Exchangeable Apolipoproteins Research Articles

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.

Quartet of APOCs and the Different Roles They Play in Diabetes.

APOA1 and APOB are the structural proteins of high-density lipoprotein and APOB-containing lipoproteins, such as low-density lipoprotein and very low-density lipoprotein, respectively. The 4 smaller APOCs (APOC1, APOC2, APOC3, and APOC4) are exchangeable apolipoproteins; they are readily transferred among high-density lipoproteins and APOB-containing lipoproteins. The APOCs regulate plasma triglyceride and cholesterol levels by modulating substrate availability and activities of enzymes interacting with lipoproteins and by interfering with APOB-containing lipoprotein uptake through hepatic receptors. Of the 4 APOCs, APOC3 has been best studied in relation to diabetes. Elevated serum APOC3 levels predict incident cardiovascular disease and progression of kidney disease in people with type 1 diabetes. Insulin suppresses APOC3 levels, and accordingly, elevated APOC3 levels associate with insulin deficiency and insulin resistance. Mechanistic studies in a mouse model of type 1 diabetes have demonstrated that APOC3 acts in the causal pathway of diabetes-accelerated atherosclerosis. The mechanism is likely due to the ability of APOC3 to slow the clearance of triglyceride-rich lipoproteins and their remnants, thereby causing an increased accumulation of atherogenic lipoprotein remnants in lesions of atherosclerosis. Less is known about the roles of APOC1, APOC2, and APOC4 in diabetes.

Formulation and Characterization of Bioactive Agent Containing Nanodisks.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodiskparticles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericinB as the hydrophobic bioactive agent.

Human Serum Amyloid a Impaired Structural Stability of High-Density Lipoproteins (HDL) and Apolipoprotein (Apo) A-I and Exacerbated Glycation Susceptibility of ApoA-I and HDL.

Human serum amyloid A (SAA) is an exchangeable apolipoprotein (apo) in high-density lipoprotein (HDL) that influences HDL quality and functionality, particularly in the acute phase of inflammation. On the other hand, the structural and functional correlations of HDL containing SAA and apoA-I have not been reported. The current study was designed to compare the change in HDL quality with increasing SAA content in the lipid-free and lipid-bound states in reconstituted HDL (rHDL). The expressed recombinant human SAA1 (13 kDa) was purified to at least 98% and characterized in the lipid-free and lipid-bound states with apoA-I. The dimyristoyl phosphatidylcholine (DMPC) binding ability of apoA-I was impaired severely by the addition of SAA, while SAA alone could not bind with DMPC. The recombinant human SAA1 was incorporated into the rHDL (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC): cholesterol: apoA-I) with various apoA-I:SAA molar ratios from 1:0 to 1:0.5, 1:1 and 1:2. With increasing SAA1 content, the rHDL particle size was reduced from 98 Å to 93 Å, and the α-helicity of apoA-I:SAA was decreased from 73% to 40% for (1:0) and (1:2), respectively. The wavelength maximum fluorescence (WMF) of tryptophan in rHDL was red-shifted from 339 nm to 345 nm for (1:0) and (1:2) of apoA-I:SAA, respectively, indicating that the addition of SAA to rHDL destabilized the secondary structure of apoA-I. Upon denaturation by urea treatment from 0 M to 8 M, SAA showed only a 3 nm red-shift in WMF, while apoA-I showed a 16 nm red-shift in WMF, indicating that SAA is resistant to denaturation and apoA-I had higher conformational flexibility than SAA. The glycation reaction of apoA-I in the presence of fructose was accelerated up to 1.8-fold by adding SAA in a dose-dependent manner than that of apoA-I alone. In conclusion, the incorporation of SAA in rHDL impaired the structural stability of apoA-I and exacerbated glycation of HDL and apoA-I.

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Circulating HDL and Non-HDL Associated Apolipoproteins and Breast Cancer Severity.

Plasma lipids are carried within lipoproteins with various apolipoprotein content. This study evaluates the interest of measuring the apolipoproteins of circulating lipoproteins in breast cancer. Patients with early-stage breast cancer (n = 140) were included. Tumors differed by the expression of estrogen and progesterone receptor (HR− and HR+ for negative and positive expression) and the proliferation marker Ki-67 (≤20% or ≥30%). Apolipoprotein concentrations were determined in plasma, HDL and non-HDL fractions, and results are given in mg/dL, median (25th–75th). Patients did not differ in their plasma and lipoprotein lipid concentrations. HDL apoC-I and non-HDL apoC-II were reduced (1.34 (1.02–1.80) vs. 1.61 (1.32–2.04), p = 0.04; 0.31 (0.18–0.65) vs. 0.63 (0.39–1.02), p = 0.01; respectively), in RH-/high Ki-67 patients in comparison to RH-/low Ki-67 patients, while plasma apoD and HDL apoD were higher (3.24 (2.99–4.16) vs. 3.07 (2.39–3.51), p = 0.04; 2.74 (2.36–3.35) vs. 2.45 (2.01–2.99), p = 0.04; respectively). When RH+/high Ki-67 patients were compared with RH+/low Ki-67 patients, HDL apoC-I and HDL apoC-III were higher (1.56 (1.20–1.95) vs. 1.35 (1.10–1.62), p = 0.02; 2.80 (2.42–3.64) vs. 2.38 (1.69–2.96), p = 0.02; respectively). The distribution of exchangeable apolipoproteins, such as apoC-I, apoC-II, apoC-III, apoD, between lipoproteins is linked to the severity of breast cancer.

Peptides as Therapeutic Agents for Atherosclerosis.

More than three decades ago, as a test for the amphipathic helix theory, an 18 amino acid residue peptide and its analogs were designed with no sequence homology to any of the exchangeable apolipoproteins. Based on the apolipoprotein A-I (the major protein component of high density lipoproteins, HDL) mimicking properties, they were termed as ApoA-I mimicking peptides. Several laboratories around the world started studying such de novo-designed peptides for their antiatherogenic properties. The present chapter describes the efforts in bringing these peptides as therapeutic agents for atherosclerosis and several lipid-mediated disorders.

Analysis of pyrene-labelled apolipoprotein A-I oligomerization in solution: Spectra deconvolution and changes in P-value and excimer formation

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.

Fragments of Locusta migratoria apoLp-III provide insight into lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein with a critical role in lipid transport in insects. The protein is composed of a bundle of five amphipathic α-helices which undergo a large conformational change upon lipid binding. To better understand the apoLp-III lipid binding interaction, the protein was cleaved by cyanogen bromide upon introduction of a S92M mutation, generating an N-terminal fragment corresponding to the first three helices (NTH1-3) and a C-terminal fragment of the last two helices (CTH4-5). MALDI-TOF analysis of the HPLC purified fragments provided masses of 9863.8 Da for NTH1-3 and 7497.0 Da for CTH4-5 demonstrating that the intended fragments were obtained. Circular dichroism spectra revealed a decrease in helical content from 82% for the intact protein to 57% for NTH1-3 and 41% for CTH4-5. The fragments adopted considerably higher α-helical structure in the presence of trifluoroethanol or phospholipids. Equimolar mixing of the two fragments did not result in changes in helical content or tryptophan fluorescence, indicating recombination into the native protein fold did not occur. The rate of protein induced dimyristoylphosphatidylcholine vesicle solubilization increased 15-fold for NTH1-3 and 100-fold for CTH4-5 compared to the intact protein. Despite the high activity in phospholipid vesicle interaction, CTH4-5 did not protect phospholipase-treated low-density lipoprotein from aggregation. In contrast, NTH1-3 provided protection to lipoprotein aggregation similar to the intact protein, indicating that specific amino acid residues in this part of apoLp-III are essential for lipoprotein binding interaction.

The impact of HDL subpopulations on the efficiency of the release of VLDL surface lipids and exchangeable apolipoproteins during lipolysis

Background and Aims: HDL plays an important role in the efficiency of VLDL-TG lipolysis (Cwiklinska et al., 2018). The aim of the study was to evaluate the impact of HDL-2 and HDL-3 on the efficiency of the surface lipids (free cholesterol, phospholipids) and exchangeable apolipoproteins (apoCs, apoE) release from VLDL during lipoprotein lipase (LPL)-mediated lipolysis.

Development of an Apolipoprotein AI Chimera for Targeted Nanodisc Drug Delivery to Breast Cancer Cells

A current challenge in cancer treatment is the ability to selectively target diseased cells with chemotherapeutics. Several promising treatments suffer this limitation and have toxic off target effects on healthy cells. Our overall objective is to generate a platform for targeted drug delivery employing self‐assembling nanodiscs comprised of phospholipids and apolipoprotein AI (apoAI). ApoAI is an exchangeable apolipoprotein located on high density lipoproteins (HDL), which promotes cholesterol efflux from macrophages and facilitates selective uptake of cholesterol by binding scavenger receptor, class B type 1 (SR‐B1), also known as the HDL receptor. We hypothesize that a chimeric protein of apoAI with MT1‐AF7p (apoAI‐AF7p), a peptide ligand that binds membrane metalloproteinase 14 (MMP14), will enhance the targeting capability of the nanodiscs to MDA‐MB‐231 breast cancer cells. MMP14 is a cell surface protein that is overexpressed in many types of cancers responsible for metastasis and linked to poor prognosis in patients. We designed a construct bearing the coding sequence for human apoAI with a hexa‐His‐tag at the N‐terminal end to facilitate protein purification by affinity chromatography, and MT1‐AF7p (HWKHLHNTKTFL) at the C‐terminal end with an intervening linker segment ([G4S]3). The construct was designed and custom‐generated with optimized codon sequence in a pCR2.1 vector. We successfully sub‐cloned the chimeric insert into a pET20b(+) expression vector employing standard molecular biology protocols and verified the resulting plasmid through Sanger sequencing. Recombinant apoAI and apoAI‐AF7p proteins were over‐expressed in E. coli and purified using a nickel affinity chromatography. The purified proteins were visualized by 15% acrylamide SDS‐PAGE, which revealed major bands at ~ 29 kDa band for apoAI and 32 kDa band for apoAI‐AF7p consistent with their expected size difference. We obtained ~ 10 mg purified protein/L culture medium, which is sufficient to carry out structural and functional characterization. Chimeric apoAI‐AF7p nanodiscs have potential not only for targeting, but also for drug delivery since MDA‐MB‐231 cells are reported to have high expression levels of both SR‐B1 and MMP14 which mediates homing and selective uptake of the hydrophobic core of HDL respectively. Preliminary data from fluorescence microscopy indicates the presence of both these proteins as well as the LDLr. Additionally, preliminary circular dichroism data shows similar helical content in the chimera as apoA1. Our next steps are to characterize the expression of MMP14, SR‐B1, and LDLr on MDA‐MB‐231 cells via FACS, qPCR and immunoblotting as well prepare nanodiscs with the chimera. Once particle formation and protein surface expression is established, cells will be incubated with particles and we’ll assess cell binding characteristics. The chimeric nanodiscs bears potential not only for targeting breast cancer cells, but also for drug delivery since these cells are reported to have high expression levels of SRB1, which mediate uptake of the contents within the hydrophobic core.Support or Funding InformationThis project is supported by the National Institute of Health (NIH) under the award numbers GM105561 (VN), R25GM071638 (DK) and T34GM008074 (RM)ApoA1‐AF7p chimera design: hexa‐His tag at the N‐terminus of ApoA1 and the affinity peptide MT1‐AF7p at the C terminus.Figure 1

Structural and Functional Analysis of Apolipoprotein E3 Modification by a Lipid Peroxidation Product, 4‐Hydroxynonenal

BackgroundPost mortem tissues from brains of Alzheimer’s disease (AD) patients show higher levels of 4‐hydroxynonenal (4‐HNE)‐modified proteins, with 4‐HNE arising as a result of oxidative stress and lipid peroxidation. The overall goal of our study is to understand the effect of 4‐HNE modification on the structure and function of apolipoprotein E3 (apoE3) and apoE4, which are 34 kDa exchangeable apolipoprotein isoforms that play a critical role in brain cholesterol homeostasis. Individuals carrying the APOE ɛ4 allele are at a higher risk of developing AD in a gene‐dose dependent manner. In the present study, we report the biophysical and functional analyses of 4‐HNE modification of apoE3 and apoE4 in terms of protein fold and conformation.MethodsRecombinant apoE3 and apoE4 were modified by 4‐HNE at concentrations typically found in pathological tissues (~20 mM), followed by Western blot and MALDI TOF mass spectrometric analyses to confirm modification. The modified samples were then subjected to circular dichroism (CD), fluorescence (intrinsic and 1‐anilinonaphthalene‐8‐sulfonic acid (ANS) fluorescence) spectroscopic, guanidine hydrochloride (GdnHCl)‐induced unfolding analyses, and lipid binding assessment.ResultsWestern blot with 4‐HNE specific antibody confirmed modification of apoE3 and apoE4, with a major band at ~36 kDa, while mass spectrometric data revealed modification of K72 and K75. 4‐HNE‐modified apoE3 and apoE4 were highly helical (~60%) comparable to that of unmodified proteins (~58%) as revealed by far UV CD spectroscopy. A significant decrease in the intrinsic fluorescence emission was noted for both 4‐HNE‐apoE3 and 4‐HNE‐apoE4, compared to the corresponding unmodified proteins. GdnHCl‐induced denaturation monitored by changes in intrinsic fluorescence revealed a notable difference in terms of increased susceptibility to unfolding for 4‐HNE‐apoE4, but not 4‐HNE‐apoE3. 4‐HNE modification significantly impaired the ability of apoE to transform DMPC vesicles to small discoidal protein/lipid complexes. The calculated t½ (time required for initial absorbance to decrease by 50%) for apoE3 was 165.6 mins, while that for 4‐HNE‐apoE3 was 516.8 mins. Similarly, the t½ for apoE4 was 4.95 min, while that for 4‐HNE‐apoE4 was 16.3 mins. Further, ANS fluorescence emission spectra revealed a 10 nm red shift in the wavelength of maximal fluorescence emission for 4‐HNE‐apoE4 (but not for 4‐HNE‐apoE3) compared to unmodified protein.ConclusionsTaken together, our data indicate that there are isoform‐specific differences in protein conformation, tertiary fold and functional ability as a consequence of modification of apoE by 4‐HNE. Assessing the differences in the susceptibility to age‐related oxidative modifications aid in understanding the molecular basis for the role of apoE4 as a risk factor for AD and amyloid pathology.Support or Funding InformationThis project was supported by the National Institutes of Health (NIH) grant award GM105561 (VN), Richard D Green Fellowship (MA), Undergraduate Research Opportunity Program (AA) and Louis Stokes Alliance for Minority Participation (AA).

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.

HCV Assembly and Egress via Modifications in Host Lipid Metabolic Systems.

Hepatitis C virus (HCV) proliferates by hijacking the host lipid machinery. In vitro replication systems revealed many aspects of the virus life cycle; in particular, viral utilization of host lipid metabolism during HCV proliferation. HCV interacts with lipid droplets (LDs) before starting the process of virus capsid formation at the lipid-rich endoplasmic reticulum (ER) membrane compartment. HCV buds into the ER via lipoprotein assembly and secretion. Exchangeable apolipoproteins, represented by apolipoprotein E (apoE), play pivotal roles in enhancing HCV-specific infectivity. HCV virions are likely to interact with other lipoproteins circulating in blood vessels and incorporate apolipoproteins as well as lipids. This review focuses on virus assembly and egress by briefly describing the recent advances in this area.

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Gain and loss events in the evolution of the apolipoprotein family in vertebrata

BackgroundVarious apolipoproteins widely distributed among vertebrata play key roles in lipid metabolism and have a direct correlation with human diseases as diagnostic markers. However, the evolutionary progress of apolipoproteins in species remains unclear. Nine human apolipoproteins and well-annotated genome data of 30 species were used to identify 210 apolipoprotein family members distributed among species from fish to humans. Our study focused on the evolution of nine exchangeable apolipoproteins (ApoA-I/II/IV/V, ApoC-I~IV and ApoE) from Chondrichthyes, Holostei, Teleostei, Amphibia, Sauria (including Aves), Prototheria, Marsupialia and Eutheria.ResultsIn this study, we reported the overall distribution and the frequent gain and loss evolutionary events of apolipoprotein family members in vertebrata. Phylogenetic trees of orthologous apolipoproteins indicated evident divergence between species evolution and apolipoprotein phylogeny. Successive gain and loss events were found by evaluating the presence and absence of apolipoproteins in the context of species evolution. For example, only ApoA-I and ApoA-IV occurred in cartilaginous fish as ancient apolipoproteins. ApoA-II, ApoE, and ApoC-I/ApoC-II were found in Holostei, Coelacanthiformes, and Teleostei, respectively, but the latter three apolipoproteins were absent from Aves. ApoC-I was also absent from Cetartiodactyla. The apolipoprotein ApoC-III emerged in terrestrial animals, and ApoC-IV first arose in Eutheria. The results indicate that the order of the emergence of apolipoproteins is most likely ApoA-I/ApoA-IV, ApoE, ApoA-II, ApoC-I/ApoC-II, ApoA-V, ApoC-III, and ApoC-IV.ConclusionsThis study reveals not only the phylogeny of apolipoprotein family members in species from Chondrichthyes to Eutheria but also the occurrence and origin of new apolipoproteins. The broad perspective of gain and loss events and the evolutionary scenario of apolipoproteins across vertebrata provide a significant reference for the research of apolipoprotein function and related diseases.

Brain-targeted drug delivery by manipulating protein corona functions

Protein corona presents a major obstacle to bench-to-bedside translation of targeted drug delivery systems, severely affecting targeting yields and directing unfavorable biodistribution. Corona-mediated targeting provides a new impetus for specific drug delivery by precisely manipulating interaction modes of functional plasma proteins on nano-surface. Here bio-inspired liposomes (SP-sLip) were developed by modifying liposomal surface with a short nontoxic peptide derived from Aβ1-42 that specifically interacts with the lipid-binding domain of exchangeable apolipoproteins. SP-sLip absorb plasma apolipoproteins A1, E and J, consequently exposing receptor-binding domain of apolipoproteins to achieve brain-targeted delivery. Doxorubicin loaded SP-sLip (SP-sLip/DOX) show significant enhancement of brain distribution and anti-brain cancer effect in comparison to doxorubicin loaded plain liposomes. SP-sLip preserve functions of the absorbed human plasma ApoE, and the corona-mediated targeting strategy works in SP modified PLGA nanoparticles. The present study may pave a new avenue to facilitate clinical translation of targeted drug delivery systems.

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.

Conformational analysis of apolipoprotein E3/E4 heteromerization.

Apolipoprotein E (apoE) is a 299 residue, exchangeable apolipoprotein that has essential roles in cholesterol homeostasis and reverse cholesterol transport. It is a two-domain protein with the C-terminal (CT) domain mediating protein self-association via helix-helix interactions. In humans, the APOE gene is polymorphic with three common alleles, ε2, ε3, and ε4, occurring in frequencies of ~5%, 77%, and 18%, respectively. Heterozygotes expressing apoE3 and apoE4 isoforms, which differ in residue at position 112 in the N-terminal domain (C112 in apoE3 and R112 in apoE4), represent the highest population of ε4 carriers, an allele highly associated with Alzheimer's disease. The objective of this study was to determine if apoE3 and apoE4 have the ability to hybridize to form a heteromer in lipid-free state. Refolding an equimolar mixture of His-apoE3 and FLAG-apoE4 (or viceversa) followed by pull-down and immunoblotting indicated formation of apoE3/apoE4 heteromers. Förster resonance energy transfer between donor fluorophore on one isoform and acceptor on the other, both located in the respective CT domains, revealed a distance of separation of ~46Å between the donor/acceptor pair. Similarly, a quencher placed on one was able to mediate significant quenching of fluorescence emission on the other, indicative of spatial proximity within collisional distance between the two. ApoE3/apoE4 heteromer association was also noted in lipid-associated state in reconstituted lipoprotein particles. The possibility of heteromerization of apoE3/apoE4 bears implications in the potential mitigating role of apoE3 on the folding and physiological behavior of apoE4 and its role in maintaining cholesterol homeostasis.

Effects of triacylglycerol on the structural remodeling of human plasma very low- and low-density lipoproteins

Very low-density lipoprotein (VLDL) is the main plasma carrier of triacylglycerol that is elevated in pathological conditions such as diabetes, metabolic syndrome, obesity and dyslipidemia. How variations in triacylglycerol levels influence structural stability and remodeling of VLDL and its metabolic product, low-density lipoproteins (LDL), is unknown. We applied a biochemical and biophysical approach using lipoprotein remodeling by lipoprotein lipase and cholesterol ester transfer protein, along with thermal denaturation that mimics key aspects of lipoprotein remodeling in vivo. The results revealed that increasing the triacylglycerol content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased triacylglycerol content in LDL promotes lipoprotein remodeling and fusion. These effects were observed in single-donor lipoproteins from healthy subjects enriched in exogenous triolein, in single-donor lipoproteins from healthy subjects with naturally occurring differences in endogenous triacylglycerol, and in LDL and VLDL from pooled plasma of diabetic and normolipidemic patients. Consequently, triacylglycerol-induced destabilization is a general property of plasma lipoproteins. This destabilization reflects a direct effect of triacylglycerol on lipoproteins. Moreover, we show that TG can act indirectly by increasing lipoprotein susceptibility to oxidation and lipolysis and thereby promoting the generation of free fatty acids that augment fusion. These in vitro findings are relevant to lipoprotein remodeling and fusion in vivo. In fact, fusion of LDL and VLDL enhances their retention in the arterial wall and, according to the response-to-retention hypothesis, triggers atherosclerosis. Therefore, enhanced fusion of triacylglycerol-rich lipoproteins suggests a new causative link between elevated plasma triacylglycerol and atherosclerosis.