Interactions of Lipid Droplets with the Intracellular Transport Machinery

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.     

Alpha-Lipoic Acid Attenuates Apoptosis and Ferroptosis in Cisplatin-Induced Ototoxicity via the Reduction of Intracellular Lipid Droplets

Alpha-lipoic acid (α-LA) is a potent antioxidant that can prevent apoptosis associated with cisplatin-induced ototoxicity through ROS. Ferroptosis is defined as an iron-dependent cell death pathway that has recently been highlighted and is associated with the accumulation of intracellular lipid droplets (LDs) due to an inflammatory process. Herein, we investigated the impact of α-LA on ferroptosis and analyzed the characteristics of LDs in auditory hair cells treated with cisplatin using high-resolution 3D quantitative-phase imaging with reconstruction of the refractive index (RI) distribution. HEI-OC1 cells were treated with 500 μM α-LA for 24 h and then with 15 μM cisplatin for 48 h. With 3D optical diffraction tomography (3D-ODT), the RI values of treated cells were analyzed. Regions with high RI values were considered to be LDs and labelled to measure the count, mass, and volume of LDs. The expression of LC3-B, P62, GPX4, 4-hydroxynonenal (4-HNE), and xCT was evaluated by Western blotting. HEI-OC1 cells damaged by cisplatin showed lipid peroxidation, depletion of xCT, and abnormal accumulation of 4-HNE. Additionally, the count, mass, and volume of LDs increased in the cells. Cells treated with α-LA had inhibited expression of 4-HNE, while the expression of xCT and GPX4 was recovered, which restored LDs to a level that was similar to that in the control group. Our research on LDs with 3D-ODT offers biological evidence of ferroptosis and provides insights on additional approaches for investigating the molecular pathways.     

In Vivo Two-Photon Imaging Analysis of Dynamic Degradation of Hepatic Lipid Droplets in MS-275-Treated Mouse Liver

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.     

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca²⁺ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca²⁺ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to G_o/q or G_s proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca²⁺ from intracellular stores was much faster than cAMP-mediated Ca²⁺ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.     

Development of Polyamine-Substituted Triphenylamine Ligands with High Affinity and Selectivity for G-Quadruplex DNA

Currently, significant efforts are devoted to designing small molecules able to bind selectively to guanine quadruplexes (G4s). These noncanonical DNA structures are implicated in various important biological processes and have been identified as potential targets for drug development. Previously, a series of triphenylamine (TPA)-based compounds, including macrocyclic polyamines, that displayed high affinity towards G4 DNA were reported. Following this initial work, herein a series of second-generation compounds, in which the central TPA has been functionalised with flexible and adaptive linear polyamines, are presented with the aim of maximising the selectivity towards G4 DNA. The acid–base properties of the new derivatives have been studied by means of potentiometric titrations, UV/Vis and fluorescence emission spectroscopy. The interaction with G4s and duplex DNA has been explored by using FRET melting assays, fluorescence spectroscopy and circular dichroism. Compared with previous TPA derivatives with macrocyclic substituents, the new ligands reported herein retain the G4 affinity, but display two orders of magnitude higher selectivity for G4 versus duplex DNA; this is most likely due to the ability of the linear substituents to embrace the G4 structure.     

Novel Antitumor L‐Arabinose Derivative of Indolocarbazole with High Affinity to DNA

Novel indolocarbazole derivative 12‐(α‐L ‐arabinopyranosyl)indolo[2,3‐α]pyrrolo[3,4‐c]carbazole‐5,7‐dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)₂₀, d(GC)₂₀ and calf thymus DNA (binding constants (1.6×10⁶) M ^?1≤K_a≤(3.3×10⁶) M ^?1). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C≤1 μM ) were identical to the concentrations that triggered p53‐dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I‐mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.     

PEGylation Effects on the Interaction of Sphingomyelin Nanoemulsions with Serum Albumin: A Thermodynamic Investigation

The recent focus in the development of novel nanosystems for biomedical applications lays firmly on their interactions with biomolecules. Thermodynamic parameters driving the interaction between nanoparticles and proteins provide insights into complex processes at bio/nanointerface. The present work aims to investigate the binding mechanisms and the dominant contributions that determine the adsorption processes during the interactions of a model protein, that is, bovine serum albumin, with a new type of drug delivery systems, Vitamin E/sphingomyelin nanoemulsions, plain and coated with polyethylene glycol, and d -ɑ-tocopheryl polyethylene glycol succinate. The binding parameters (binding constant, binding stoichiometry, enthalpy, Gibbs energy, and entropy changes of binding) are evaluated by the isothermal titration calorimetry with a MicroCaliTC200 equipment. The effect of nanoemulsions on the protein stability is examined by measuring the thermodynamic parameters for the protein's unfolding (heat capacity; enthalpy, entropy, and free energy changes) with a NanoDSC (TA Instrument) apparatus. The thermodynamic profile shows for all compositions an entropy-driven interaction dominated by hydrophobic forces due to the rearrangements/displacement of the surrounding water molecules, while maintaining the native conformation of the protein. All the information acquired by thermodynamic approach may significantly enhance the knowledge with special focus on PEGylated nanoemulsions used for biomedical applications.     

Live Imaging and Quantitation of Lipid Droplets and Mitochondrial Membrane Potential Changes with Aggregation-Induced Emission Luminogens in an in Vitro Model of Liver Steatosis

High specificity, low background, good biocompatibility and photostability are common properties of aggregation-induced emission luminogens (AIEgens). In this study, an AIEgen FAS was used in live HepG2 cells, an in vitro model of liver steatosis, to quantify lipid droplet number and size instead of the traditional method of only measuring fluorescence intensity emitted from fluorescence dye stained in lipid droplet. In parallel, another AIEgen, TPE-Ph-In, was used to perform continuous monitoring and quantitation of mitochondrial membrane potential in the same batch of live HepG2 cells. The data show a significant increase in lipid droplet numbers after 24 h treatment by amiodarone and a significant increase in both lipid droplet numbers and size after 48 h amiodarone treatment. Moreover, the data suggest a significant increase in mitochondria membrane potential in cells treated with amiodarone for 24 and 48 h, with restoration to pre-treatment level 24 h after removal of the amiodarone. Further investigation is needed to fully understand the underlying mechanism.     

Loss of swiss cheese in Neurons Contributes to Neurodegeneration with Mitochondria Abnormalities,Reactive Oxygen Species Acceleration and Accumulation of Lipid Droplets in Drosophila Brain

Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. In this study, we used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)—the evolutionarily conserved ortholog of human NTE/PNPLA6—in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. Our results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. We propose that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.     

无水体系中环氧氯丙烷活化琼脂糖凝胶制备高载量固定化金属亲和层析介质

在以二甲基亚砜为溶剂的无水体系中,利用环氧氯丙烷对琼脂糖凝胶Sepharose 6 Fast Flow进行活化,并偶联亚氨基二乙酸和Cu2+制备了固定化金属亲和层析介质. 结果表明,该体系中环氧氯丙烷对琼脂糖凝胶的活化效率大幅度提高,在40%(j)环氧氯丙烷、0.02 g/mL NaOH及50℃、反应时间4 h的优化条件下,环氧基活化密度最高达165 mmol/mL,较目前报道的最高值提高50%以上. 最终所制介质的Cu2+螯合密度为128.3 mmol/mL,对BSA的平衡吸附容量达2.05 mmol/L. 以0.5 mol/L咪唑为洗脱剂,被吸附的BSA洗脱率可达90%以上.     

Nerve Growth Factor Peptides Bind Copper(II) with High Affinity: A Thermodynamic Approach to Unveil Overlooked Neurotrophin Roles

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aβ) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1–15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aβ and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aβ, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer’s disease onset.     

Multi-Omic Profiling of Macrophages Treated with Phospholipids Containing Omega-3 and Omega-6 Fatty Acids Reveals Complex Immunomodulatory Adaptations at Protein,Lipid and Metabolic Levels

In recent years, several studies have demonstrated that polyunsaturated fatty acids have strong immunomodulatory properties, altering several functions of macrophages. In the present work, we sought to provide a multi-omic approach combining the analysis of the lipidome, the proteome, and the metabolome of RAW 264.7 macrophages supplemented with phospholipids containing omega-3 (PC 18:0/22:6; ω3-PC) or omega-6 (PC 18:0/20:4; ω6-PC) fatty acids, alone and in the presence of lipopolysaccharide (LPS). Supplementation of macrophages with ω3 and ω6 phospholipids plus LPS produced a significant reprogramming of the proteome of macrophages and amplified the immune response; it also promoted the expression of anti-inflammatory proteins (e.g., pleckstrin). Supplementation with the ω3-PC and ω6-PC induced significant changes in the lipidome, with a marked increase in lipid species linked to the inflammatory response, attributed to several pro-inflammatory signalling pathways (e.g., LPCs) but also to the pro-resolving effect of inflammation (e.g., PIs). Finally, the metabolomic analysis demonstrated that supplementation with ω3-PC and ω6-PC induced the expression of several metabolites with a pronounced inflammatory and anti-inflammatory effect (e.g., succinate). Overall, our data show that supplementation of macrophages with ω3-PC and ω6-PC effectively modulates the lipidome, proteome, and metabolome of these immune cells, affecting several metabolic pathways involved in the immune response that are triggered by inflammation.     

Crosslinked-Chelating Porous Sheet with High Dynamic Binding Capacity of Metal Ions

A crosslinked chelating porous sheet was prepared by cografting ethylene glycol dimethacrylate (EGDMA) with glycidyl methacrylate onto an electron-beam-irradiated porous polyethylene sheet, followed by the introduction of an iminodiacetate group. At a molar percentage of EGDMA of 1.0 mol%, the sheet exhibited a maximum dynamic binding capacity for copper ions of 0.93 mmol/g, while the equilibrium binding capacity remained the same (1.2 mmol/g) as that of a non-crosslinked chelating porous sheet. The crosslinking of the grafted chain causes copper ions to lower their diffusion rate along the sheet thickness driven by the gradient of the amount of copper ions adsorbed.     

高性能浇注型聚氨酯弹性体的动态性能研究

通过高性能浇注型聚氨酯弹性体的合成，研究了二异氰酸酯结构、多元醇结构对浇注型聚氨酯弹性体动态性能的影响，结果表明，异氰酸酯基团含量增加，聚氨酯弹性体的硬度、撕裂强度和耐热性提高。     

高压均质法制备载有维甲酸的纳米结构脂质载体

采用高压均质法制备了载有维甲酸(RA)的纳米结构脂质载体(Nanostructured Lipid Carriers, NLC),并用透射电镜(TEM)、粒度分析仪、 电位仪和高效液相色谱仪(HPLC)对其进行了表征. 结果表明,随高压均质循环次数的增加,NLC的均粒径从120 nm减小到10 nm,同时单分散性变差;NLC的微观形貌呈球形,表明其未结晶具有较好的载药能力;NLC将维甲酸浓度从20 g/mL(溶解于硅油)提高到56.26 g/mL,并能提高其光稳定性;NLC体系的稳定性良好, 电位高达( 30.9 0.6) mV,于4℃以10000 r/min冷冻离心120 min或避光保存6个月后粒径分布仍然基本不变,且可冷冻干燥( 40℃, 0.01 Pa)后长期保存.     

高产油绿球藻GIEC-38转录本和基因表达谱分析

通过对天然条件下生长好且含油高的绿球藻(Chlorococcum sp.) GIEC-38细胞内转录本的测定,发现:在获得的74 605条转录本、65 984个基因中共得到344条代谢途径,其中核糖体、蛋白质、核苷酸、核糖核酸、碳固定、光合作用以及脂代谢等通路都非常活跃。通过缺N培养发现GIEC-38含油脂可达50%以上,通过对比细胞表达谱发现相比于原始藻株,缺N条件下培养的GIEC-38细胞中有868个基因显著上调、1 157个基因显著下调,分布在41个生物学功能、71条代谢途径中。其中,为脂肪酸的合成提供大量原料的中间产物合成酶,以及脂类代谢相关的几个通路中关键酶的基因,表达都有明显上调,促使细胞脂类的合成,提高了细胞的油脂产量。     

Distinctive Lipid Composition of the Copepod Limnocalanus macrurus with a High Abundance of Polyunsaturated Fatty Acids

We studied the copepod Limnocalanus macrurus for seasonal variation in the composition of fatty acids, wax esters and sterols in large boreal lakes, where it occurs as a glacial‐relict. Vast wax ester reserves of Limnocalanus were accumulated in a period of only two months, and comprised mono‐ and polyunsaturated fatty acids (PUFA) and saturated fatty alcohols. In winter, the mobilization of wax esters was selective, and the proportion of long‐chain polyunsaturated wax esters declined first. PUFA accounted for >50 % of all fatty acids throughout the year reaching up to ca. 65 % during late summer and fall. Long‐chain PUFA 20:5n‐3 and 22:6n‐3 together comprised 17–40 % of all fatty acids. The rarely reported C24 and C26 very‐long‐chain PUFA (VLC‐PUFA) comprised 6.2 ± 3.4 % of all fatty acids in August and 2.1 ± 1.7 % in September. The VLC‐PUFA are presumably synthesized by Limnocalanus from shorter chain‐length precursors because they were not found in the potential food sources. We hypothesize that these VLC‐PUFA help Limnocalanus to maximize lipid reserves when food is abundant. Sterol content of Limnocalanus, consisting ca. 90 % of cholesterol, did not show great seasonal variation. As a lipid‐rich copepod with high abundance of PUFA, Limnocalanus is excellent quality food for fish. The VLC‐PUFA were also detected in planktivorous fish, suggesting that these compounds can be used as a trophic marker indicating feeding on Limnocalanus.