An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Identification of a cryptic functional apolipophorin-III domain within the Prominin-1 gene of Litopenaeus vannamei.

The Apolipophorin-III (apoLp-III) is reported as an essential protein element in lipids transport and incorporation in lepidopterans. Structurally, apoLp-III has an α-helix bundle structure composed of five α-helices. Interestingly, classic studies proposed a structural switch triggered by its interaction with lipids, where the α-helix bundle opens. Currently, the study of the apoLp-III has been limited to insects, with no homologs identified in other arthropods. By implementing a structure-based search with the Phyre2 algorithm surveying the shrimp Litopenaeus vannamei's transcriptome, we identified a putative apoLp-III in this farmed penaeid (LvApoLp-III). Unlike canonical apoLp-III, the LvApoLp-III was identified as an internal domain within the transmembrane protein Prominin-1. Structural modeling using the template-based Phyre2 and template-free AlphaFold algorithms rendered two distinct structural topologies: the α-helix bundle and a coiled-coil structure. Notably, the secondary structure composition on both models was alike, with differences in the orientation and distribution of the α-helices and hydrophobic moieties. Both models provide insights into the classical structural switch induced by lipids in apoLp-III. To corroborate structure/function inferences, we cloned the synthetic LvApoLp-III domain, overexpressed, and purified the recombinant protein. Circular dichroism measurements with the recombinant LvApoLp-III agreed with the structural models. In vitro liposome interaction demonstrated that the apoLp-III domain within the PROM1 of L.vannamei associated similarly to exchangeable apolipoproteins. Altogether, this work reports the presence of an apolipophorin-III domain in crustaceans for the first time and opens questions regarding its function and importance in lipid metabolism or the immune system.

Molecular modeling of apoE in complexes with Alzheimer's amyloid-ß fibrils from human brain suggests a structural basis for apolipoprotein co-deposition with amyloids.

Apolipoproteins co-deposit with amyloids, yet apolipoprotein-amyloid interactions are enigmatic. To understand how apoE interacts with Alzheimer's amyloid-ß (Aß) peptide in fibrillary deposits, the NMR structure of full-length human apoE was docked to four structures of patient-derived Aß1-40 and Aß1-42 fibrils determined previously using cryo-electron microscopy or solid-state NMR. Similar docking was done using the NMR structure of human apoC-III. In all complexes, conformational changes in apolipoproteins were required to expose large hydrophobic faces of their amphipathic α-helices for sub-stoichiometric binding to hydrophobic surfaces on sides or ends of fibrils. Basic residues flanking the hydrophobic helical faces in apolipoproteins interacted favorably with acidic residue ladders in some amyloid polymorphs. Molecular dynamics simulations of selected apoE-fibril complexes confirmed their stability. Amyloid binding via cryptic sites, which became available upon opening of flexibly linked apolipoprotein α-helices, resembled apolipoprotein-lipid binding. This mechanism probably extends to other apolipoprotein-amyloid interactions. Apolipoprotein binding alongside fibrils could interfere with fibril fragmentation and secondary nucleation, while binding at the fibril ends could halt amyloid elongation and dissolution in a polymorph-specific manner. The proposed mechanism is supported by extensive prior experimental evidence and helps reconcile disparate reports on apoE's role in Aß aggregation. Furthermore, apoE domain opening and direct interaction of Arg/Cys158 with amyloid potentially contributes to isoform-specific effects in Alzheimer's disease. In summary, current modeling supported by prior experimental studies suggests similar mechanisms for apolipoprotein-amyloid and apolipoprotein-lipid interactions; explains why apolipoproteins co-deposit with amyloids; and helps reconcile conflicting reports on the chaperone-like apoE action in Aß aggregation.

Insights into the C-terminal domain of apolipoprotein E from chimera studies with apolipophorin III.

Apolipoprotein E3 (apoE) is a critical cholesterol transport protein in humans and is composed of two domains: a well characterized N-terminal (NT) domain that harbors the low-density lipoprotein LDL receptor, and a less understood C-terminal (CT) domain that is the site of protein oligomerization and initiation of lipid binding. To better understand the domain structure of apoE, the CT domain was fused to apolipophorin III (apoLp-III), a single-domain, monomeric apolipoprotein of insect origin, to yield a chimeric protein, apoLp-III/CT-apoE. Recombinant apoLp-III/CT-apoE maintained an overall helical content similar to that of the parent proteins, while chemical induced unfolding studies indicated that its structural integrity was not compromised. Analysis using 1-anilinonaphthalene-8-sulfonic acid (ANS), a sensitive fluorescent indicator of exposed hydrophobic sites and protein folding, demonstrated that whereas apoLp-III provided few ANS binding sites, apoLp-III/CT-apoE harbored an abundance of ANS binding sites. Thus, this indicated tertiary structure formation in CT-apoE when part of the chimera. Size-exclusion chromatography and chemical crosslinking analysis demonstrated that while apoLp-III is monomeric, the chimeric protein formed large oligomeric complexes, similar to native apoE3. Compared to apoLp-III, the chimera showed a two-fold enhancement in phospholipid vesicle solubilization rates and a significantly improved ability to bind to lipolyzed low-density lipoprotein, preventing the onset of lipoprotein aggregation at concentrations comparable to that of parent CT-apoE. These results confirm that high lipid binding and self-association sites are located in the CT domain of apoE, and that these properties can be transferred to an unrelated apolipoprotein, demonstrating that these properties operate independently from the NT domain.

Transcriptome analysis based on dietary beta-carotene supplement reveals genes potentially involved in carotenoid metabolism in Crassostrea gigas.

Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster.

Poly (N-vinylpyrrolidone) modification mitigates plasma protein corona formation on phosphomolybdate-based nanoparticles.

Phosphomolybdate-based nanoparticles (PMo12-based NPs) have been commonly applied in nanomedicine. However, upon contact with biofluids, proteins are quickly adsorbed onto the NPs surface to form a protein corona, which induces the opsonization and facilitates the rapid clearance of the NPs by macrophage uptake. Herein, we introduce a family of structurally homologous PMo12-based NPs (CDS-PMo12@PVPx(x = 0 ~ 1) NPs) capping diverse content of zwitterionic polymer poly (N-vinylpyrrolidone) (PVP) to regulate the protein corona formation on PMo12-based NPs. The fluorescence quenching data indicate that the introduction of PVP effectively reduces the number of binding sites of proteins on PMo12-based NPs. Molecular docking simulations results show that the contact surface area and binding energy of proteins to CDS-PMo12@PVP1 NPs are smaller than the CDS-PMo12@PVP0 NPs. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further applied to analyze and quantify the compositions of the human plasma corona formation on CDS-PMo12@PVPx(x = 0 ~ 1) NPs. The number of plasma protein groups adsorption on CDS-PMo12@PVP1 NPs, compared to CDS-PMo12@PVP0 NPs, decreases from 372 to 271. In addition, 76 differentially adsorption proteins are identified between CDS-PMo12@PVP0 and CDS-PMo12@PVP1 NPs, in which apolipoprotein is up-regulated in CDS-PMo12@PVP1 NPs. The apolipoprotein adsorption onto the NPs is proposed to have dysoponic activity and enhance the circulation time of NPs. Our findings demonstrate that PVP grafting on PMo12-based NPs is a promising strategy to improve the anti-biofouling property for PMo12-based nanodrug design.

Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl2 resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl2-induced effects. Centrifugation of CaCl2-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl2 at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl2 under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca2+ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.

Identification and characterization of Staphylococcus spp. and their susceptibility to insect apolipophorin III.

Aim: This study investigated the effect of an insect antimicrobial protein, apolipophorin III (apoLp-III), against two newly isolated, identified and characterized clinical strains of Staphylococcus spp. Materials & methods: Both strains were identified by 16S rRNA sequencing and metabolic and phenotypic profiling. The antibacterial activity of apoLp-III was tested using a colony counting assay. ApoLp-III interaction with bacterial cell surface was analyzed by Fourier transform IR spectroscopy. Results:Staphylococcus epidermidis and Staphylococcus capitis were identified. ApoLp-III exerted a dose-dependent bactericidal effect on the tested strains. The differences in the Staphylococcus spp. surface components may contribute to the various sensitivities of these strains to apoLp-III. Conclusion: ApoLp-III may provide a baseline for development of antibacterial preparations against Staphylococcus spp. involved in dermatological problems.

Cyclodextrin-micellar electrokinetic chromatography of apolipoproteins on human very low-density lipoprotein.

The apolipoproteins (APOs) of human very low-density lipoprotein (VLDL) were investigated by an optimized cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method. The separation buffer consisted of 20 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-ß-cyclodextrin (CM-ß-CD) (pH 7.0). For CD-MEKC separation, a sample injection time of 12 s, a separation voltage of 15 KV, and a capillary temperature of 15°C were chosen. The optimal CD-MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R2 ) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01-0.54 mg/mL, and the concentration limits of detection (LODs) were lower than 0.02 mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD-MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.

Host Defence Cryptides from Human Apolipoproteins: Applications in Medicinal Chemistry.

Several eukaryotic proteins with defined physiological roles may act as precursors of cryptic bioactive peptides released upon protein cleavage by the host and/or bacterial proteases. Based on this, the term "cryptome" has been used to define the unique portion of the proteome encompassing proteins with the ability to generate bioactive peptides (cryptides) and proteins (crypteins) upon proteolytic cleavage. Hence, the cryptome represents a source of peptides with potential pharmacological interest. Among eukaryotic precursor proteins, human apolipoproteins play an important role, since promising bioactive peptides have been identified and characterized from apolipoproteins E, B, and A-I sequences. Human apolipoproteins derived peptides have been shown to exhibit antibacterial, anti-biofilm, antiviral, anti-inflammatory, anti-atherogenic, antioxidant, or anticancer activities in in vitro assays and, in some cases, also in in vivo experiments on animal models. The most interesting Host Defence Peptides (HDPs) identified thus far in human apolipoproteins are described here with a focus on their biological activities applicable to biomedicine. Altogether, reported evidence clearly indicates that cryptic peptides represent promising templates for the generation of new drugs and therapeutics against infectious diseases.

Apolipophorin III interaction with phosphatidylglycerol and lipopolysaccharide: A potential mechanism for antimicrobial activity.

Apolipophorin III (apoLp-III) is a model insect apolipoprotein to study structure-function relationships of exchangeable apolipoproteins. The protein associates with lipoproteins to aid in the transport of neutral lipids, and also interacts with the bacterial membrane. To better understand a potential role as an antimicrobial protein, the binding interaction of apoLp-III from Locust migratoria and Galleria mellonella with phosphatidylglycerol and lipopolysaccharides was analyzed. ApoLp-III from either species induced a robust release of calcein from phosphatidylglycerol vesicles, but was ineffective for phosphatidylcholine vesicles with comparable side-chain architecture. Acetylation of L. migratoria apoLp-III lysine residues greatly reduced the calcein release from phosphatidylglycerol vesicles, indicating a critical role of lysine side-chains in phosphatidylglycerol vesicles interaction. Isothermal calorimetry provided Kd values of 0.26 µM (L. migratoria) and 0.50 µM (G. mellonella) for binding to dimyristoylphosphatidylglycerol vesicles, which is an order of magnitude stronger compared to zwitterionic vesicles. A strong preference of apoLp-III for dimyristoylphosphatidylglycerol vesicles was also observed with differential scanning calorimetry with a concentration dependent shift in the lipid phase transition temperature. Native PAGE analysis showed that LPS binding was significantly weaker for L. migratoria apoLp-III compared to G. mellonella apoLp-III. This difference was confirmed by fluorescence titration analysis of L. migratoria apoLp-III, which also indicated that acetylation of the apolipoprotein did not affect LPS binding. Taken together, the results indicate that apoLp-III phosphatidylglycerol interaction may follow a detergent model with an important electrostatic binding component. Since lipopolysaccharide binding was not affected by neutralization of apoLp-III lysine-side chains, the binding interaction may be distinctly different from that of phosphatidylglycerol.

An MRM-Based Multiplexed Quantification Assay for Human Adipokines and Apolipoproteins.

Adipokines and apolipoproteins are key regulators and potential biomarkers in obesity and associated diseases and their quantitative assessment is crucial for functional analyses to understand disease mechanisms. Compared to routinely used ELISAs, multiple reaction monitoring (MRM)-based mass spectrometry allows multiplexing and detection of proteins for which antibodies are not available. Thus, we established an MRM method to quantify 9 adipokines and 10 apolipoproteins in human serum. We optimized sample preparation by depleting the two most abundant serum proteins for improved detectability of low abundant proteins. Intra-day and inter-day imprecision were below 16.5%, demonstrating a high accuracy. In 50 serum samples from participants with either normal weight or obesity, we quantified 8 adipokines and 10 apolipoproteins. Significantly different abundances were observed for five adipokines (adipsin, adiponectin, chemerin, leptin, vaspin) and four apolipoproteins (apo-B100/-C2/-C4/-D) between the body mass index (BMI) groups. Additionally, we applied our MRM assay to serum samples from normal weight children and human adipocyte cell culture supernatants to proof the feasibility for large cohort studies and distinct biological matrices. In summary, this multiplexed assay facilitated the investigation of relationships between adipokines or apolipoproteins and phenotypes or clinical parameters in large cohorts, which may contribute to disease prediction approaches in the future.

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA.

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.

Understanding the Lipid and Protein Corona Formation on Different Sized Polymeric Nanoparticles.

When in contact with biological fluids, nanoparticles dynamically absorb biomolecules like proteins and lipids onto their surface, forming a "corona". This biocorona is a dynamic and complex structure that determines how host cells respond to nanoparticles. Despite the common use of mouse models in pre-clinical and toxicological experiments, the impact of corona formed in mouse serum on the biophysical and biological properties of different size NP has not been thoroughly explored. Furthering the knowledge on the corona formed on NP exposed to mouse serum proteins can help in understanding what role it might have in in vivo studies at systemic, tissue, and cellular levels. To investigate biocorona formation, different sized polystyrene NP were exposed to mouse serum. Our data show a size- and time-dependent protein and lipid corona formation. Several proteins were identified and apolipoproteins were by far the most common group on the NPs surfaces. Moreover, we observed that cholesterol and triglycerides effectively bind to NP emphasizing that proteins are not the only biomolecules with high-affinity binding to nanomaterial surfaces. These results highlight that further knowledge on NP interactions with mouse serum is necessary regarding the common use of this model to predict the in vivo efficiency of NP.

The effect of acetylation on the protein stability of BmApoLp-III in the silkworm, Bombyx mori.

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin-III (BmApoLp-III). First, the expression of BmApoLp-III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp-III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp-III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp-III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp-III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition.

The role of apolipoprotein- and vitronectin-enriched protein corona on lipid nanoparticles for in vivo targeted delivery and transfection of oligonucleotides in murine tumor models.

The application of lipid-based nanoparticle (LNP) delivery systems remains a popular strategy for the systemic delivery of gene therapies to specific disease targets, including solid tumors. It is now well acknowledged that upon systemic administration, biomolecules from blood will adsorb onto nanoparticles' surfaces, forming a "protein corona", affording nanoparticles a "biological identity" on top of their "synthetic identity". Detailed analysis of nanoparticle protein corona is gradually revealing the "missing link" between nanoparticle chemical properties and the biological identity. Nevertheless, the discovery of nanoparticle protein corona's impact on tumor delivery is limited. In this study, we demonstrate that protein corona can be manipulated by formulation composition and particle surface charge changes, and a single lipid switch could switch the nanoparticle protein corona profile. The protein corona composition differences had a profound impact on cell transfection, in vivo biodistribution as well as tumor-specific delivery efficiency. Nanoparticles with apolipoprotein-rich corona showed better delivery to hepatocellular carcinoma (HepG2) as compared to those with vitronectin-rich corona. In addition, we found that, the PEG conjugated lipid chain length and PEG amount in LNPs were key factors to consider in successful RNA interference therapy for solid tumors.

Analysis of apolipoprotein multigene family in spotted sea bass (Lateolabrax maculatus) and their expression profiles in response to Vibrio harveyi infection.

Apolipoproteins (Apos), which are the protein components of plasma lipoproteins, play important roles in lipid transport in vertebrates. It has been demonstrated that in teleosts, several Apos display antimicrobial activity and play crucial roles in innate immunity. Despite their importance, apo genes have not been systematically characterized in many aquaculture fish species. In our study, a complete set of 23 apo genes was identified and annotated from spotted sea bass (Lateolabrax maculatus). Phylogenetic and homology analyses provided evidence for their annotation and evolutionary relationships. To investigate their potential roles in the immune response, the expression patterns of 23 apo genes were determined in the liver and intestine by qRT-PCR after Vibrio harveyi infection. After infection, a total of 20 differentially expressed apo genes were observed, and their expression profiles varied among the genes and tissues. 5 apo genes (apoA1, apoA4a.1, apoC2, apoF and apoO) were dramatically induced or suppressed (log2 fold change >4, P < 0.05), suggesting their involvement in the immune response of spotted sea bass. Our study provides a valuable foundation for future studies aimed at uncovering the specific roles of each apo gene during bacterial infection in spotted sea bass and other teleost species.

Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein.

Apolipophorin III (apoLp-III) is an insect apolipoprotein that is predominantly present in a lipid-free state in the hemolymph. ApoLp-III from Galleria mellonella is able to interact with membrane components of Gram-negative bacteria, as part of an innate immune response to infection. The protein also exists in a lipoprotein-associated state when large amounts of lipids are mobilized. Therefore, lipid-bound apoLp-III was generated to analyze the binding interaction with lipopolysaccharides and phosphatidylglycerol, both abundantly present in membranes of Gram-negative bacteria. G. mellonella apoLp-III was lipidated with palmitoyl-2-oleoyl-glycero-3-phosphocholine to form lipid-protein complexes. The particle shape was discoidal with a 16.4 nm diameter, a molecular mass of 460 kDa, and contained 4 apoLp-III molecules. These discoidal lipoproteins were used to compare the lipopolysaccharide and phosphatidylglycerol binding activity with lipid-free apoLp-III. Lipopolysaccharide binding interaction was analyzed by non-denaturing PAGE, showing reduced ability of the lipid-bound protein to form lipopolysaccharide-protein complexes and to disaggregate lipopolysaccharide micelles. The apoLp-III-induced release of calcein from phosphatidylglycerol vesicles was decreased approximately fivefold when the protein was in the lipid-bound form, indicating reduced binding interaction with the phosphatidylglycerol membrane surface. These results show that when apoLp-III adopts a lipid-bound conformation, it is markedly less effective in interacting with lipopolysaccharides and phosphatidylglycerol vesicles. Thus, in order to be an effective antimicrobial protein, apoLp-III needs to be in a lipid-free state.

Could ornithine supplementation be beneficial to prevent the formation of pro-atherogenic carbamylated low-density lipoprotein (c-LDL) particles?

Carbamylation (or carbamoylation) is a non-enzymatic post-translational modification process of lysine residues and protein N-termini, which occurs throughout the lifespan of both various plasma proteins and low-density lipoprotein (LDL) particles. Carbamylation results from the binding of isocyanates spontaneously derived from high levels of blood urea, environmental pollutants, nutritional sources and leads to the formation of potentially atherogenic carbamylated-LDL (c-LDL) particles. The carbamylation of LDL apolipoproteins is associated unfavorable downstream effects. Ornithine is a non-proteinogenic amino acid, which plays a central role at the urea cycle function. The primary use of ornithine in supplements is to support athletic performance, liver function and wound recovery. Ornithine is structurally highly similar to lysine, and is only one carbon atom shorter in its side-chain. Therefore, we hypothesize that supplemented ornithine could compete with ε-amino groups of lysine residues found in apolipoproteins of native LDL particles in their binding to isocyanates and decrease c-LDL formation. This issue still remains unresolved in current literature and needs to be elucidated in experimental studies.

The Exchangeable Apolipoprotein Nplp2 Sustains Lipid Flow and Heat Acclimation in Drosophila.

In ectotherms, increased ambient temperature requires the organism to consume substantial amounts of energy to sustain a higher metabolic rate, prevent cellular damage, and respond to heat stress. Here, we identify a heat-inducible apolipoprotein required for thermal acclimation in Drosophila. Neuropeptide-like precursor 2 (Nplp2) is an abundant hemolymphatic protein thought to be a neuropeptide. In contrast, we show that Nplp2 contributes to lipid transport, functioning as an exchangeable apolipoprotein. More precisely, Nplp2-deficient flies accumulate lipids in their gut, have reduced fat stores, and display a dyslipoproteinemia, showing that Nplp2 is required for dietary lipid assimilation. Importantly, Nplp2 is induced upon thermal stress and contributes to survival upon heat stress. We propose that Nplp2 associates with lipoprotein particles under homeostatic and high energy-demand conditions to optimize fat transport and storage. Our study also shows that modulation of the lipid uptake and transport machinery is part of an integrated cytoprotective response.