Exchangeable Apolipoproteins Research Articles

Effects of lipid composition on the structural properties of human serum amyloid A in reconstituted high-density lipoprotein particles.

Serum amyloid A (SAA) is a member of exchangeable apolipoproteins that predominantly exists as a component of high-density lipoproteins (HDL). During inflammation, SAA displaces apolipoprotein A-I from HDL and becomes the major protein constituents of HDL. In addition, HDL lipid composition is altered in response to inflammation, which may induce the structural reorganization of SAA and affect its function. Therefore, the physiological roles of HDL can be influenced by changes in their protein and lipid compositions that are triggered by inflammatory diseases. Here, the effect of HDL lipid composition on the structural properties of SAA was examined. Uniformly sized reconstituted HDL (rHDL) was prepared and mainly composed of phosphatidylcholine with a single additional lipid species. Results showed that changes in lipid composition had no significant impact on the helical content of SAA and its thermodynamic stability. However, rHDL lipid composition affected other structural properties of SAA, such as its tryptophan microenvironment and kinetic stability, and thus influenced the susceptibility of SAA to enzymatic digestion. Therefore, changes in HDL lipid composition may affect the physiological function of SAA and the pathogenesis of SAA-related diseases.

Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1–191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.

Abstract 548: Separation of Lipoprotein Particles Utilizing Asymmetric Flow Field-flow Fractionation and Quantitation of Apolipoprotein L1 in Human Plasma with IMER-UPLC-MS/MS

Apolipoprotein L1 (ApoL1) is an intriguing protein and was shown to be involved in numerous diseases states such as chronic kidney disease (CKD), cardiovascular diseases(CVD), Human Immunodeficiency Virus associated Nephropathy (HIVAN), systemic lupus erythematosus (SLE) (associated collapsing glomerulopathy), and type 2 diabetes(especially for people of African descent). People of African ancestry were shown to have either one or two alleles which contribute to their susceptibility of diseases related to CKD. ApoL1 is characterized as a HDL binding protein. In this work we studied ApoL1 binding to HDL by using a gentle size fractionation technique, asymmetric flow field-flow fractionation (AF4), and liquid chromatography (LC) with quantitative tandem mass spectrometry (MS/MS) detection. We injected 50 uL of whole plasma onto the AF4 system, where smaller HDL particles eluted first followed by the larger HDL, and then by LDL, IDL and VLDL particles. The lipoproteins were separated into 40 fractions and quantitatively analyzed using on-line trypsin digestion coupled with LC-MS/MS analysis. We analyzed plasma samples both with and without purification by UC. When HDL particles were separated by UC, apoL1 was found in the higher density HDL fractions, as was also found by previous studies. When HDL separated using AF4 with or without UC, apoL1 eluted in the larger HDL size region (12-13 nm). With UC vs. without UC, the apoL1 peptide signal intensities were reduced by more than 50%, indicating significant loss of apoL1 from the surface of HDL particles during UC. Our data on apoL1 provides further evidence for the fact that size and density are not directly interconvertible physical characteristics of lipoproteins. Furthermore, our data shows that apoL1 is also an example of exchangeable apolipoproteins whose binding can be significantly diminished due to the effect of intense shear forces during UC separation. This work also demonstrates the advantages of utilizing AF4-IMER-UPLC-MS/MS methodology to separate lipoprotein particles and study of their apolipoprotein composition.

Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a β-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.-Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Hepatic tropism of hepatitis C virus infection

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a major cause of life-threatening liver diseases such as liver cirrhosis and hepatocellular carcinoma. In current research, we aimed to clarify the mechanism of hepatic tropism of HCV infection. Although non-hepatic cells could not permit replication of HCV RNA, exogenous expression of liver specific miRNA, miR-122 facilitated efficient replication of viral RNA through direct interaction with 5'UTR of viral genome, indicating that miR-122 is one of the key determinants for hepatic tropism of HCV infection. In spite of efficient replication of viral RNA, formation of infectious particles was not observed in non-hepatic cells exogenously expressing miR-122. We found that expression of apolipoprotein E (ApoE) facilitated the formation of infectious HCV particles in non-hepatic cells, indicating that not only miR-122 but also ApoE participate in tissue tropism of HCV infection. To understand the exact roles of miR-122 and apolipoproteins in hepatic tropism of HCV, we established miR-122 and ApoB/ApoE knockout (KO) Huh7 cells, respectively. Although slight increase of intracellular HCV RNA and infectious titers in the culture supernatants was observed, propagation of HCV was impaired in miR-122 KO Huh7 cells. After serial passages of HCV in miR-122 KO cells, we obtained an adaptive mutant that possessed G28A substitutions in the 5'UTR of the HCV genome and exhibited efficient translation and replication in both miR-122 KO Huh7 and non-hepatic cells without exogenous expression of miR-122. These results suggest that HCV mutants replicating in non-hepatic cells in an miR-122-independent manner participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. Deficiency of both ApoB and ApoE strongly inhibited the formation of infectious HCV particles. Interestingly, expression not only of ApoE but also of ApoA or ApoC could rescue the production of infectious HCV particles in ApoB/ApoE KO cells, suggesting that exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified invitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

Abstract 20984: Transfer of Functional Properties From the Two-Domain Apolipoprotein E3 to a Single Domain 5-Helix Bundle Apolipoprotein

Apolipoprotein E3 (apoE3) is a 34 kDa exchangeable apolipoprotein critical for lipid transport and associates with chylomicrons, VLDL, and a subclass of HDL. The protein contains two independently folded domains: a 22 kDa N-terminal domain and a 10 kDa C-terminal domain. The N-terminal domain contains LDL receptor binding sites and is tightly folded into a 4-helix bundle. The smaller C-terminal (CT) domain contains helical segments that: (i) promote protein-protein interaction and apoE3 self-association, (ii) bear high lipid binding affinity, and, (iii) facilitate cholesterol efflux from macrophages. In contrast, insect apolipophorin III (apoLp-III) is a one-domain protein bearing a 5-helix bundle, does not self-associate and binds to diacylglycerol enriched lipoproteins. To better understand the domain organization of apolipoproteins, a chimeric protein was constructed of apoLp-III and the CT domain of apoE3 (residues 201-299). The structure and function of the 28 kDa apoLp-III/CT-apoE3 chimera was determined and compared to that of parent proteins apoLp-III and apoE3. The novel chimeric construct was expressed in E. coli and purified using an N-terminal poly-histidine tag. Electrophoresis and Western blot analysis showed that the apoLp-III/CT-apoE3 chimera was of the expected size and reacted with apoE3 specific antibodies. Circular dichroism demonstrated that the chimera was α-helical, similar to both parent proteins. While apoLp-III was monomeric, the chimera formed tetramers similar to apoE3. Probing for hydrophobic pockets on the protein surface with anilinonaphthalene-8-sulfonic acid demonstrated that the CT domain provided increased binding sites for the probe. Addition of the CT domain to apoLp-III conferred an increased ability to promote cholesterol efflux from J774 macrophages, comparable to that of parent apoE3. Thus, the fold of apoLp-III/CT-apoE3 chimera was similar to that of apoE3, promoted self-association and cholesterol efflux. This demonstrated that functional characteristics of CT domain of apoE3 can be transferred independently to another apolipoprotein, allowing us to further increase our understanding of apolipoprotein domains.

Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

ABSTRACTHepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans. All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry.IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry.

Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae.

Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.

Unravelling the multiple roles of apolipoprotein E in the hepatitis C virus life cycle.

Worldwide, about 3% of the population is infected with HCV, and chronic infection is associated with a risk of serious progressive liver disease. The recent introduction of direct-acting antiviral agents (DAAs) has considerably improved the treatment of chronic HCV infection, and most patients are now cured without major side effects.1 However, subgroups of patients difficult-to-treat and requiring additional therapeutic approaches will probably emerge. Furthermore, DAAs are very expensive, limiting their use in some countries.1 Thus, in the absence of prophylactic vaccines, improvements to our understanding of the mechanisms underlying virus assembly, maturation and secretion from the cell are required, to facilitate the development of new treatment options. In patients, HCV particles circulate as hybrid particles, known as lipoviroparticles (LVPs), combining components of both HCV and very low-density lipoprotein (VLDL).2 LVPs have a low density (<1.06 g/mL) and are highly infectious. Their maturation and release are tightly associated with VLDL synthesis, leading to cholesterol and triglyceride export from hepatocytes. Similar characteristics have been described for viruses produced in cell culture (HCVcc) by human Huh7.5 hepatoma cells.2 LVPs are rich in triacylglycerol and total cholesterol. They contain the viral RNA, capsid protein, envelope glycoproteins and apolipoproteins. They carry highly variable amounts of lipids and are therefore highly heterogeneous in size and density. LVPs have been shown to associate with apolipoprotein B and apolipoprotein E (ApoE).3 ApoE is a soluble and exchangeable apolipoprotein. It plays a critical role in VLDL assembly and is also involved in cellular lipid transport.2 The importance of ApoE for the life cycle of HCV has been confirmed by studies showing ApoE to be required for LVP production4 ,5 HCV infectivity being associated with the presence of ApoE in …

Analysis of apolipoprotein genes and their involvement in disease response of channel catfish after bacterial infection

Apolipoproteins are protein component of plasma lipoproteins. They exert crucial roles in lipoprotein metabolism and serve as enzyme cofactors, receptor ligands, and lipid transfer carriers in mammals. In teleosts, apolipoproteins are also involved in diverse processes including embryonic and ontogenic development, liver and digestive system organogenesis, and innate immunity. In this study, we identified a set of 19 apolipoprotein genes in channel catfish (Ictalurus punctatus). Phylogenetic analysis and syntenic analysis were conducted to determine their identities and evolutionary relationships. The expression signatures of apolipoproteins in channel catfish were determined in healthy tissues and after infections with two major bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. In healthy channel catfish, most apolipoprotein genes exhibited tissue-specific expression patterns in channel catfish. After ESC and columnaris infections, 5 and 7 apolipoprotein genes were differentially expressed respectively, which presented a pathogen-specific and time-dependent pattern of regulation. After ESC infection, three exchangeable apolipoproteins (apoA-IB, apoC-I, and apoE-B) were suppressed in catfish intestine, while two nonexchangeable apolipoproteins (apoB-A and apoB-B) were slightly up-regulated. After columnaris infection, apoB-B, apoD-B, and apoE-A were significantly down-regulated in catfish gill, while apoF, apoL-IV, apoO-like, and apo-14 kDa showed significantly up-regulation. Taken together, these results suggested that apolipoprotein genes may play significant roles in innate immune responses to bacterial pathogens in channel catfish.

Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation.

Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic α-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic α-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic α-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic α-helices such as CAMP. Various exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic α-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic α-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic α-helices without the expression of apolipoproteins.

Involvement of a versatile pattern recognition receptor, apolipophorin-III in prophenoloxidase activation and antibacterial defense of the Chinese oak silkworm, Antheraea pernyi

Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi.

Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein.

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein found in insects and plays an important function in lipid transport. The protein has an unusual five-helix bundle architecture, deviating from the common four-helix bundle motif. To understand the role of the additional helix in apoLp-III, the N-terminal or C-terminal helix was deleted to create a putative four-helix bundle protein. While the protein lacking helix-1 could be expressed in bacteria albeit at reduced yields, apoLp-III lacking helix-5 could not be produced. Mutational analysis by truncating helix-5 showed that a minimum segment of approximately one-third of the C-terminal helix is required for protein expression. The variant lacking helix-5 was produced by inserting a methionine residue between helix-4 and -5; subsequent cyanogenbromide cleavage generated the four-helix variant. Both N- and C-terminal helix deletion variants displayed significantly reduced helical content, protein stability, and tertiary structure. Despite the significantly altered structure, the variants were still fully functional. The rate of dimyristoylphosphatidylcholine vesicle solubilization was enhanced 4-5-fold compared to the wild-type protein, and the deletion variants were effective in binding to lipolyzed low density lipoprotein thereby preventing lipoprotein aggregation. These results show that the additional helix of apoLp-III is not essential for lipid binding but is required for proper folding to keep the protein into a stable conformation.

Abstract 391: Apolipoprotein E3, Apolipoprotein AI, and Apolipophorin III Chimeras Provide Insight Into the Domain Organization of Apolipoproteins

Apolipoproteins (apo) A-I and E are exchangeable apolipoproteins that play a dominant role in regulating lipoprotein metabolism. They are composed of a series of amphipathic α-helices, which are organized into an N-terminal (NT) helix bundle domain and a smaller C-terminal (CT) domain. The objective of the current study is to understand the functional and structural role of the domains of these proteins by “domain swapping” using apoE3 and apoAI, both of which bear a 4-helix bundle in their NT domain, and apolipophorin III (apoLp-III), a monomeric one-domain model apolipoprotein. A series of chimeric apolipoproteins were generate: apoE3-NT/apoAI-CT, apoAI-NT/apoE-CT and apoLp-III/apoA-I-CT. The α-helical content of the chimeras was comparable to that of the parent proteins. Unfolding studies indicated that addition of CT-apoE3 to NT-apoAI conferred more stability to apoAI. While addition of CT-apoAI to NT-apoE3 had no significant effect on the stability of apoE3, addition of NT-apoAI to apoLp-III increased the stability of apoLp-III. Moreover, apoLp-III switched from a monomeric to an oligomeric protein upon addition of CT-apoAI, showing that CT-apoAI has a strong tendency to self-associate. Addition of CT-apoAI to NT-apoE or apoLp-III increased the lipid binding activity by ~ 10-fold, while addition of apoE-CT to apoA-I-NT had no measurable effect on the lipid binding activity. All three chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being highest for apoAI-NT/apoE-CT. Whereas apoE3-NT/apoAI-CT elicits LDLr binding ability, apoAI-NT/apoE-CT did not. These results suggest that CT of apoAI is a strong promoter of lipid binding, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

Abstract 199: The Regulation of Intestinal apoC-III by Dietary Fat: Key Differences From Hepatic apoC-III Regulation and Implications for Lipoprotein Synthesis and Secretion

Apolipoprotein C-III (apoC-III) influences both plasma triglycerides and inflammation, and as such is a particularly important target for mediating cardiovascular disease (CVD). ApoC-III is an exchangeable apolipoprotein expressed in both liver and intestine; in humans, apoC-III levels in plasma are an independent predictor of CVD risk. ApoC-III is a potent regulator of plasma triglycerides through effects on intestinal lipid absorption, plasma triglyceride clearance, hepatic lipoprotein uptake, and VLDL secretion. ApoC-III also acts as a signaling molecule modulating vascular function. Hepatic apoC-III is regulated by insulin via the transcription factor, forkhead box protein O1 (FOXO1), and by glucose via carbohydrate-responsive element-binding protein (ChREBP). Though much is known about hepatic apoC-III, little is known about the regulation of intestinal apoC-III (including the primary stimulus for its expression). We have found that apoC-III is secreted from the intestine in response to dietary carbohydrate, and that a fish oil-enriched diet lowers intestinal apoC-III mRNA expression. Our findings suggest that ChREBP is the primary modulator of apoC-III expression in the intestine. While dietary fish oil lowers both hepatic and intestinal apoC-III mRNA levels, it appears to do so through distinctive mechanisms. This is a potentially important difference since apoC-III is a key regulator of post-prandial plasma lipid levels and inflammation.

Mechanism of Lipid Binding of Human Apolipoprotein E3 by Hydrogen/Deuterium Exchange/Mass Spectrometry and Fluorescence Polarization.

Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding. The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out. HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain. Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.

Examining Properties of the C-Terminal Domain of Human Apolipoprotein A-I Utilizing a Chimeric Apolipoprotein

Human apolipoprotein A-I (apoA-I) is a 28 kDa exchangeable apolipoprotein that constitutes the primary protein of high density lipoprotein and plays a vital role in reverse cholesterol transport. ApoA-I comprises two structural domains: an α-helical N-terminal domain (NT, residues 1-189) and a less structured C-terminal domain (CT, residues 190-243). To evaluate the properties of the CT domain of apoA-I independently from the NT domain, a chimeric apolipoprotein was engineered in which residues 179-243 of apoA-I were attached to apolipophorin III (apoLp-III). ApoLp-III is a well-characterized insect apolipoprotein with a one domain structure composed of a bundle of α-helices. Two cysteine residues were introduced in apoLp-III to control opening of the helix bundle. ApoLp-III is monomeric but cross-linking studies with dimethyl suberimidate showed that the chimera self-associates similar to apoA-I. Far UV circular dichroism showed that the chimera displayed less α-helical content compared to apoLp-III, which is consistent with the addition of a less structured domain. Denaturation analysis using guanidine HCl showed that the stability of the chimera increased compared to apoLp-III. Functional analysis showed that the chimera was able to solubilize phospholipid vesicles at a faster rate than apoLp-III and that the percent calcein released from large unilamellar vesicles made of phosphatidylglycerol was substantially higher. Oxidized chimera protected LDL from phospholipase C-induced aggregation to a similar extent as apoLp-III but was far superior in its reduced state. Since the attachment of the CT domain of apoA-I to apoLp-III resulted in oligomerization and enhanced lipid binding, this demonstrated that these properties reside in the CT domain of apoA-I, and were transferable to an unrelated apolipoprotein. This demonstrates the potential to use the chimera as a tool to study the CT domain of apoA-I.