Serum amyloid A sequesters diverse phospholipids and their hydrolytic products, hampering fibril formation and proteolysis in a lipid-dependent manner.

Serum amyloid A action in immune response and deposition in inflammation-linked amyloidosis involve SAA-lipid interactions. We show that SAA sequesters neutral and anionic phospholipids and their hydrolytic products to form nanoparticles, suggesting a synergy with phospholipase A2. The lipid charge and shape affect SAA protection from proteolysis, aggregation and fibrillogenesis.

Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis.

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to ß-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

Serum amyloid A self-assembles with phospholipids to form stable protein-rich nanoparticles with a distinct structure: A hypothetical function of SAA as a "molecular mop" in immune response.

Serum amyloid A (SAA) is an acute-phase protein whose action in innate immunity and lipid homeostasis is unclear. Most circulating SAA binds plasma high-density lipoproteins (HDL) and reroutes lipid transport. In vivo SAA binds existing lipoproteins or generates them de novo upon lipid uptake from cells. We explored the products of SAA-lipid interactions and lipoprotein remodeling in vitro. SAA complexes with palmitoyl-oleoyl phosphocholine (POPC) were analyzed for structure and stability using circular dichroism and fluorescence spectroscopy, electron microscopy, gel electrophoresis and gel filtration. The results revealed the formation of 8-11nm lipoproteins that were∼50% α-helical and stable at near-physiological conditions but were irreversibly remodeled at Tm∼52°C. Similar HDL-size nanoparticles formed spontaneously at ambient conditions or upon thermal remodeling of parent lipoproteins containing various amounts of proteins and lipids, including POPC and cholesterol. Therefore, such HDL-size particles formed stable kinetically accessible structures in a wide range of conditions. Based on their size and stoichiometry, each particle contained about 12 SAA and 72 POPC molecules, with a protein:lipid weight ratio circa 2.5:1, suggesting a structure distinct from HDL. High stability of these nanoparticles and their HDL-like size suggest that similar lipoproteins may form in vivo during inflammation or injury when SAA concentration is high and membranes from dead cells require rapid removal. We speculate that solubilization of membranes by SAA to generate lipoproteins in a spontaneous energy-independent process constitutes the primordial function of this ancient protein, providing the first line of defense in clearing cell debris from the injured sites.

Thermal stability of human plasma electronegative low-density lipoprotein: A paradoxical behavior of low-density lipoprotein aggregation.

Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(-), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(-). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(-) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(-) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(-). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(-) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(-) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(-) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo.

Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I.

High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis.

Fusion of modified LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the first quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique among lipoproteins, suggesting that fusion is preceded by other structural changes. High activation energy of denaturation, E(a) = 100 ± 8 kcal/mol, indicates disruption of extensive packing interactions in LDL. Size-exclusion chromatography, nondenaturing gel electrophoresis, and negative-stain electron microscopy suggest that LDL dimerization is an early step in thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH < 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the first kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase the particle size may have opposite effects on LDL fusion.

The aminoterminal 1-185 domain of human apolipoprotein E suffices for the de novo biogenesis of apoE-containing HDL-like particles in apoA-I deficient mice.

AIMS: Recently we showed that apolipoprotein E promotes the de novo biogenesis of apoE-containing HDL particles in a process that requires the function of the lipid transporter ABCA1. Here, we sought to identify the domain of apoE that is responsible for its functional interactions with ABCA1 and the formation of apoE-rich HDL-like particles. METHODS AND RESULTS: Recombinant attenuated adenoviruses expressing carboxy-terminal truncated forms of apoE4 (apoE4[1-259], apoE4[1-229], apoE4[1-202], and apoE4[1-185]) were administered to apoA-I-deficient mice at a low dose of 8×10(8) pfu and five days post-infection plasma samples were isolated and analyzed for HDL formation. Fractionation of plasma lipoproteins of the infected mice by density gradient ultracentrifugation and FPLC revealed that all forms were capable of promoting HDL formation. Negative staining electron microscopy analysis of the HDL density fractions confirmed that all C-terminal truncated forms of apoE4 promoted the formation of particles with diameters in the HDL region. Interestingly, apoE4[1-259], apoE4[1-229], and apoE4[1-202] led to the formation of spherical particles while plasma from apoE4[1-185] expressing mice contained a mixture of spherical and discoidal particles. CONCLUSIONS: Taken together, our data establish that the aminoterminal 1-185 region of apoE suffices for the formation of HDL particles in vivo. Our findings may have important ramifications in the design of new biological drugs for the treatment of dyslipidemia, atherosclerosis and coronary heart disease.

Effects of phospholipase A(2) and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets.

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial ß-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins.

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia.

In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles compared with WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared with those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared with WT apoE4.

Effects of acyl chain length, unsaturation, and pH on thermal stability of model discoidal HDLs.

HDLs prevent atherosclerosis by removing excess cell cholesterol. Lipid composition affects HDL functions in cholesterol removal, yet its effects on the disk stability remain unclear. We hypothesize that reduced length or increased cis-unsaturation of phosphatidylcholine acyl chains destabilize discoidal HDL and promote protein dissociation and lipoprotein fusion. To test this hypothesis, we determined thermal stability of binary complexes reconstituted from apoC-I and diacyl PCs containing 12-18 carbons with 0-2 cis-double bonds. Kinetic analysis using circular dichroism shows that, for fully saturated PCs, chain length increase by two carbons stabilizes lipoprotein by deltaDeltaG* (37 degrees C) congruent with 1.4 kcal/mol, suggesting that hydrophobic interactions dominate the disk stability; distinct effects of pH and salt indicate contribution of electrostatic interactions. Similarly, apoA-I-containing disks show increased stability with increasing chain length. Acyl chain unsaturation reduces disk stability. In summary, stability of discoidal HDL correlates directly with fatty acyl chain length and saturation: the longer and more fully saturated are the chains, the more extensive are the stabilizing lipid-protein and lipid-lipid interactions and the higher is the free energy barrier for protein dissociation and lipoprotein fusion. This sheds new light on the existing data of cholesterol efflux to discoidal HDL and suggests that moderate lipoprotein destabilization facilitates cholesterol insertion.

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.

Effects of oxidation on the structure and stability of human low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.

Interactions between fatty acids and alpha-synuclein.

alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.

Effects of salt on the thermal stability of human plasma high-density lipoprotein.

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.

Kinetic stabilization and fusion of apolipoprotein A-2:DMPC disks: comparison with apoA-1 and apoC-1.

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E(a). CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E(a,slow) = 60 kcal/mol and a faster phase with E(a,fast) = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.

Poly(ethylene glycol)-induced fusion and destabilization of human plasma high-density lipoproteins.

High-density lipoproteins (HDL) are macromolecular complexes of specific proteins and lipids that mediate the removal of cholesterol from peripheral tissues. Chemical unfolding revealed that HDL fusion and rupture are the two main kinetic steps in HDL denaturation. Here we test the hypothesis that lipid fusogens such as poly(ethylene glycol) (PEG) may promote lipoprotein fusion and rupture and thereby destabilize HDL. We analyze thermal disruption of spherical HDL in 0-15% PEG-8000 by calorimetric, spectroscopic, electron microscopic, and light scattering techniques. We demonstrate that the two irreversible high-temperature endothermic HDL transitions involve particle enlargement and show a heating rate dependence characteristic of kinetically controlled reactions with high activation energy. The first calorimetric transition reflects HDL fusion and dissociation of lipid-poor apolipoprotein A-1 (apoA-1), and the second transition reflects HDL rupture and release of the apolar lipid core. Neither transition involves substantial protein unfolding; thus, the transition heat originates from lipid and/or protein dissociation and repacking. At room temperature, PEG-8000 induces HDL fusion that is distinct from the heat-, denaturant-, or enzyme-induced fusion since it leads to formation of larger particles and does not involve apoA-1 dissociation. Increasing the PEG concentration in solution from 0 to 15% leads to low-temperature shifts by approximately -18 degrees C in the two calorimetric HDL transitions without altering their nature. Thus, consistent with our hypothesis, PEG-8000 induces fusion and reduces the thermal stability of HDL. Our results suggest that PEG is useful for the analysis of the molecular events involved in metabolic HDL remodeling and fusion.

Nickel (Ni2+) enhancement of microtubule assembly in vitro is dependent on GTP function.

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni2+ damaging actions on MT, we have focused on dissecting the role of the Ni2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A350 at 27 degrees C using purified bovine brain MT proteins containing 162 microM each of Mg2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 microM, respectively. Replacement of GTP (25-87%) with increasing [NiCl2] while keeping the sum of [GTP] and [Ni2+] constant at 500 microM enabled MT assembly to proceed with shortened "lags" but reaching the same maximum plateau levels or elongation rates as with 500 microM GTP only. However, in reactions with Ni2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca2+ disruption than those assembled with 500 microM GTP only. Notably, in the presence of 500 microM Ni2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni2+ replacements of GTP. These results point to an important role of GTP/Ni2+ interaction in modulating the Ni2+ enhancement of MT assembly in vitro.

Human plasma high-density lipoproteins are stabilized by kinetic factors.

High-density lipoproteins (HDL) are heterogeneous complexes of proteins and lipids that mediate cholesterol removal from the body. Our thermal and chemical denaturation studies of mature spherical HDL isolated from human plasma show that, contrary to the widely held assumption, the particle stability has a kinetic rather than thermodynamic origin. Guanidinum hydrochloride (GdmHCl) concentration jumps at 25 degrees C monitored by circular dichroism (CD) at 222 nm reveal two dominant irreversible kinetic phases in HDL denaturation. The slower phase (relaxation time tau(1) approximately 2 x 10(4) seconds) is observed in 1-6 M GdmHCl, and the faster phase (tau(2) approximately 2 x 10(3) seconds) is detected in 3-6 M GdmHCl. Comparison of the free energy barriers associated with these phases, deltaG* = 16-17 kcal mol(-1), with the near-zero apparent thermodynamic stability inferred from the spectroscopic measurements after prolonged incubation in 0-6 M GdmHCl at 22 degrees C indicates the kinetic origin for HDL stabilization. Electron microscopic analysis of HDL incubated in 0-6 M GdmHCl suggests that the slower kinetic phase involves HDL fusion, while the faster phase involves particle rupture and release of the apolar lipid core. Thermal denaturation experiments indicate high enthalpic barriers for the particle rupture that may arise from the transient disruption of lipid and/or protein packing interactions. These results corroborate our earlier analysis of model discoidal HDL and indicate that a kinetic mechanism provides a universal natural strategy for lipoprotein stabilization. Such a mechanism may facilitate structural integrity of the heterogeneous lipoprotein particles, slow their spontaneous interconversions, and thereby modulate lipoprotein lifetime and functions.

Effects of mutations in apolipoprotein C-1 on the reconstitution and kinetic stability of discoidal lipoproteins.

To probe the role of protein conformation in the formation and kinetic stability of discoidal lipoproteins, thermal unfolding and refolding studies were carried out using model lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and selected mutants of human apolipoprotein C-1 (apoC-1). Circular dichroism (CD) spectroscopy and electron microscopy show that the Q31P mutant, which has alpha-helical content in solution (33%) and on DMPC disks (67%) similar to that of the wild type (WT), forms disks of smaller diameter, = 13 nm, compared to 17 nm of the WT-DMPC disks. The L34P mutant, which is largely unfolded in solution, forms disks with alpha-helix content and diameter similar to those of the WT. The R23P mutant, which is fully unfolded in solution, forms disks that have similar diameter but reduced alpha-helix content (40%) compared to the WT-DMPC disks (65%). Remarkably, despite large variations in the alpha-helix content or the disk diameter among different mutant-DMPC complexes, the mutations have no significant effect on the unfolding rates or the Arrhenius activation energy of the disk denaturation, E(a) = 25-29 kcal/mol. This suggests that the kinetic stability of the discoidal complexes is dominated by the lipid-lipid rather than the protein-lipid interactions. In contrast to the heat denaturation, the lipoprotein reconstitution upon cooling monitored by CD and light scattering is significantly affected by mutations, with Q31P forming disks in the broadest and R23P in the narrowest temperature range. Our results suggest that the apolipoprotein helical structure in solution facilitates reconstitution of discoidal lipoproteins but has no significant effect on their kinetic stability.