Apolipoprotein C1: Its Pleiotropic Effects in Lipid Metabolism and Beyond.

Apolipoprotein C1 (apoC1), the smallest of all apolipoproteins, participates in lipid transport and metabolism. In humans, APOC1 gene is in linkage disequilibrium with APOE gene on chromosome 19, a proximity that spurred its investigation. Apolipoprotein C1 associates with triglyceride-rich lipoproteins and HDL and exchanges between lipoprotein classes. These interactions occur via amphipathic helix motifs, as demonstrated by biophysical studies on the wild-type polypeptide and representative mutants. Apolipoprotein C1 acts on lipoprotein receptors by inhibiting binding mediated by apolipoprotein E, and modulating the activities of several enzymes. Thus, apoC1 downregulates lipoprotein lipase, hepatic lipase, phospholipase A2, cholesterylester transfer protein, and activates lecithin-cholesterol acyl transferase. By controlling the plasma levels of lipids, apoC1 relates directly to cardiovascular physiology, but its activity extends beyond, to inflammation and immunity, sepsis, diabetes, cancer, viral infectivity, and-not last-to cognition. Such correlations were established based on studies using transgenic mice, associated in the recent years with GWAS, transcriptomic and proteomic analyses. The presence of a duplicate gene, pseudogene APOC1P, stimulated evolutionary studies and more recently, the regulatory properties of the corresponding non-coding RNA are steadily emerging. Nonetheless, this prototypical apolipoprotein is still underexplored and deserves further research for understanding its physiology and exploiting its therapeutic potential.

The structure of human apolipoprotein C-1 in four different crystal forms.

Human apolipoprotein C1 (APOC1) is a 57 amino acid long polypeptide that, through its potent inhibition of cholesteryl ester transferase protein, helps regulate the transfer of lipids between lipid particles. We have now determined the structure of APOC1 in four crystal forms by X-ray diffraction. A molecule of APOC1 is a single, slightly bent, α-helix having 13-14 turns and a length of about 80 Å. APOC1 exists as a dimer, but the dimers are not the same in the four crystals. In two monoclinic crystals, two helices closely engage one another in an antiparallel fashion. The interactions between monomers are almost entirely hydrophobic with sparse electrostatic complements. In the third monoclinic crystal, the two monomers spread at one end of the dimer, like a scissor opening, and, by translation along the crystallographic a axis, form a continuous, contiguous sheet through the crystal. In the orthorhombic crystals, two molecules of APOC1 are related by a noncrystallographic 2-fold axis to create an arc of about 120 Å length. This symmetrical dimer utilizes interactions not present in dimers of the monoclinic crystals. Versatility of APOC1 monomer association shown by these crystals is suggestive of physiological function.

Identification of Apolipoprotein C-I Peptides as a Potential Biomarker and its Biological Roles in Breast Cancer.

BACKGROUND: Breast cancer (BC) is one of the most common cancers and is among the main causes of death in females around the world. Although several serum biomarkers have been identified for breast cancer, due to lack of adequate sensitivity and specificity they do not adequately distinguish BC from confounding conditions. New approaches are urgently needed to improve BC detection and treatment. MATERIAL/METHODS: Eighty serum samples from 20 healthy individuals and 60 patients with BC (22 triple-negative breast cancer, TNBC; 38 non-triple-negative breast cancer, NTNBC) were included. Protein profiling of serum samples was analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS). Candidate biomarkers were purified by SDS-PAGE electrophoresis and identified by MALDI-TOF/TOF. RESULTS: The candidate biomarker positioned at 6447.9 m/z was significantly decreased in BC patients. Moreover, the expression intensity of the candidate biomarker was weaker in the TNBC and pre-surgery group compared with the NTNBC and post-surgery group. We ultimately identified the biomarker as apolipoprotein C-I (ApoC-I). Furthermore, we found that ApoC-I peptides inhibited proliferation of human breast cancer cells in vitro and suppressed tumor growth in vivo. CONCLUSIONS: These results suggest that ApoC-I peptides may be a potential diagnostic biomarker and therapeutic approach for BC.

Apolipoprotein C1 regulates epiboly during gastrulation in zebrafish.

Apolipoprotein C1 (Apoc1) is associated with lipoprotein metabolism, but its physiological role during embryogenesis is largely unknown. We reveal a new function of Apoc1b, a transcript isoform of Apoc1, in epiboly during zebrafish gastrulation. Apoc1b is expressed in yolk syncytial layers and in deep cells of the ventral and lateral region of the embryos. It displays a radial gradient with high levels in the interior layer and low levels in the superficial layer. Knockdown of Apoc1b by injecting antisense morpholino (MO) caused the epiboly arrest in deep cells. Moreover, we show that the radial intercalation and the radial gradient distribution of E-cadherin are disrupted both in Apoc1b knockdown and overexpressed embryos. Therefore, Apoc1b controls epiboly via E-cadherin-mediated radial intercalation in a gradient-dependent manner.

Apolipoproteins C-I and C-III inhibit lipoprotein lipase activity by displacement of the enzyme from lipid droplets.

Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces.

Amphipathic α-helices mediate binding of exchangeable apolipoproteins to lipoproteins. To probe the role of α-helical structure in protein-lipid interactions, we used oil-drop tensiometry to characterize the interfacial behavior of apolipoprotein C-I (apoC-I) variants at triolein/water (TO/W) and 1-palmitoyl-2-oleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. ApoC-I, the smallest apolipoprotein, has two amphipathic α-helices. Mutants had single Pro or Ala substitutions that resulted in large differences in helical content in solution and on phospholipids. The ability of apoC-I to bind TO/W and POPC/TO/W interfaces correlated strongly with α-helical propensity. On binding these interfaces, peptides with higher helical propensity increased surface pressure to a greater extent. Likewise, peptide exclusion pressure at POPC/TO/W interfaces increased with greater helical propensity. ApoC-I retention on TO/W and POPC/TO/W interfaces correlated strongly with phospholipid-bound helical content. On compression of these interfaces, peptides with higher helical content were ejected at higher pressures. Substitution of Arg for Pro in the N-terminal α-helix altered net charge and reduced apoC-I affinity for POPC/TO/W interfaces. Our results suggest that peptide-lipid interactions drive α-helix binding to and retention on lipoproteins. Point mutations in small apolipoproteins could significantly change α-helical propensity or charge, thereby disrupting protein-lipid interactions and preventing the proteins from regulating lipoprotein catabolism at high surface pressures.

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II.

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.

Apolipoprotein C-I binds more strongly to phospholipid/triolein/water than triolein/water interfaces: a possible model for inhibiting cholesterol ester transfer protein activity and triacylglycerol-rich lipoprotein uptake.

Apolipoprotein C-I (apoC-I) is an important constituent of high-density lipoprotein (HDL) and is involved in the accumulation of cholesterol ester in nascent HDL via inhibition of cholesterol ester transfer protein and potential activation of lecithin:cholesterol acyltransferase (LCAT). As the smallest exchangeable apolipoprotein (57 residues), apoC-I transfers between lipoproteins via a lipid-binding motif of two amphipathic α-helices (AαHs), spanning residues 7-29 and 38-52. To understand apoC-I's behavior at hydrophobic lipoprotein surfaces, oil drop tensiometry was used to compare the binding to triolein/water (TO/W) and palmitoyloleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. When apoC-I binds to either interface, the surface tension (γ) decreases by ~16-18 mN/m. ApoC-I can be exchanged at both interfaces, desorbing upon compression and readsorbing on expansion. The maximal surface pressures at which apoC-I begins to desorb (Π(max)) were 16.8 and 20.7 mN/m at TO/W and POPC/TO/W interfaces, respectively. This suggests that apoC-I interacts with POPC to increase its affinity for the interface. ApoC-I is more elastic on POPC/TO/W than TO/W interfaces, marked by higher values of the elasticity modulus (ε) on oscillations. At POPC/TO/W interfaces containing an increasing POPC:TO ratio, the pressure at which apoC-I begins to be ejected increases as the phospholipid surface concentration increases. The observed increase in apoC-I interface affinity due to higher degrees of apoC-I-POPC interactions may explain how apoC-I can displace larger apolipoproteins, such as apoE, from lipoproteins. These interactions allow apoC-I to remain bound to the interface at higher Π values, offering insight into apoC-I's rearrangement on triacylglycerol-rich lipoproteins as they undergo Π changes during lipoprotein maturation by plasma factors such as lipoprotein lipase.

Identification of apolipoprotein C-I in rainbow trout seminal plasma.

Three distinct bands with high electrophoretic migration rates were isolated and purified from rainbow trout seminal plasma. The molecular masses of these bands were determined to be 5158.8, 4065.9 and 4929.0 Da. The N-terminal amino acids sequences were elucidated and were found to have high homology with Atlantic salmon apolipoprotein C-I. It can be concluded that apolipoprotein C-I is a major component of rainbow trout seminal plasma. Further studies are necessary to confirm the protective effects of apolipoprotein C-I on spermatozoa in terms of the stabilisation of the sperm membrane.

Detection of two distinct forms of apoC-I in great apes.

ApoC-I, the smallest of the soluble apolipoproteins, associates with both TG-rich lipoproteins and HDL. Mass spectral analyses of human apoC-I previously had demonstrated that in the circulation there are two forms, either a 57 amino acid protein or a 55 amino acid protein, due to the loss of two amino acids from the N-terminus. In our analyses of the apolipoproteins of the other great apes by mass spectrometry, four forms of apoC-I were detected. Two of these showed a high degree of identity to the mature and truncated forms of human apoC-I. The other two were homologous to the virtual protein and its truncated form that are encoded by a human pseudogene. In humans, the genes for apoC-I and its pseudogene are located on chromosome 19, the pseudogene being 2.5 kb downstream from the apoC-I gene. Based on the similarity between the apoC-I gene and the pseudogene, it has been concluded that the latter arose from the former as a result of gene duplication approximately 35 million years ago. Interestingly, the virtual protein encoded by the pseudogene is acidic, not basic like apoC-I. In the chimpanzee, there also are two genes for apoC-I, the one upstream encodes a basic protein and the downstream gene, rather than being a pseudogene, encodes an acidic protein (P86336). In addition to reporting on the molecular masses of great ape apoC-I, we were able to clearly demonstrate by "Top-down" sequencing that the acidic form arose from a separate gene. In our analyses, we have measured the molecular masses of apoC-I associated with the HDL of the following great apes: bonobo (Pan paniscus), chimpanzee (Pan troglodytes), and the Sumatran orangutan (Pongo abelii). Genomic variations in chromosome 19 among great apes, baboons and macaques as they relate to both genes for apoC-I and the pseudogene are compared and discussed.

Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix.

Timely sensing of lipopolysaccharide (LPS) is critical for the host to fight invading Gram-negative bacteria. We recently showed that apolipoprotein CI (apoCI) (apoCI1-57) avidly binds to LPS, involving an LPS-binding motif (apoCI48-54), and thereby enhances the LPS-induced inflammatory response. Our current aim was to further elucidate the structure and function relationship of apoCI with respect to its LPS-modulating characteristics and to unravel the mechanism by which apoCI enhances the biological activity of LPS. We designed and generated N- and C-terminal apoCI-derived peptides containing varying numbers of alternating cationic/hydrophobic motifs. ApoCI1-38, apoCI1-30, and apoCI35-57 were able to bind LPS, whereas apoCI1-23 and apoCI46-57 did not bind LPS. In line with their LPS-binding characteristics, apoCI1-38, apoCI1-30, and apoCI35-57 prolonged the serum residence of 125I-LPS by reducing its association with the liver. Accordingly, both apoCI1-30 and apoCI35-57 enhanced the LPS-induced TNFalpha response in vitro (RAW 264.7 macrophages) and in vivo (C57Bl/6 mice). Additional in vitro studies showed that the stimulating effect of apoCI on the LPS response resembles that of LPS-binding protein (LBP) and depends on CD14/ Toll-like receptor 4 signaling. We conclude that apoCI contains structural elements in both its N-terminal and C-terminal helix to bind LPS and to enhance the proinflammatory response toward LPS via a mechanism similar to LBP.

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins.

High-density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic alpha-helices that have high content (> or = 1/3) and distinct distribution of charged and apolar residues, adopt molten globule-like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA-I, shows relatively large volume contraction, DeltaV(unf) = -0.33%, and an apparent reduction in thermal expansivity upon unfolding, Deltaalpha(unf) < or = 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon alpha-helical unfolding, which may result from disruption of multiple salt bridges. At 5 degrees C, apoA-I shows large thermal expansion coefficient, alpha(5 degrees) = 15.10(-4) K(-1), that rapidly declines upon heating from 5 to 40 degrees C, alpha(40 degrees) - alpha(5 degrees) = -4.10(-4) K(-1); apolipoprotein C-I shows similar values of alpha(5 degrees) and alpha(40 degrees). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein-lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA-I or apoC-I, dimyristoyl phosphatidylcholine, and 0-20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport.

Discovery and identification of potential biomarkers of papillary thyroid carcinoma.

BACKGROUND: Thyroid carcinoma is the most common endocrine malignancy and a common cancer among the malignancies of head and neck. Noninvasive and convenient biomarkers for diagnosis of papillary thyroid carcinoma (PTC) as early as possible remain an urgent need. The aim of this study was to discover and identify potential protein biomarkers for PTC specifically. METHODS: Two hundred and twenty four (224) serum samples with 108 PTC and 116 controls were randomly divided into a training set and a blind testing set. Serum proteomic profiles were analyzed using SELDI-TOF-MS. Candidate biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays. RESULTS: A total of 3 peaks (m/z with 9190, 6631 and 8697 Da) were screened out by support vector machine (SVM) to construct the classification model with high discriminatory power in the training set. The sensitivity and specificity of the model were 95.15% and 93.97% respectively in the blind testing set. The candidate biomarker with m/z of 9190 Da was found to be up-regulated in PTC patients, and was identified as haptoglobin alpha-1 chain. Another two candidate biomarkers (6631, 8697 Da) were found down-regulated in PTC and identified as apolipoprotein C-I and apolipoprotein C-III, respectively. In addition, the level of haptoglobin alpha-1 chain (9190 Da) progressively increased with the clinical stage I, II, III and IV, and the expression of apolipoprotein C-I and apolipoprotein C-III (6631, 8697 Da) gradually decreased in higher stages. CONCLUSION: We have identified a set of biomarkers that could discriminate PTC from non-cancer controls. An efficient strategy, including SELDI-TOF-MS analysis, HPLC purification, MALDI-TOF-MS trace and LC-MS/MS identification, has been proved successful.

Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology.

Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.

Elucidation of potential bortezomib response markers in mutliple myeloma patients.

Liquid chromatography coupled to mass spectrometry (LC/MS) was used to elucidate early biomarkers of bortezomib response in multiple myeloma patients. The change in serum myeloma M-protein level, maintained for a minimum of 6 weeks, is used as one of the main criteria to evaluate patient clinical response to therapy. The objective of this study was to identify biomarkers using LC/MS in order to predict patient response to bortezomib sooner and more accurately compared to serum M-protein levels. The plasma LC/MS biomolecular/biochemical profiles, comprised of thousands of endogenous small molecules, peptides and proteins, were determined for 10 multiple myeloma patients at predose and 24 h after initial dosing with bortezomib. The comparative analysis of the metabolic profiles of non-responders and partial responders provided an opportunity to investigate mechanisms related to disease progression and identify biomarkers related to drug response. The plasma levels of two potential efficacy response markers were significantly more abundant in the non-responsive patients compared to the responders at 24-h postdose. The potential response biomarkers, apolipoprotein C-I and apolipoprotein C-I', were identified by mass spectral analyses and confirmed by authentic protein standards based on MALDI-TOF MS/MS sequencing of proteolytic peptides.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper.

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Effects of protein oxidation on the structure and stability of model discoidal high-density lipoproteins.

High-density lipoproteins (HDLs) prevent atherosclerosis by removing cholesterol from macrophages and by providing antioxidants for low-density lipoproteins. Oxidation of HDLs affects their functions via the complex mechanisms that involve multiple protein and lipid modifications. To differentiate between the roles of oxidative modifications in HDL proteins and lipids, we analyzed the effects of selective protein oxidation by hypochlorite (HOCl) on the structure, stability, and remodeling of discoidal HDLs reconstituted from human apolipoproteins (A-I, A-II, or C-I) and phosphatidylcholines. Gel electrophoresis and electron microscopy revealed that, at ambient temperatures, protein oxidation in discoidal complexes promotes their remodeling into larger and smaller particles. Thermal denaturation monitored by far-UV circular dichroism and light scattering in melting and kinetic experiments shows that protein oxidation destabilizes discoidal lipoproteins and accelerates protein unfolding, dissociation, and lipoprotein fusion. This is likely due to the reduced affinity of the protein for lipid resulting from oxidation of Met and aromatic residues in the lipid-binding faces of amphipathic alpha-helices and to apolipoprotein cross-linking into dimers and trimers on the particle surface. We conclude that protein oxidation destabilizes HDL disk assembly and accelerates its remodeling and fusion. This result, which is not limited to model discoidal but also extends to plasma spherical HDL, helps explain the complex effects of oxidation on plasma lipoproteins.

Lipid dependant disorder-to-order conformational transitions in apolipoprotein CI derived peptides.

In contrast to the notion established for many years that protein function depends on rigid 3D structures, nowadays there is important evidence suggesting that non-structured segments of proteins play important roles in protein function. Therefore, disorder-to-order dynamic conformational transitions have been proposed as an attractive mechanism involved in protein-protein recognition. Our laboratory using Langmuir monolayers of apolipoproteins has previously shown that upon lateral compression at the air/water and phospholipid/water interfaces, there is an important movement of the C-terminal segment of apolipoprotein CI towards the air, considered the hydrophobic region of the monolayer and the acyl-chain region of the interface when phospholipids are used. Here, in an attempt to define secondary structure changes that might occur within this C-terminal segment of apoCI while moving from the monolayer interface back and forth its hydrophobic region, employing three peptides derived from apoCI we studied by circular dichroism and dynamic light scattering their conformational properties when associated to a series of amphipathic lipids and lipid-like molecules. Our results show that a series of lysophospholipids present the ability to modulate the formation of an alpha helix at the C-terminal peptide of apoCI through a disorder-to-order transition while forming small lipid/peptide aggregates below 10nm in diameter.

Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification.

Mice that overexpress human apolipoprotein C-I (apoC-I) homozygously (APOC1(+/+) mice) are protected against obesity and show cutaneous abnormalities. Although these effects can result from our previous observation that apoC-I inhibits FFA generation by LPL, we have also found that apoC-I impairs the uptake of a FFA analog in adipose tissue. In this study, we tested the hypothesis that apoC-I interferes with cellular FFA uptake independent of LPL activity. The cutaneous abnormalities of APOC1(+/+) mice were not affected after transplantation to wild-type mice, indicating that locally produced apoC-I prevents lipid entry into the skin. Subsequent in vitro studies with apoC-I-deficient versus wild-type macrophages revealed that apoC-I reduced the cell association and subsequent esterification of [(3)H]oleic acid by approximately 35% (P < 0.05). We speculated that apoC-I binds FFA extracellularly, thereby preventing cell association of FFA. We showed that apoC-I was indeed able to mediate the binding of oleic acid to otherwise protein-free VLDL-like emulsion particles involving electrostatic interaction. We conclude that apoC-I binds FFA in the circulation, thereby reducing the availability of FFA for uptake by cells. This mechanism can serve as an additional mechanism behind the resistance to obesity and the cutaneous abnormalities of APOC1(+/+) mice.

Role of secondary structure in protein-phospholipid surface interactions: reconstitution and denaturation of apolipoprotein C-I:DMPC complexes.

Binding of protein to a phospholipid surface is commonly mediated by amphipathic alpha-helices. To understand the role of alpha-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kDa) or its mutants containing single Pro substitutions along the sequence and differing in their alpha-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher alpha-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at Tc = 24 degrees C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g., at the lipid phase boundary), insertion of protein into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion are aided by other surface-bound proteins and depend on their helical propensity: the larger the propensity, the faster the binding and the broader its temperature range. This positive cooperativity in binding of alpha-helices to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding.