic cell membranes. Diffusion of PH domains plays a critical role in biological reactions on membrane surfaces. Although diffusivity can be estimated by long-time measurements, it lacks information on the short-time diffusive nature. We reveal two diffusive properties of a PH domain bound to the surface of a PIP-containing membrane using molecular dynamics simulations. One is fractional Brownian motion, attributed to the motion of the lipids with which the PH domain interacts. The other is temporally fluctuating diffusivity; that is, the short-time diffusivity of the bound protein changes substantially with time. Moreover, the diffusivity for short-time measurements is intrinsically different from that for long-time measurements. This fluctuating diffusivity results from dynamic changes in interactions between the PH domain and PIP molecules. Our results provide evidence that the complexity of protein-lipid interactions plays a crucial role in the diffusion of proteins on biological membrane surfaces. Changes in the diffusivity of PH domains and related membrane-bound proteins may in turn contribute to the formation/dissolution of protein complexes in membranes.
Project description:Dietary lipids impact development, homeostasis, and disease, but links between specific dietary fats and cell fates are poorly understood. Ferroptosis is an iron-dependent form of nonapoptotic cell death associated with oxidized polyunsaturated phospholipids. Here, we show that dietary ingestion of the polyunsaturated fatty acid (PUFA) dihomogamma-linolenic acid (DGLA; 20:3n-6) can trigger germ-cell ferroptosis and sterility in the nematode Caenorhabditis elegans. Exogenous DGLA is also sufficient to induce ferroptosis in human cells, pinpointing this omega-6 PUFA as a conserved metabolic instigator of this lethal process. In both C. elegans and human cancer cells, ether-lipid synthesis protects against ferroptosis. These results establish C. elegans as a powerful animal model to study the induction and modulation of ferroptosis by dietary fats and indicate that endogenous ether lipids act to prevent this nonapoptotic cell fate.
Project description:Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke phenotypic changes in lung fibroblasts, forming a supportive metastatic niche. Colonization of the lungs confers an inflammatory phenotype in metastasis-associated fibroblasts. Specifically, IL-1α and IL-1β secreted by breast cancer cells induce CXCL9 and CXCL10 production in lung fibroblasts via NF-κB signaling, fueling the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells, which exhibits tumor-initiating ability when co-transplanted with fibroblasts and has high JNK signaling that drives IL-1α/β expression. Importantly, disruption of the intercellular JNK-IL-1-CXCL9/10-CXCR3 axis reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for the molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.
Project description:Extracellular vesicles (ECV) are heterogeneous membrane-enclosed structures containing proteins, nucleic acids and lipids that participate in intercellular communication by transferring their content to recipient cells. Although most of the attention has been directed at the biological effect of proteins and microRNA, the contribution of phospholipids present in ECV on cellular activation has not been extensively addressed. Here we investigated the biological effect of phosphatidylserine (PS) and phosphatidylcholine (PC), two phospholipids highly abundant in ECV. A transcriptomic analysis revealed that about 4,700 genes were specifically modified by exposing peritoneal macrophages to PS or PC liposomes in vivo. Among them, the expression of several chemokines and cytokines was highly upregulated by PS liposome treatment, translating into a massive neutrophil infiltration into the peritoneum capable of neutralizing a septic polymicrobial insult. Both the L and D stereoisomers of PS induced the same response, suggesting that the effect was related to the negative charge of the phospholipid head. We concluded that an increase in the internal negative charge of the cell triggers a signaling cascade activating an innate immune response capable of controlling infection.
Project description:Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.
Project description:Crohn's disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth cell function. Paneth cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear cells (PBMCs) from healthy controls restored the attenuated Paneth cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived cells and epithelial secretory Paneth cells. Defective Paneth cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.
Project description:Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.
Project description:The intracellular unbound drug concentration (Cu,cell) drives pharmacological and toxicological responses for targets inside cells. We have previously shown that Cu,cell is not always equal to extracellular unbound drug concentrations (e.g. plasma concentrations). However, the underlying mechanisms that drive cellular drug binding are poorly understood. The aim of this study was to evaluate drug binding to individual cellular components, such as neutral lipids (NL) and phospholipids (PL). The fibroblastic 3T3-L1 cell line was used as a cellular model. The cells were either used as wild-type (WT cells) or were treated with a differentiation cocktail that induced an adipocyte-like phenotype with large NL droplets (NL+PL+ cells), or treated with propranolol to induce accumulation of PL (PL+ cells). The three cell types were analysed for morphology, global protein expression, lipid content, cellular volume and lysosomal appearance. The cells were exposed to 23 drugs with diverse physicochemical properties, e.g., with regard to lipophilicity, charge, molecular weight and polar surface area. The cellular binding (fu,cell) and the total accumulation ratio (Kp) were measured and used to obtain the intracellular bioavailability (Fic)—defined as the unbound fraction of the drug concentration added to the cells that is available for intracellular target interaction. The NL+PL+ cells had an adipocyte-like morphology, whereas the PL+ were not visually distinguishable from WT cells. Analysis of the global proteome combined with pathway analysis supported the morphological appearances and confirmed the adipocyte-like phenotype of NL+PL+ cells and the normal morphology of PL+ cells. Fic was significantly altered in both treated cell types as compared to WT. A strong negative correlation between fu,cell and PL content in the cell homogenates was observed, whereas the increased NL content in the NL+PL+ cells did not increase binding further. The importance of PLs for drug binding was confirmed by affinities to beads coated with purified PLs. The NL+PL+ cells lacked acidic subcellular compartments (i.e., endo-lysosomal space), which further influenced the subcellular distribution of cationic drugs. In conclusion, our results suggest that PL content, but not NL content, is a major determinant of drug binding in cells, and that PL beads may constitute a simple alternative to more cumbersome cell distribution studies.