The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

miRNAs as Regulators of the Early Local Response to Burn Injuries.

In burn injuries, risk factors and limitations to treatment success are difficult to assess clinically. However, local cellular responses are characterized by specific gene-expression patterns. MicroRNAs (miRNAs) are single-stranded, non-coding RNAs that regulate mRNA expression on a posttranscriptional level. Secreted through exosome-like vesicles (ELV), miRNAs are intracellular signalers and epigenetic regulators. To date, their role in the regulation of the early burn response remains unclear. Here, we identified 43 miRNAs as potential regulators of the early burn response through the bioinformatics analysis of an existing dataset. We used an established human ex vivo skin model of a deep partial-thickness burn to characterize ELVs and miRNAs in dermal interstitial fluid (dISF). Moreover, we identified miR-497-5p as stably downregulated in tissue and dISF in the early phase after a burn injury. MiR-218-5p and miR-212-3p were downregulated in dISF, but not in tissue. Target genes of the miRNAs were mainly upregulated in tissue post-burn. The altered levels of miRNAs in dISF of thermally injured skin mark them as new biomarker candidates for burn injuries. To our knowledge, this is the first study to report miRNAs altered in the dISF in the early phase of deep partial-thickness burns.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Artificial High Density Lipoprotein Nanoparticles in Cardiovascular Research.

Lipoproteins are endogenous nanoparticles which are the major transporter of fats and cholesterol in the human body. They play a key role in the regulatory mechanisms of cardiovascular events. Lipoproteins can be modified and manipulated to act as drug delivery systems or nanocarriers for contrast agents. In particular, high density lipoproteins (HDL), which are the smallest class of lipoproteins, can be synthetically engineered either as nascent HDL nanodiscs or spherical HDL nanoparticles. Reconstituted HDL (rHDL) particles are formed by self-assembly of various lipids and apolipoprotein AI (apo-AI). A variety of substances including drugs, nucleic acids, signal emitting molecules, or dyes can be loaded, making them efficient nanocarriers for therapeutic applications or medical diagnostics. This review provides an overview about synthesis techniques, physicochemical properties of rHDL nanoparticles, and structural determinants for rHDL function. We discuss recent developments utilizing either apo-AI or apo-AI mimetic peptides for the design of pharmaceutical rHDL formulations. Advantages, limitations, challenges, and prospects for clinical translation are evaluated with a special focus on promising strategies for the treatment and diagnosis of atherosclerosis and cardiovascular diseases.

Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

The effect of saliva on the fate of nanoparticles.

OBJECTIVES: The design of nanocarriers for local drug administration to the lining mucosa requires a sound knowledge of how nanoparticles (NPs) interact with saliva. This contact determines whether NPs agglomerate and become immobile due to size- and interaction-filtering effects or adsorb on the cell surface and are internalized by epithelial cells. The aim of this study was to examine the behavior of NPs in saliva considering physicochemical NP properties. MATERIALS AND METHODS: The salivary pore-size distribution was determined, and the viscosity of the fluid inside of the pores was studied with optical tweezers. Distinct functionalized NPs (20 and 200 nm) were dispersed in saliva and salivary buffers and characterized, and surface-bound MUC5B and MUC7 were analyzed by 1D electrophoresis and immunoblotting. NP mobility was recorded, and cellular uptake studies were performed with TR146 cells. RESULTS: The mode diameter of the salivary mesh pores is 0.7 µm with a peak width of 1.9 µm, and pores are filled with a low-viscosity fluid. The physicochemical properties of the NPs affected the colloidal stability and mobility: compared with non-functionalized particles, which did not agglomerate and showed a cellular uptake rate of 2.8%, functionalized particles were immobilized, which was correlated with agglomeration and increased binding to mucins. CONCLUSION: The present study showed that the salivary microstructure facilitates NP adsorption. However, NP size and surface functionalization determine the colloidal stability and cellular interactions. CLINICAL RELEVANCE: The sound knowledge of NP interactions with saliva enables the improvement of current treatment strategies for inflammatory oral diseases.

Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity.

Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide-membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide promoted concentration-dependent disorder on the membrane arrangement. Perturbation effects were observed on both membrane systems although most likely induced by different modes of action. These results suggest that membrane activity critically depends on the peptide's inherent ability to form highly cohesive supramolecular structures.

Antibacterial activity of silver and zinc nanoparticles against Vibrio cholerae and enterotoxic Escherichia coli.

Vibrio cholerae and enterotoxic Escherichia coli (ETEC) remain two dominant bacterial causes of severe secretory diarrhea and still a significant cause of death, especially in developing countries. In order to investigate new effective and inexpensive therapeutic approaches, we analyzed nanoparticles synthesized by a green approach using corresponding salt (silver or zinc nitrate) with aqueous extract of Caltropis procera fruit or leaves. We characterized the quantity and quality of nanoparticles by UV-visible wavelength scans and nanoparticle tracking analysis. Nanoparticles could be synthesized in reproducible yields of approximately 10(8) particles/ml with mode particles sizes of approx. 90-100 nm. Antibacterial activity against two pathogens was assessed by minimal inhibitory concentration assays and survival curves. Both pathogens exhibited similar resistance profiles with minimal inhibitory concentrations ranging between 5×10(5) and 10(7) particles/ml. Interestingly, zinc nanoparticles showed a slightly higher efficacy, but sublethal concentrations caused adverse effects and resulted in increased biofilm formation of V. cholerae. Using the expression levels of the outer membrane porin OmpT as an indicator for cAMP levels, our results suggest that zinc nanoparticles inhibit adenylyl cyclase activity. This consequently deceases the levels of this second messenger, which is a known inhibitor of biofilm formation. Finally, we demonstrated that a single oral administration of silver nanoparticles to infant mice colonized with V. cholerae or ETEC significantly reduces the colonization rates of the pathogens by 75- or 100-fold, respectively.

A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles.

Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections.

Archaeosomes for Oral Drug Delivery: From Continuous Microfluidics Production to Powdered Formulations.

Archaeosomes were manufactured from natural archaeal lipids by a microfluidics-assisted single-step production method utilizing a mixture of di- and tetraether lipids extracted from Sulfolobus acidocaldarius. The primary aim of this study was to investigate the exceptional stability of archaeosomes as potential carriers for oral drug delivery, with a focus on powdered formulations. The archaeosomes were negatively charged with a size of approximately 100 nm and a low polydispersity index. To assess their suitability for oral delivery, the archaeosomes were loaded with two model drugs: calcein, a fluorescent compound, and insulin, a peptide hormone. The archaeosomes demonstrated high stability in simulated intestinal fluids, with only 5% of the encapsulated compounds being released after 24 h, regardless of the presence of degrading enzymes or extremely acidic pH values such as those found in the stomach. In a co-culture cell model system mimicking the intestinal barrier, the archaeosomes showed strong adhesion to the cell membranes, facilitating a slow release of contents. The archaeosomes were loaded with insulin in a single-step procedure achieving an encapsulation efficiency of approximately 35%. These particles have been exposed to extreme manufacturing temperatures during freeze-drying and spray-drying processes, demonstrating remarkable resilience under these harsh conditions. The fabrication of stable dry powder formulations of archaeosomes represents a promising advancement toward the development of solid dosage forms for oral delivery of biological drugs.

Interleukin-10: an anti-inflammatory marker to target atherosclerotic lesions via PEGylated liposomes.

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF-) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Renewal of the air-water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface-active compounds.

Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air-water interface. To gain deepened understanding and improved quantification of the air-water interface effect on rhGH stability, we analyzed the protein's aggregation rate (r(agg)) at controlled specific air-water surface areas (a(G/L)) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a(G/L) (≈ 100 m(2)/m(3)), aeration gave a 40-fold higher r(agg) than stirring. The enhanced r(agg) under aeration was ascribed to faster macroscopic regeneration of free a(G/L) during aeration as compared to stirring. We also show that r(agg) was independent of the rhGH concentration in the range 0.67 - 6.7 mg/mL, and that it increased linearly dependent on the available a(G/L). The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r(agg). Foam formation was not a factor influencing r(agg). Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein-surfactant interactions that prevented rhGH from becoming attached to the air-water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air-water interface as the key limiting step. Proteins or protein aggregates released back into the bulk liquid appear to be essentially insoluble.

Lipid Nanoparticles as a Shuttle for Anti-Adipogenic miRNAs to Human Adipocytes.

Obesity and type 2 diabetes are major health burdens for which no effective therapy is available today. One treatment strategy could be to balance the metabolic functions of adipose tissue by regulating gene expressions using miRNAs. Here, we have loaded two anti-adipogenic miRNAs (miR26a and miR27a) into a pegylated lipid nanoparticle (PEG-LNP) formulation by a single-step microfluidic-assisted synthesis step. For the miRNA-loaded LNPs, the following system properties were determined: particle size, zeta potential, miRNA complexation efficiency, and cytotoxicity. We have used a human preadipocyte cell line to address the transfection efficiency and biological effects of the miRNA candidates at the gene and protein level. Our findings revealed that the upregulation of miR27a in preadipocytes inhibits adipogenesis by the downregulation of PPARγ and the reduction of lipid droplet formation. In contrast, miR26a transfection in adipocytes induced white adipocyte browning detected as the upregulation of uncoupling protein 1 (UCP1) as a marker of non-shivering thermogenesis. We conclude that the selective delivery of miRNAs by PEG-LNPs to adipocytes could offer new perspectives for the treatment of obesity and related metabolic diseases.

Targeting structural flexibility in low density lipoprotein by integrating cryo-electron microscopy and high-speed atomic force microscopy.

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein.

Targeting properties of peptide-modified radiolabeled liposomal nanoparticles.

Radiolabeled PEGylated liposomal nanoparticles (NPs) open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and monitoring treatment effects. Here we describe the characterization of liposomal NPs (liposomes and micelles) derivatized with the somatostatin analogue tyrosine-3-octreotide as a proof of concept for tumor targeting. NPs were radiolabeled with indium-111, and targeting properties were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC(50) values in the low nanomolar range. Biodistribution studies were performed in Lewis rats and compared to single-photon emission computed tomography images. Moderate tumor uptake was found in xenografted nude mice (<2.5% ID/g tissue) as compared to control. Micelles and liposomes revealed comparable pharmacokinetics and targeting properties. This study provides insight into tumor-targeting characteristics of peptide-derivatized liposomal NPs and can serve as a basis for further improvement of these constructs. FROM THE CLINICAL EDITOR: The authors investigated tumor-targeting characteristics of peptide-derivatized liposomal NPs. Similar radiolabeled PEGylated liposomal NPs open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and treatment monitoring.

Delivery of miRNAs to the adipose organ for metabolic health.

Despite the increasing prevalence of obesity and diabetes, there is no efficient treatment to combat these epidemics. The adipose organ is the main site for energy storage and plays a pivotal role in whole body lipid metabolism and energy homeostasis, including remodeling and dysfunction of adipocytes and adipose tissues in obesity and diabetes. Thus, restoring and balancing metabolic functions in the adipose organ is in demand. MiRNAs represent a novel class of drugs and drug targets, as they are heavily involved in the regulation of many cellular and metabolic processes and diseases, likewise in adipocytes. In this review, we summarize key regulatory activities of miRNAs in the adipose organ, discuss various miRNA replacement and inhibition strategies, promising delivery systems for miRNAs and reflect the future of novel miRNA-based therapeutics to target adipose tissues with the ultimate goal to combat metabolic disorders.

Can Liposomes Survive Inkjet Printing? The Effect of Jetting on Key Liposome Attributes for Drug Delivery Applications.

Purpose: Inkjet printing has the potential to enable novel personalized and tailored drug therapies based on liposome and lipid nanoparticles. However, due to the significant shear force exerted on the jetted fluids, its suitability for shear-sensitive materials such as liposomes, has not been verified. We have conducted a proof-of-concept study to examine whether the particle concentration and size distribution of placebo liposomes are affected by common inkjet/dispensing technologies. Methods: We have subjected three types of liposome-containing fluids ("inks") to two different commercial dispensing/jetting technologies, which are relevant to most drug printing approaches. The liposome jetting processes were observed in real-time using strobographic imaging techniques. The phospholipid concentrations and particle size distributions were determined before and after jetting via enzymatic colorimetric and dynamic light scattering methods, respectively. Results: Our results have shown that the jetting dynamics of the liposome inks are well predicted by the established inkjet printing regime map based on their physical properties and the jetting conditions. Importantly, although significant shear forces were confirmed during jetting, the liposome concentrations and particle size distributions in the collected samples remain largely unaffected. Conclusion: These findings, we believe, provide the essential proof-of-concept to encourage further development in this highly topical research area.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS).

Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity.

Dynamics of Apolipoprotein B-100 in Interaction with Detergent Probed by Incoherent Neutron Scattering.

Apolipoprotein B-100 (apo B-100) is the protein moiety of both low- and very-low-density lipoproteins, whose role is crucial to cholesterol and triglyceride transport. Aiming at the molecular dynamics' details of apo B-100, scarcely studied, we performed elastic and quasi-elastic incoherent neutron scattering (EINS, QENS) experiments combining different instruments and time scales. Similar to classical membrane proteins, the solubilization results in remaining detergent, here Nonidet P-40 (NP40). Therefore, we propose a framework for QENS studies of protein-detergent complexes, with the introduction of a combined model, including the experimental apo B-100/NP40 ratio. Relying on the simultaneous analysis of all QENS amplitudes, this approach is sensitive enough to separate both contributions. Its application identified two points: (i) apo B-100 slow dynamics and (ii) the acceleration of NP40 dynamics in the presence of apo B-100. Direct translation of the exposed methodology now makes the investigation of more membrane proteins by neutron spectroscopy achievable.