Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry

The interactions between phospholipid molecules in suspensions have been studied by using mass spectrometry. Electrospray mass spectra of homogeneous preparations formed from three different phospholipid molecules demonstrate that under certain conditions interactions between 90 and 100 lipid molecules can be preserved. In the presence of apolipoprotein C-II, a phospholipid binding protein, a series of lipid molecules and the protein were observed in complexes. The specificity of binding was demonstrated by proteolysis; the resulting mass spectra reveal lipid-bound peptides that encompass the proposed lipid-binding domain. The mass spectra of heterogeneous suspensions and their complexes with apolipoprotein C-II demonstrate that the protein binds simultaneously to two different phospholipids. Moreover, when apolipoprotein C-II is added to lipid suspensions formed with local concentrations of the same lipid molecule, the protein is capable of remodeling the distribution to form one that is closer to a statistical arrangement. These observations demonstrate a capacity for apolipoprotein C-II to change the topology of the phospholipid surface. More generally, these results highlight the fact that mass spectrometry can be used to probe lipid interactions in both homogeneous and heterogeneous suspensions and demonstrate reorganization of the distribution of lipids upon surface binding of apolipoprotein C-II.     

An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.     

Total synthesis of human plasma apolipoprotein C-II

Apolipoprotein C-II (apoC-II), a protein of 79 amino acid residues present in very low density lipoproteins, enhances the lipoprotein lipase (LpL)-catalyzed hydrolysis of triacylglycerols transported in plasma triglyceride-rich lipoproteins. To elucidate the structure-activity relationship of this activator protein, the complete amino acid sequence of apoC-II has been synthesized by the solid-phase method with Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 10% yield by a combination of ion-exchange and preparative high-performance liquid chromatography (HPLC). The purified peptide had the expected amino-terminal sequence and amino acid composition. Synthetic and native apoC-II were indistinguishable by cochromatography on analytical HPLC, peptide mapping of tryptic digest, radioimmunoassay, and activation of LpL with both artificial and lipoprotein substrates.     

Identification and metabolic characteristics of an apolipoprotein C-II variant isolated from a hypertriglyceridemic subject

The very low density lipoprotein (VLDL) apolipoproteins from a Type IV hypertriglyceridemic Caucasian subject (plasma TG: 645 mg/dl) and his brother (plasma TG: 328 mg/dl) were separated by isoelectric focusing gel electrophoresis (IEF) and found to contain two isoforms of apoC-II, identified by immunoblot. These corresponded to normal apoC-II-1 (isoelectric point: pI 4.88) and a variant isoform (apoC-II-v, pI 4.74). The pI of C-II-v was not altered by neuraminidase treatment, indicating that it was not sialylated. The concentration of total immunoreactive C-II in VLDL was elevated (18 mg/dl vs normal; 5.0 +/- 2 mg/dl) but similar to that in other Type IV subjects. In VLDL, which contained 90% of the plasma immunoreactive apoC-II, the ratio (by IEF) of C-II-1:C-II-v was 2:1, whereas in high density lipoproteins (HDL) the ratio was 1:1. VLDL apoB turnover was measured after the pulse injection of 125I-labeled VLDL. VLDL apoB kinetic parameters for the proband and four Type IV subjects were similar: production rate, 28 mg/kg per day versus 30 mg/kg per day; fractional catabolic rate, 1.62.day-1 versus 1.96.day-1; and pool size, 17 mg/kg versus 18 mg/kg. The decline in VLDL triglyceride (TG) after the infusion of heparin (9,000 IU over 4 h) was also similar to that observed in Type IV subjects. In VLDL, the fractional catabolic rates of apoC-II-1 and C-II-v were similar (C-II-1: 0.31.day-1, C-II-v: 0.29.day-1) whereas in HDL, although similar to each other, the rates were greater than in VLDL (C-II-1: 0.48.day-1, C-II-v: 0.44.day-1). VLDL and HDL from the proband were normal in their ability to activate bovine skim milk lipase, compared to Type IV VLDL and HDL without C-II-v. Purified apoC-II-1 and apoC-II-v activated the milk lipase to a similar extent (at 1 microgram of C-II; C-II-1: 34 units/h, C-II-v: 35 units/h). Thus, apoC-II-v is a newly recognized isoform of apoC-II-1. It remains to be determined whether this mutation plays a role in the genesis of hypertriglyceridemia.     

Genetic studies of human apolipoproteins. III. Polymorphism of apolipoprotein C-II

Using a simple and rapid one-dimensional isoelectric focusing technique followed by immunoblotting, we have detected genetic polymorphism of human apolipoprotein C-II (APO C-II) in normal unfractionated plasma samples of individuals of black ancestry. Two common autosomal codominantly expressed alleles, designated APO C-II*1 and APO C-II*2, at the APO C-II structural locus have been observed with frequencies of 0.975 and 0.025 in US blacks and 0.943 and 0.049 in Nigerian blacks. In addition, the gene product of a rare allele designated APO C-II*3 was observed in a single Nigerian black. Apart from a single example of an APO C-II 2-1 phenotype in plasma samples from 187 whites, which was electrophoretically identical to the 2-1 phenotype observed in blacks, it appears that APO C-II*2 is a unique black marker of potential importance in anthropogenetic and atherosclerosis studies.     

Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II.

Human apolipoprotein C-II (apoC-II) slowly forms amyloid fibers in lipid-free solutions at physiological pH and salt concentrations (Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., and Howlett, G. J. (2000) Biochemistry 39, 8276--8283). Measurements of the time dependence of solution turbidity, thioflavin T reactivity, and the amount of sedimentable aggregate reveal that the rate and extent of amyloid formation are significantly increased by the addition of an inert polymer, dextran T10, at concentrations exceeding 20 g/liter. High dextran concentrations do not alter the secondary structure of the protein, fiber morphology, or the thioflavin T and Congo Red binding capacity of apoC-II amyloid. Analytical ultracentrifugation studies show that monomeric apoC-II does not associate significantly with dextran. The observed dependence of the overall rate of amyloid formation on dextran concentration may be accounted for quantitatively by a simple model for nonspecific volume exclusion. The model predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibers by any amyloidogenic protein.     

Visualization of polymorphism in apolipoprotein C-II amyloid fibrils

The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.     

Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix.

We describe here a systematic study to determine the effect on secondary structure of d-amino acid substitutions in the nonpolar face of an amphipathic alpha-helical peptide. The helix-destabilizing ability of 19 d-amino acid residues in an amphipathic alpha-helical model peptide was evaluated by reversed-phase HPLC and CD spectroscopy. l-Amino acid and d-amino acid residues show a wide range of helix-destabilizing effects relative to Gly, as evidenced in melting temperatures (DeltaTm) ranging from -8.5 degrees C to 30.5 degrees C for the l-amino acids and -9.5 degrees C to 9.0 degrees C for the d-amino acids. Helix stereochemistry stability coefficients defined as the difference in Tm values for the l- and d-amino acid substitutions [(DeltaTm' = TmL and TmD)] ranging from 1 degrees C to 34.5 degrees C. HPLC retention times [DeltatR(XL-XD)] also had values ranging from -0.52 to 7.31 min at pH 7.0. The helix-destabilizing ability of a specific d-amino acid is highly dependent on its side-chain, with no clear relationship to the helical propensity of its corresponding l-enantiomers. In both CD and reversed-phase HPLC studies, d-amino acids with beta-branched side-chains destabilize alpha-helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure-activity studies and de novo design of novel biologically active peptides.     

An immunodominant epitope of myelin basic protein is an amphipathic alpha-helix

Myelin basic protein is a candidate autoantigen in multiple sclerosis. One of its dominant antigenic epitopes is segment Pro85 to Pro96 (human sequence numbering, corresponding to Pro82 to Pro93 in the mouse). There have been several, contradictory predictions of secondary structure in this region; either beta-sheet, alpha-helix, random coil, or combinations thereof have all been proposed. In this paper, molecular dynamics and site-directed spin labeling in aqueous solution indicate that this segment forms a transient alpha-helix, which is stabilized in 30% trifluoroethanol. When bound to a myelin-like membrane surface, this antigenic segment exhibits a depth profile that is characteristic of an amphipathic alpha-helix, penetrating up to 12 A into the bilayer. The alpha-helix is tilted approximately 9 degrees, and the central lysine is in an ideal snorkeling position for side-chain interaction with the negatively charged phospholipid head groups.     

No severe bottleneck during human evolution: evidence from two apolipoprotein C-II deficiency alleles.

The DNA sequences of a Japanese and a Venezuelan apolipoprotein (apo) C-II deficiency allele, of a normal Japanese apo C-II gene, and of a chimpanzee apo C-II gene were amplified by PCR, and their nucleotide sequences were determined on multiple clones of the PCR products. The normal Japanese sequence is identical to--and the chimpanzee sequence differs by only three nucleotides from--a previously published normal Caucasian sequence. In contrast, the two human mutant sequences each differ from the normal apo C-II gene sequence by several nucleotides, including deletions. The data suggest that both mutant alleles arose greater than 500,000 years ago. It is shown that a defective allele can persist in a population for only a short time if a bottleneck occurs. Therefore, the antiquity of the two alleles suggests no severe bottleneck during human evolution. Moreover, the fact that one allele is from Japan and the other is from a Venezuelan Caucasian family is more consistent with the multiregional evolution model of modern human origins than with the complete replacement or "out of Africa" model.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

The circularization of amyloid fibrils formed by apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.     

Far-u.v. CD-spectroscopy and immunological properties of synthetic sequential peptides derived from cardiotoxin VII1 of Naja nivea venom: an amphipathic alpha-helix formed by sequence 15-25 of a beta-protein

1. Immunological properties of cardiotoxin V(II)1 of Naja nivea were investigated. 2. Polyvalent antiserum raised against the cardiotoxin was tested for its interaction with synthetic peptides of overlapping sequence in order to locate possible sequential epitopes. 3. The conformation of each synthetic peptide in various solvents was determined by circular dichroism spectroscopy for relating immunological to structural properties. 4. It was found that sequential epitopes are absent in this cardiotoxin, but that region 15-25, although part of a beta-structured region, could be a possible T-cell epitope through the formation of an amphipathic helix.     

Apolipoprotein C-II deficiency: detection of immunoreactive apolipoprotein C-II in the intestinal mucosa of two patients

Recent data suggest that mutant immunoreactive forms of apolipoprotein C-II (apoC-II) can be detected in the plasma of patients with the apoC-II deficiency syndrome. We studied the possible presence of apoC-II mutants in the plasma of two patients with apoC-II deficiency by immunological means. The patients were hypertriglyceridemic, and apoC-II was undetectable in plasma as determined by radial immunodiffusion, electroimmunoassay, and immunonephelometry. Furthermore, apoC-II was undetectable either by electrophoresis or by immunoblotting in the plasma of the probands, while apoC-II was present in the plasma of their parents, although at less than half-normal concentration. Immunochemical localization of apoC-II, however, showed that the apoprotein could be detected within the enterocytes obtained from the intestinal mucosa of the patients. From these data we conclude that the patients synthesize apoC-II, at least in the intestine.     

Activation of lipoprotein lipase by native and acylated peptides of apolipoprotein C-II.

Apolipoprotein C-II, a protein found associated with all major classes of plasma lipoproteins, is a potent activator of the enzyme lipoprotein lipase. We have prepared the maleyl, citraconyl and succinyl derivatives of apolipoprotein C-II, and compared the capacities of the intact and tryptically cleaved proteins to activate lipoprotein lipase. The NH2-terminal 50 residue peptide proved virtually inactive, even after removal of the masking groups from the citraconyl derivative. The COOH-terminal 29 residue peptides of maleyl and citraconyl apolipoprotein C-II were more active than the corresponding succinylated peptide. After deacylation of the citraconyl derivative, the COOH-terminal peptide had maximal activity as great as apolipoprotein C-II, although the profile of activation remained dissimilar at low activator concentrations.     

Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin.

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.     

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.     

Immunochemistry of human plasma apolipoprotein C-II as studied by radioimmunoassay

The immunoreactivity of human plasma apolipoprotein C-II was investigated using a specific radioimmunoassay. In whole plasma, the mean value quantitated was 2.21 ± 0.415 mg/dl, while in delipidated plasma, a mean value of 3.84 ± 1.186 mg/dl was obtained, suggesting that the antigenic sites of the apolipoprotein were not fully detected in unmodified plasma by our antibody preparation. Two detergents, Tween-20 and Triton X-100, were studied to determine if they could enhance the immunoreactivity of apolipoprotein C-II in whole plasma. At concentrations of 0.012–0.06%, Tween-20 markedly increased the immunoreactivity of whole plasma, but not of delipidated plasma, indicating that antigenic sites of plasma apolipoprotein C-II has been exposed by Tween-20. In contrast, Triton X-100 had no effect on the immunoreactivity of whole plasma apolipoprotein C-II. A radioimmunoassay conducted in the presence of 0.06% Tween-20, resulted in a mean value in whole plasma (3.39 ± 1.11 mg/dl) that was not significantly different from that obtained when the assay was done on delipidated samples. The immunoreactivity of VLDL apolipoprotein C-II was also drastically enhanced following lipolysis by bovine milk lipoprotein lipase, supporting the hypothesis that antigenic sites are masked by the lipids. Finally, the mechanism responsible for the effect of Tween-20 on apolipoprotein C-II immunoreactivity was investigated. The results obtained from circular dichroism and ultracentrifugation suggest that the detergent may dissociate the apolipoprotein from lipoprotein particles, thus fully exposing the antigenic sites for reaction with antibodies.