The Role of Lipid Domains in Bacterial Cell Processes

Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.     

Validated Growth Rate-Dependent Regulation of Lipid Metabolism in Yarrowia lipolytica

Given the strong potential of Yarrowia lipolytica to produce lipids for use as renewable fuels and oleochemicals, it is important to gain in-depth understanding of the molecular mechanism underlying its lipid accumulation. As cellular growth rate affects biomass lipid content, we performed a comparative proteomic analysis of Y. lipolytica grown in nitrogen-limited chemostat cultures at different dilution rates. After confirming the correlation between growth rate and lipid accumulation, we were able to identify various cellular functions and biological mechanisms involved in oleaginousness. Inspection of significantly up- and downregulated proteins revealed nonintuitive processes associated with lipid accumulation in this yeast. This included proteins related to endoplasmic reticulum (ER) stress, ER–plasma membrane tether proteins, and arginase. Genetic engineering of selected targets validated that some genes indeed affected lipid accumulation. They were able to increase lipid content and were complementary to other genetic engineering strategies to optimize lipid yield.     

The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites

The conserved VPS13 proteins constitute a new family of lipid transporters at membrane contact sites. These large proteins are suspected to bridge membranes and form a direct channel for lipid transport between organelles. Mutations in the 4 human homologs (VPS13A–D) are associated with a number of neurological disorders, but little is known about their precise functions or the relevant contact sites affected in disease. In contrast, yeast has a single Vps13 protein which is recruited to multiple organelles and contact sites. The yeast model system has proved useful for studying the function of Vps13 at different organelles and identifying the localization determinants responsible for its membrane targeting. In this review we describe recent advances in our understanding of VPS13 proteins with a focus on yeast research.     

Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-phosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.     

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q₁₀) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q₁₀. Q₁₀ is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q₁₀. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q₁₀ is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q₁₀, as well as lipid composition, on enzyme binding.     

Characterization of a Cyclic Nucleotide‐Activated K+ Channel and its Lipid Environment by Using Solid‐State NMR Spectroscopy

Voltage‐gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain‐related mobility, structural characterization has proved challenging. We analyzed high‐resolution solid‐state NMR data on different isotope‐labeled protein constructs of a bacterial cyclic nucleotide‐activated K⁺ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage‐sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope‐labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane‐embedded proteins, even in the presence of local disorder.     

The Role of Short-Chain Conjugated Poly-(R)-3-Hydroxybutyrate (cPHB) in Protein Folding

Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli.     

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.     

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane‐associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH₂) into thromboxane A₂ (TXA₂), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER‐bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug‐metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small‐molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.     

Differential Membrane Lipid Profiles and Vibrational Spectra of Three Edaphic Algae and One Cyanobacterium

The membrane glycerolipids of four phototrophs that were isolated from an edaphic assemblage were determined by UPLC–MS after cultivation in a laboratory growth chamber. Identification was carried out by 18S and 16S rDNA sequencing. The algal species were Klebsormidium flaccidum (Charophyta), Oocystis sp. (Chlorophyta), and Haslea spicula (Bacillariophyta), and the cyanobacterium was Microcoleus vaginatus (Cyanobacteria). The glycerolipid profile of Oocystis sp. was dominated by monogalactosyldiacylglycerol (MGDG) species, with MGDG(18:3/16:4) accounting for 68.6%, whereas MGDG(18:3/16:3) was the most abundant glycerolipid in K. flaccidum (50.1%). A ratio of digalactosyldiacylglycerol (DGDG) species to MGDG species (DGDG/MGDG) was shown to be higher in K. flaccidum (0.26) than in Oocystis sp. (0.14). This ratio increased under high light (HL) as compared to low light (LL) in all the organisms, with its highest value being shown in cyanobacterium (0.38–0.58, LL−HL). High contents of eicosapentaenoic acid (EPA, C20:5) and hexadecenoic acid were observed in the glycerolipids of H. spicula. Similar Fourier transform infrared (FTIR) and Raman spectra were found for K. flaccidum and Oocystis sp. Specific bands at 1629.06 and 1582.78 cm⁻¹ were shown by M. vaginatus in the Raman spectra. Conversely, specific bands in the FTIR spectrum were observed for H. spicula at 1143 and 1744 cm⁻¹. The results of this study point out differences in the membrane lipid composition between species, which likely reflects their different morphology and evolutionary patterns.     

Recombinant Bovine Growth Hormone-Induced Metabolic Remodelling Enhances Growth of Gilthead Sea-Bream (Sparus aurata): Insights from Stable Isotopes Composition and Proteomics

Growth hormone and insulin-like growth factors (GH/IGF axis) regulate somatic growth in mammals and fish, although their action on metabolism is not fully understood in the latter. An intraperitoneal injection of extended-release recombinant bovine growth hormone (rbGH, Posilac^®) was used in gilthead sea bream fingerlings and juveniles to analyse the metabolic response of liver and red and white muscles by enzymatic, isotopic and proteomic analyses. GH-induced lipolysis and glycogenolysis were reflected in liver composition, and metabolic and redox enzymes reported higher lipid use and lower protein oxidation. In white and red muscle reserves, rBGH increased glycogen while reducing lipid. The isotopic analysis of muscles showed a decrease in the recycling of proteins and a greater recycling of lipids and glycogen in the rBGH groups, which favoured a protein sparing effect. The protein synthesis capacity (RNA/protein) of white muscle increased, while cytochrome-c-oxidase (COX) protein expression decreased in rBGH group. Proteomic analysis of white muscle revealed only downregulation of 8 proteins, related to carbohydrate metabolic processes. The global results corroborated that GH acted by saving dietary proteins for muscle growth mainly by promoting the use of lipids as energy in the muscles of the gilthead sea bream. There was a fuel switch from carbohydrates to lipids with compensatory changes in antioxidant pathways that overall resulted in enhanced somatic growth.     

Lipid Self-Assemblies under the Atomic Force Microscope

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.     

Fatty Acid Elongation in Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma

Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.     

Insight into the Impact of Oxidative Stress on the Barrier Properties of Lipid Bilayer Models

As a new field of oxidative stress-based therapy, cold physical plasma is a promising tool for several biomedical applications due to its potential to create a broad diversity of reactive oxygen and nitrogen species (RONS). Although proposed, the impact of plasma-derived RONS on the cell membrane lipids and properties is not fully understood. For this purpose, the changes in the lipid bilayer functionality under oxidative stress generated by an argon plasma jet (kINPen) were investigated by electrochemical techniques. In addition, liquid chromatography-tandem mass spectrometry was employed to analyze the plasma-induced modifications on the model lipids. Various asymmetric bilayers mimicking the structure and properties of the erythrocyte cell membrane were transferred onto a gold electrode surface by Langmuir-Blodgett/Langmuir-Schaefer deposition techniques. A strong impact of cholesterol on membrane permeabilization by plasma-derived species was revealed. Moreover, the maintenance of the barrier properties is influenced by the chemical composition of the head group. Mainly the head group size and its hydrogen bonding capacities are relevant, and phosphatidylcholines are significantly more susceptible than phosphatidylserines and other lipid classes, underlining the high relevance of this lipid class in membrane dynamics and cell physiology.     

Lipid Estimation of Surfactant-Extracted Microalgae Oil Using Nile Red

Nile red staining has been used as a lipid quantification technique in many microalgae growth and oil accumulation studies. However, its application in lysed microalgae cells is limited. Therefore, this study focused on lysed microalgae cells and utilized the Nile red staining technique to evaluate oil content and extraction. This study aims to provide a rapid and high-throughput alternative method particularly in the microalgae extraction screening process. Potential interferences such as chlorophyll, β-carotene, soluble protein, and phospholipids were evaluated. The hydrophobic Nile red dye was found to quench in water, therefore the fluorometric measurement has to be completed immediately or within 5 min of dye addition. The fluorescence intensity was also found to be Nile red concentration dependent. The optimum Nile red concentration of 656 ppb was used throughout the study. In microalgae samples containing chlorophyll and carotenoids (such as Nannochloropsis sp.), Nile red fluorescence intensity was significantly reduced in comparison to non-chlorophyll microalgae (Schizochytrium limacinum). Soluble proteins from defatted microalgae did not fluoresce significantly relative to lipids, therefore did not interfere with the method to a high degree. Comparing the optimized Nile red staining method with the gravimetric lipid quantification method, a good linear correlation was found in all three materials tested (soybean oil, Nannochloropsis sp., and Schizochytrium limacinum).     

Structural Diversity of PDZ–Lipid Interactions

PDZ domains are globular protein modules that are over‐and‐above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ–phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin‐1, syntenin‐2, PTP‐Bas, PAR‐3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ–lipid interactions and the consequences for peptide interactions.     

Certain,but Not All,Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLM_PLFE can be attributed to strong PLFE–PLFE and PLFE–substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLM_PLFE to values recorded in BLM_Sa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLM_PLFE and exhibit channel activity, but not in BLM_Sa-MV. Additionally, the opening/closing of the MthK in BLM_PLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLM_PLFE and BLM_Sa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.     

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.