Imaging of Neutral Lipids and Neutral Lipid Associated Proteins

Abstract Background Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite’s survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization. Results Here, we show that one of the parasite phospholipase (P. falciparum lysophospholipase, PfLPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the PfLPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. PfLPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of PfLPL1 to rescue the parasite growth as well as restore hemozoin levels. Conclusions We found that the transient downregulation of PfLPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.

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The genetics of neutral lipid biosynthesis: an evolutionary perspective

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90898.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. E19-E27 ◽

Cited By ~ 26

Author(s):

Aaron R. Turkish ◽

Stephen L. Sturley

Keyword(s):

Neutral Lipid ◽

Metabolic Pathways ◽

Fatty Liver Disease ◽

Molecular Mechanisms ◽

Neutral Lipids ◽

Lipid Synthesis ◽

Therapeutic Interventions ◽

Wax Ester ◽

Evolutionary Perspective ◽

Nonalcoholic Fatty Liver

The storage of fatty acids and fatty alcohols in the form of neutral lipids such as triacylglycerol (TAG), cholesteryl ester (CE), and wax ester (WE) serves to provide reservoirs for membrane formation and maintenance, lipoprotein trafficking, lipid detoxification, evaporation barriers, and fuel in times of stress or nutrient deprivation. This ancient process likely originated in actinomycetes and has persisted in eukaryotes, albeit by different molecular mechanisms. A surfeit of neutral lipids is strongly, perhaps causally, related to several human diseases such as diabetes mellitus, obesity, atherosclerosis and nonalcoholic fatty liver disease. Therefore, understanding the metabolic pathways of neutral lipid synthesis and the roles of the enzymes involved may facilitate the development of new therapeutic interventions for these syndromes.

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The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

The Journal of Cell Biology ◽

10.1083/jcb.201010111 ◽

2011 ◽

Vol 192 (6) ◽

pp. 1043-1055 ◽

Cited By ~ 167

Author(s):

Oludotun Adeyo ◽

Patrick J. Horn ◽

SungKyung Lee ◽

Derk D. Binns ◽

Anita Chandrahas ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Neutral Lipid ◽

Strong Evidence ◽

Lipid Droplets ◽

Neutral Lipids ◽

Glucose Medium ◽

Wild Type ◽

Lipid Levels ◽

Total Neutral Lipid ◽

A Site

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.

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Pairing and aggregation of Amblosoma suwaense (Trematoda: Brachylaimidae) metacercariae in vitro and partial characterization of lipids involved in chemo-attraction

Parasitology ◽

10.1017/s003118200005695x ◽

1981 ◽

Vol 82 (2) ◽

pp. 225-229 ◽

Cited By ~ 15

Author(s):

B. Fried ◽

G. A. Robinson

Keyword(s):

Thin Layer ◽

Neutral Lipid ◽

Sterol Ester ◽

Neutral Lipids ◽

Sterol Esters ◽

Free Sterols ◽

Lipid Fractions ◽

Oil Red O

SUMMARYHistochemical and thin-layer chromatographic (t.l.c.) analyses were made on neutral lipids in the free (unencysted) metacercariae of Amblosoma suwaense (Brachylaimidae). As determined by t.l.c. the major neutral lipid fractions in metacercariae removed directly from Campeloma decisum snails were free sterols and sterol esters. Metacercariae incubated for 1 h at 37±1° C in sterile Locke's solution released mainly sterol esters and a lesser amount of free sterols into the medium. As determined by Oil Red O (ORO) staining, metacercariae accumulated neutral lipid in the intestinal caeca during incubation and the excretory system was ORO negative. Behavioural studies showed that metacereariae paired and aggregated in vitro and were attracted to lipophilic but not to hydrophilic worm products. Following t.l.c. preparative analysis it was demonstrated that metacercariae were attracted to sterol ester worm products but not to free sterol products.

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Neutral lipids regulate amphipathic helix affinity for model lipid droplets

The Journal of Cell Biology ◽

10.1083/jcb.201907099 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 8

Author(s):

Aymeric Chorlay ◽

Abdou Rachid Thiam

Keyword(s):

Neutral Lipid ◽

Lipid Membranes ◽

Lipid Droplets ◽

Neutral Lipids ◽

Aqueous Environment ◽

Major Step ◽

Amphipathic Helices ◽

Binding Preference ◽

Hydrophobic Nature ◽

Binding Level

Cellular lipid droplets (LDs) have a neutral lipid core shielded from the aqueous environment by a phospholipid monolayer containing proteins. These proteins define the biological functions of LDs, and most of them bear amphipathic helices (AH), which can selectively target to LDs, or to LD subsets. How such binding preference happens remains poorly understood. Here, we found that artificial LDs made of different neutral lipids but presenting equal phospholipid packing densities differentially recruit AHs. Varying the phospholipid density shifts the binding levels, but the differential recruitment is unchanged. We found that the binding level of AHs is defined by their interaction preference with neutral lipids and ability to decrease surface tension. The phospholipid packing level regulates mainly the amount of neutral lipid accessible. Therefore, it is the hydrophobic nature of the phospholipid packing voids that controls the binding level of AHs. Our data bring us a major step closer to understanding the binding selectivity of AHs to lipid membranes.

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Protein Quality Control and Lipid Droplet Metabolism

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-031320-101827 ◽

2020 ◽

Vol 36 (1) ◽

pp. 115-139 ◽

Cited By ~ 1

Author(s):

Melissa A. Roberts ◽

James A. Olzmann

Keyword(s):

Quality Control ◽

Protein Quality ◽

Metabolic Diseases ◽

Neutral Lipids ◽

Protein Quality Control ◽

Cellular Protein ◽

Nutrient Sensing ◽

Nonalcoholic Fatty Liver ◽

Triacylglycerol Synthesis ◽

Associated Proteins

Lipid droplets (LDs) are endoplasmic reticulum–derived organelles that consist of a core of neutral lipids encircled by a phospholipid monolayer decorated with proteins. As hubs of cellular lipid and energy metabolism, LDs are inherently involved in the etiology of prevalent metabolic diseases such as obesity and nonalcoholic fatty liver disease. The functions of LDs are regulated by a unique set of associated proteins, the LD proteome, which includes integral membrane and peripheral proteins. These proteins control key activities of LDs such as triacylglycerol synthesis and breakdown, nutrient sensing and signal integration, and interactions with other organelles. Here we review the mechanisms that regulate the composition of the LD proteome, such as pathways that mediate selective and bulk LD protein degradation and potential connections between LDs and cellular protein quality control.

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Starvation-induced lipid reserve depletion in Pratylenchus brachyurus leads to decreased infectivity in maize roots

Nematology ◽

10.1163/15685411-bja10032 ◽

2020 ◽

Vol 23 (1) ◽

pp. 103-111

Author(s):

Paula S. Alves ◽

Willian C. Terra ◽

Giselle B. Pinto ◽

Paulo V.M. Pacheco ◽

Bárbhara J.R. Fatobene ◽

...

Keyword(s):

Neutral Lipid ◽

Lipid Content ◽

Consumption Rate ◽

Neutral Lipids ◽

Control Strategies ◽

Maize Roots ◽

Neutral Lipid Content ◽

Pratylenchus Brachyurus ◽

Waiting Periods ◽

Oil Red O

Summary Nematode body neutral lipid (triacylglycerol) content has been related to infectivity and has direct implications in control strategies. In this study, Pratylenchus brachyurus populations were split into two groups: i) freshly hatched second-stage juveniles (J2) containing lipids stored during embryogenesis; ii) third- and fourth-stage juveniles (J3/J4) plus females that replenished their lipid reserves by feeding on maize (Zea mays) roots. These groups were subjected to starvation to study their lipid consumption dynamics by staining with Oil Red O, which binds specifically to neutral lipids. Before starvation, freshly hatched J2 had 27% of their body area stained, whereas J3/J4 and females had 75%. Freshly hatched J2 starved for 28 days at 25°C in water lost 63.8% of the original neutral lipid content, which caused a reduction of 91% of infectivity in maize roots. By contrast, J3/J4 and females exposed to the same conditions lost 56.7% of the original neutral lipid content, which resulted in less than 50% reduction in infectivity. During the period of food deprivation, J2 had a mean daily neutral lipid consumption rate of 0.63% and the other infectious stages (J3/J4 and females) had a mean daily neutral lipid consumption rate of 1.46% per day. This study adds information on the dynamics of lipid utilisation that supports the use of longer waiting periods for planting crops after fallow in soils infested with P. brachyurus as compared to Meloidogyne spp.-infested soils.

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The effect of long term storage on the lipid reserves and fatty acid composition of cysts and hatched juveniles of Globodera rostochiensis and G. pallida

Journal of Helminthology ◽

10.1017/s0022149x0001631x ◽

1998 ◽

Vol 72 (2) ◽

pp. 133-141 ◽

Cited By ~ 2

Author(s):

R.A. Holz ◽

D.J. Wright ◽

R.N. Perry

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Free Fatty Acid ◽

Neutral Lipid ◽

Lipid Content ◽

Neutral Lipids ◽

Lipid Fraction ◽

Neutral Lipid Fraction ◽

Fatty Acid Profiles ◽

Acidic Phospholipids

AbstractThe lipid composition of three batches of single generation cysts of Globodera rostochiensis, stored dry at 4°C for 1,7 and 13 years, comprised 81%, 74% and 53% neutral lipids, 14%, 18% and 27% non-acidic phospholipids and 5%, 8% and 20% free fatty acids, respectively. Lipids in eggs from two batches of G. pallida cysts, stored for 3 and 7 years, comprised 80% and 67% neutral lipids, 15% and 23% non-acidic phospholipids and 5% and 10% free fatty acids, respectively. All batches contained the same fatty acids which were dominated by C18:l, C20:l and C20:4. The fatty acid profiles of hatched J2 of G. rostochiensis from two batches, stored for 1 and 9 years, differed only in their free fatty acid fractions. Thus, while it is not possible to determine the age of cysts by their fatty acid profile, it may be possible to use the relative amounts of the main lipid classes as an indicator of age. Four batches of hatched J2 of G. pallida were investigated, with sample A hatched during the second week in potato root diffusate, B during week 3, C during week 4 and D during weeks 5 and 6 and stored for 3.5 days (on average) after hatching. Total lipid content was 27.2%, 31.5%, 18.5% and 6.3% of the dry weight for A, B, C and D, respectively. In the neutral lipid fraction of D an increase in C18:l and to a lesser extent C18:2 was observed. In the free fatty acid fraction of sample D, the percentages of C18:l, C18:2 and C18:3 were greater but the percentages of C20:3 and C20:4 were smaller compared with sample C. Fresh early hatched J2 of G. rostochiensis were compared with later hatched and stored (for 13 days on average) individuals for their lipid content and fatty acid composition. The lipid content was 26.1% and 11.4% in fresh and stored J2, respectively. Total lipid consisted of 77% and 70% neutral lipid, 18% and 26% non-acidic phospholipid and 6% and 4% free fatty acid in fresh and stored J2, respectively. In the neutral lipid fraction of stored J2 C18:l, C16:0 and C18:0 increased, whereas C20:4, C20:l and C20:3 decreased. Therefore, both neutral lipid and free fatty acid fractions showed changes in their fatty acid profiles after long delayed hatching and/or storage in both PCN species.

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The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

Biochemical Journal ◽

10.1042/bj20060986 ◽

2007 ◽

Vol 402 (1) ◽

pp. 197-204 ◽

Cited By ~ 142

Author(s):

John M. Pisciotta ◽

Isabelle Coppens ◽

Abhai K. Tripathi ◽

Peter F. Scholl ◽

Joel Shuman ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Neutral Lipid ◽

Membrane Lipids ◽

Neutral Lipids ◽

Close Association ◽

Subcellular Fractionation ◽

Digestive Vacuole ◽

Lipid Mixture ◽

Intracellular Mechanism

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

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Married at Birth: Regulation of Cellular Fat Metabolism by ER–Lipid Droplet Crosstalk

Contact ◽

10.1177/2515256420934671 ◽

2020 ◽

Vol 3 ◽

pp. 251525642093467

Author(s):

Zhe Cao ◽

Ho Yi Mak

Keyword(s):

Neutral Lipid ◽

Neutral Lipids ◽

Close Contact ◽

Low Density Lipoprotein ◽

Lipid Synthesis ◽

Density Lipoprotein ◽

Very Low Density Lipoprotein ◽

Low Density ◽

Acid Modification

The endoplasmic reticulum (ER) is a hub that coordinates neutral lipid synthesis, storage, and export. To fulfill this role, the ER maintains close contact with lipid droplets (LDs), which are evolutionarily conserved organelles for the storage of neutral lipids. Decades of biochemical evidence points to fatty acid modification and neutral lipid synthesis in the ER. Conceptually, lipid export into extracellular space or lipid retention intracellularly require the subsequent remodeling of an ER membrane leaflet that faces the lumen or cytoplasm, respectively. This is because LDs and very-low-density lipoprotein particles are all structures surrounded by a phospholipid monolayer. While the export of neutral lipids via very-low-density lipoprotein production is well characterized, there has been increasing interest in the mechanisms that underlie neutral lipid retention in LDs. Structural determination, in vitro reconstitution, and localization of key proteins by advanced microscopy techniques collectively enrich models of ER-LD engagement. In this review, we consider current concepts on how LDs emerge from the ER in a directional manner and how sustained ER-LD contacts support LD expansion.

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