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.

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.

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.

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.

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.

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.

Architectured Therapeutic and Diagnostic Nanoplatforms for Combating SARS-CoV-2: Role of Inorganic, Organic, and Radioactive Materials.

Although extensive research is being done to combat SARS-CoV-2, we are yet far away from a robust conclusion or strategy. With an increased amount of vaccine research, nanotechnology has found its way into vaccine technology. Researchers have explored the use of various nanostructures for delivering the vaccines for enhanced efficacy. Apart from acting as delivery platforms, multiple studies have shown the application of inorganic nanoparticles in suppressing the growth as well as transmission of the virus. The present review gives a detailed description of various inorganic nanomaterials which are being explored for combating SARS-CoV-2 along with their role in suppressing the transmission of the virus either through air or by contact with inanimate surfaces. The review further discusses the use of nanoparticles for development of an antiviral coating that may decrease adhesion of SARS-CoV-2. A separate section has been included describing the role of nanostructures in biosensing and diagnosis of SARS-CoV-2. The role of nanotechnology in providing an alternative therapeutic platform along with the role of radionuclides in SARS-CoV-2 has been described briefly. Based on ongoing research and commercialization of this nanoplatform for a viral disease, the nanomaterials show the potential in therapy, biosensing, and diagnosis of SARS-CoV-2.

Black Phosphorus as Multifaceted Advanced Material Nanoplatforms for Potential Biomedical Applications.

Black phosphorus is one of the emerging members of two-dimensional (2D) materials which has recently entered the biomedical field. Its anisotropic properties and infrared bandgap have enabled researchers to discover its applicability in several fields including optoelectronics, 3D printing, bioimaging, and others. Characterization techniques such as Raman spectroscopy have revealed the structural information of Black phosphorus (BP) along with its fundamental properties, such as the behavior of its photons and electrons. The present review provides an overview of synthetic approaches and properties of BP, in addition to a detailed discussion about various types of surface modifications available for overcoming the stability-related drawbacks and for imparting targeting ability to synthesized nanoplatforms. The review further gives an overview of multiple characterization techniques such as spectroscopic, thermal, optical, and electron microscopic techniques for providing an insight into its fundamental properties. These characterization techniques are not only important for the analysis of the synthesized BP but also play a vital role in assessing the doping as well as the structural integrity of BP-based nanocomposites. The potential role of BP and BP-based nanocomposites for biomedical applications specifically, in the fields of drug delivery, 3D printing, and wound dressing, have been discussed in detail to provide an insight into the multifunctional role of BP-based nanoplatforms for the management of various diseases, including cancer therapy. The review further sheds light on the role of BP-based 2D platforms such as BP nanosheets along with BP-based 0D platforms-i.e., BP quantum dots in the field of therapy and bioimaging of cancer using techniques such as photoacoustic imaging and fluorescence imaging. Although the review inculcates the multimodal therapeutic as well as imaging role of BP, there is still research going on in this field which will help in the development of BP-based theranostic platforms not only for cancer therapy, but various other diseases.

Potential therapeutic targets for combating SARS-CoV-2: Drug repurposing, clinical trials and recent advancements.

The present pandemic of SARS-CoV-2 has been a tough task for the whole world to deal with. With the absence of specific drugs or vaccines against SARS-CoV-2, the situation is very difficult to control. Apart from the absence of specific therapies, the lack of knowledge about potential therapeutic targets and individual perception is adding to the complications. The present review describes the novel SARS-CoV-2 structure, surface proteins, asymptomatic and symptomatic transmission in addition to the genotype and phenotype of SARS-CoV-2 along with genetic strains and similarity between SARS, MERS and SARS-CoV-2. Therapeutic strategies such as inhibition of the endocytic pathway and suppressing RNA polymerase activity by metal ions, which could be quite beneficial for controlling COVID-19, are outlined. The drug repurposing for SARS-CoV-2 is discussed in detail along with therapeutic classes such as antivirals, antibiotics, and amino quinolones and their probable role in suppressing SARS-CoV-2 with reference to case studies. The ongoing clinical trials both with respect to drug repurposing and vaccines are summarized along with a brief description. The recent advancements and future perspective of ongoing research for therapy and detection of SARS-CoV-2 are provided. The review, in brief, summarizes epidemiology, therapy and the current scenario for combating SARS-CoV-2.

Lipid nanocarriers for microRNA delivery.

Non-coding RNAs (ncRNAs) like microRNAs (miRNAs) or small interference RNAs (siRNAs) with their power to selectively silence any gene of interest enable the targeting of so far 'undruggable' proteins and diseases. Such RNA molecules have gained much attention from biotech and pharmaceutical companies, which led to the first Food and Drug Administration (FDA) approved ncRNA therapeutic in 2018. However, the main barrier in clinical practice of ncRNAs is the lack of an effective delivery system that can protect the RNA molecules from nuclease degradation, deliver them to specific tissues and cell types, and release them into the cytoplasm of the targeted cells, all without inducing adverse effects. For that reason, drug delivery approaches, formulations, technologies and systems for transporting pharmacological ncRNA compounds to achieve a diagnostic or therapeutic effect in the human body are in demand. Here, we review the development of therapeutic lipid-based nanoparticles for delivery of miRNAs, one class of endogenous ncRNAs with specific regulatory functions. We outline challenges and opportunities for advanced miRNA-based therapies, and discuss the complexity associated with the delivery of functional miRNAs. Novel strategies are addressed how to deal with the most critical points in miRNA delivery, such as toxicity, specific targeting of disease sites, proper cellular uptake and endosomal escape of miRNAs. Current fields of application and various preclinical settings involving miRNA therapeutics are discussed, providing an outlook to future clinical approaches. Following the current trends and technological developments in nanomedicine exciting new delivery systems for ncRNA-based therapeutics can be expected in upcoming years.

GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A.

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Biological Activity Of miRNA-27a Using Peptide-based Drug Delivery Systems.

BACKGROUND: Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. METHODS: In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. RESULTS: The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. CONCLUSION: The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.

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.

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.

High Hydrostatic Pressure Induces a Lipid Phase Transition and Molecular Rearrangements in Low-Density Lipoprotein Nanoparticles.

Low-density lipoproteins (LDL) are natural lipid transporter in human plasma whose chemically modified forms contribute to the progression of atherosclerosis and cardiovascular diseases accounting for a vast majority of deaths in westernized civilizations. For the development of new treatment strategies, it is important to have a detailed picture of LDL nanoparticles on a molecular basis. Through the combination of X-ray and neutron small-angle scattering (SAS) techniques with high hydrostatic pressure (HHP) this study describes structural features of normolipidemic, triglyceride-rich and oxidized forms of LDL. Due to the different scattering contrasts for X-rays and neutrons, information on the effects of HHP on the internal structure determined by lipid rearrangements and changes in particle shape becomes accessible. Independent pressure and temperature variations provoke a phase transition in the lipid core domain. With increasing pressure an inter-related anisotropic deformation and flattening of the particle are induced. All LDL nanoparticles maintain their structural integrity even at 3000 bar and show a reversible response toward pressure variations. The present work depicts the complementarity of pressure and temperature as independent thermodynamic parameters and introduces HHP as a tool to study molecular assembling and interaction processes in distinct lipoprotein particles in a nondestructive manner.

Comparative analysis of nanosystems' effects on human endothelial and monocytic cell functions.

The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0-400 µg/mL) on endothelial-monocytic cell interactions in response to TNF-α was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0-100 µg/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0-400 µg/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25-50 µg/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60-70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.

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.

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.

Peptide self-assembly into lamellar phases and the formation of lipid-peptide nanostructures.

Lipids exhibit an extraordinary polymorphism in self-assembled mesophases, with lamellar phases as biologically most relevant representative. To mimic lipid lamellar phases with amphiphilic designer peptides, seven systematically varied short peptides were engineered. Indeed, four peptide candidates (V4D, V4WD, V4WD2, I4WD2) readily self-assembled into lamellae in aqueous solution: small-angle X-ray scattering patterns (SAXS) revealed ordered lamellar structures with a repeat distance of ~4-5 nm. Transmission electron microscopy (TEM) images confirmed the presence of stacked sheets. Two derivatives (V3D and V4D2) remained as loose aggregates dispersed in solution; one peptide (L4WD2) formed twisted tapes with internal lamellae and an antiparallel ß-type monomer alignment. To understand the interaction of peptides with lipids they were mixed with phosphatidylcholines. Low peptide concentrations (1.1 mM) induced the formation of a heterogeneous mixture of vesicular structures: large multilamellar vesicles (d-spacing ~6.3 nm) coexisted with oligo- or unilamellar vesicles (~50 nm in diameter) and bicelle-like structures (~45 nm length, ~18 nm width). High peptide concentrations (11 mM) led to unilamellar vesicles (ULV, diameter ~260-280 nm) with a homogeneous mixing of lipids and peptides. SAXS revealed the temperature-dependent fine structure of these ULVs: at 25 °C the bilayer is in a fully interdigitated state (headgroup-to-headgroup distance dhh ~2.9 nm), whereas at 50 °C this interdigitation opens up (dhh ~3.6 nm). Our results highlight the versatility of self-assembled peptide superstructures: subtle changes in the amino acid composition are key design elements in creating peptide- or lipid-peptide nanostructures with the same richness in morphology as known from the lipid-world.