Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins

Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein particles, cofactors for enzymes and ligands for cell-surface receptors. Recent findings with apoA-I and apoE suggest that the tertiary structures of these two members of the human exchangeable apolipoprotein gene family are related. Characteristically, these proteins contain a series of proline-punctuated, 11- or 22-amino acid, amphipathic alpha-helical repeats that can adopt a helix bundle conformation in the lipid-free state. The amino- and carboxyl-terminal regions form separate domains with the latter being primarily responsible for lipid binding. Interaction with lipid induces changes in the conformation of the amino-terminal domain leading to alterations in function; for example, opening of the amino-terminal four-helix bundle in apolipoprotein E upon lipid binding is associated with enhanced receptor-binding activity. The concept of a two-domain structure for the larger exchangeable apolipoproteins is providing new molecular insights into how these apolipoproteins interact with lipids and other proteins, such as receptors. The ways in which structural changes induced by lipid interaction modulate the functionality of these apolipoproteins are reviewed.     

The interfacial properties of ApoA-I and an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at the triolein/water interface

Apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein (HDL). During lipid metabolism, apoA-I moves among HDL and triacylglycerol-rich lipoproteins. The main structure and the major lipid binding motif of apoA-I is the amphipathic alpha-helix. To understand how apoA-I behaves at hydrophobic lipoprotein interfaces, the interfacial properties of apoA-I and an amphipathic alpha-helical consensus sequence peptide (CSP) were studied at the triolein/water (TO/W) interface. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-residue tandem repeat sequences that form amphipathic alpha-helices modeling the central part of apoA-I. ApoA-I or CSP added into the aqueous phase surrounding a triolein drop lowered the interfacial tension (gamma) of TO/W in a concentration- and time-dependent fashion. The gamma(TO/W) was lowered approximately 16 millinewtons (mN)/m by apoA-I at 1.4 x 10(-6) m and approximately 15 mN/m by CSP at 2.6 x 10(-6) m. At equilibrium gamma, both apoA-I and CSP desorbed from the interface when compressed and readsorbed when expanded. The maximum surface pressure CSP could withstand without being ejected (PiMAX) was 16 mN/m. The PiMAX) of apoA-I was only 14.8 mN/m, but re-adsorption kinetics suggested that only part of the apoA-I desorbed at Pi between 14.8 and 19 mN/m. However, above approximately 19 mN/m (PiOFF) the entire apoA-I molecule desorbed into the water. ApoA-I was more flexible at the TO/W interface than CSP and showed more elasticity at oscillation periods 4-128 s even at high compression, whereas CSP was elastic only at faster periods (4 and 8 s) and moderate compression. Flexibility and surface pressure-mediated desorption and re-adsorption of apoA-I probably provides lipoprotein stability during metabolic-remodeling reactions in plasma.     

Temperature-induced transitions in the structure and interfacial rheology of human meibum

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.     

Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements.

The dynamics of protein adsorption at an oil/water interface are examined over time scales ranging from seconds to several hours. The pendant drop technique is used to determine the dynamic interfacial tension of several proteins at the heptane/aqueous buffer interface. The kinetics of adsorption of these proteins are interpreted from tension/log time plots, which often display three distinct regimes. (I) Diffusion and protein interfacial affinity determine the duration of an initial induction period of minimal tension reduction. A comparison of surface pressure profiles at the oil/water and air/water interface reveals the role of interfacial conformational changes in the early stages of adsorption. (II) Continued rearrangement defines the second regime, where the resulting number of interfacial contacts per protein molecule causes a steep tension decline. (III) The final regime occurs upon monolayer coverage, and is attributed to continued relaxation of the adsorbed layer and possible build-up of multilayers. Denaturation of proteins by urea in the bulk phase is shown to affect early regimes.     

The adsorption characteristics, activity and stability of trypsin onto mesoporous silicates

The immobilization of the hydrolytic enzyme trypsin onto various mesoporous silicates (MPS) was studied. MPS were prepared using cationic or non-ionic surfactants (average pore diameters were in the range of 28–300 Å). All MPS were characterised by X-ray diffraction, scanning electron microscopy and nitrogen porosimetry. Enzyme purity strongly influenced loading. Trypsin adsorbed on MPS was found to be desorbed more readily by polyethylene glycol than by ammonium sulphate, suggesting that hydrophobic–hydrophilic interactions were important. Immobilized trypsin showed 10–20 times higher activity than the free trypsin and was stable for 4–6 weeks when stored at 4 or 25 °C. The trypsin–MPS catalyst was successfully reused for up to 6 cycles     

Role of organic solvents on Pa-hydroxynitrile lyase interfacial activity and stability

Catalytic activity and adsorption of Pa-hydroxynitrile lyase (Pa-Hnl) was investigated at various organic solvent/water interfaces. We focused on the role of solvent polarity in promoting activity and stability in two-phase systems, specifically for the solvents heptane, dibutyl ether (DBE), diisopropyl ether (DIPE), butylmethyl ether (BME), and methyl tert-butyl ether (MTBE). Enzyme activity towards mandelonitrile cleavage was determined in a recycle reactor with a well-defined interfacial area as described by Hickel, et al. 1999. Here the recycle reactor was modified to permit exchange of the aqueous phase. With this modification, irreversibility of enzyme adsorption was determined as a function of the adsorption time at the interface. Irreversibility of enzyme adsorption was also investigated by measuring the surface pressure of a sessile-drop upon washout. We find that Pa-Hnl exhibits the highest stability but the lowest initial catalytic activity at the aqueous/organic solvent interface with the most polar organic solvents. Thus, DIPE and MTBE display no loss in enzyme activity over a period of several hours. However, the more apolar the solvent is the higher the initial Pa-Hnl activity, but the faster the loss of activity. Dynamic tensiometry reveals that Pa-Hnl adsorbs more strongly at the interface of the more apolar solvents. Surprisingly, Pa-Hnl develops some irreversible adsorption after 30 min at the DIPE/water interface, but does not lose catalytic activity.     

The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.

Site-directed mutagenesis and other molecular biology-based techniques are now available for probing the amphipathic alpha helix structural motif in the exchangeable apolipoproteins. Here we survey the published literature on lipid-binding and functional domains in apolipoproteins A-I, A-II, A-IV, C-I, C-II, C-III, and E and compare these results with recently developed computer methods for analysis of the location and properties of amphipathic helixes. This comparison suggests that there are at least three distinct classes of amphipathic helixes (classes A, Y, and G*) in the exchangeable apolipoproteins whose distribution varies within and between the seven apolipoproteins. This comparison further suggests that lipid affinity resides largely in class A amphipathic helixes (Segrest, J. P., et al. 1990. Proteins. 8: 103) and that variations in structure and/or numbers of class A domains in individual apolipoproteins allow a range of lipid affinities from high to low. The positions of the four alpha helixes recently shown to form a 4-helix bundle globular structure in apoE (Wilson, C., et al. 1991. Science. 252: 1817) correspond closely to the four amino-terminal class G* amphipathic helixes of apoE identified by our computer analysis. It is of particular interest, therefore, that all of the exchangeable apolipoproteins except apoA-II and C-I, contain amphipathic helixes of class G*. Additional implications of amphipathic helix heterogeneity for the structure and function of the exchangeable apolipoproteins will be discussed.     

Intracellular role of exchangeable apolipoproteins in energy homeostasis,obesity and non‐alcoholic fatty liver disease

Exchangeable apolipoproteins play an important role in systemic lipid metabolism, especially for lipoproteins with which they are associated. Recently, emerging evidence has suggested that exchangeable apolipoproteins, such as apolipoprotein A4 (apoA4), apolipoprotein A5 (apoA5), apolipoprotein C3 (apoC3) and apolipoprotein E (apoE), also exert important effects on intracellular lipid homeostasis. There is a close link between lipid metabolism in adipose tissue and liver because the latter behaves as the metabolic sensor of dysfunctional adipose tissue and is a main target of lipotoxicity. Given that the energy balance between these two major lipogenic organs is intimately involved in the pathogenesis of obesity and non‐alcoholic fatty liver disease (NAFLD), we here review recent findings concerning the intracellular function of exchangeable apolipoproteins in triglyceride metabolism in adipocytes and hepatocytes. These apolipoproteins may act as mediators of crosstalk between adipose tissue and liver, thus influencing development of obesity and hepatosteatosis. This review provides new insights into the physiological role of exchangeable apolipoproteins and identifies latent targets for therapeutic intervention of obesity and its related disorders.     

In vitro studies of disease-linked variants of human tRNA nucleotidyltransferase reveal decreased thermal stability and altered catalytic activity

Mutations in the human TRNT1 gene encoding tRNA nucleotidyltransferase (tRNA-NT), an essential enzyme responsible for addition of the CCA (cytidine-cytidine-adenosine) sequence to the 3′-termini of tRNAs, have been linked to disease phenotypes including congenital sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD) or retinitis pigmentosa with erythrocyte microcytosis. The effects of these disease-linked mutations on the structure and function of tRNA-NT have not been explored. Here we use biochemical and biophysical approaches to study how five SIFD-linked amino acid substitutions (T154I, M158V, L166S, R190I and I223T), residing in the N-terminal head and neck domains of the enzyme, affect the structure and activity of human tRNA-NT in vitro. Our data suggest that the SIFD phenotype is linked to poor stability of the T154I and L166S variant proteins, and to a combination of reduced stability and altered catalytic efficiency in the M158 V, R190I and I223T variants.     

Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area - tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 3 10(-)(3) sec(-)(1), whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 3 10(-)(3) sec(-)(1). Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure.We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles.     

Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability.

Surface tension-area relations from pulmonary surfactant were obtained with a new apparatus that contains a leak free captive bubble of controllable size. Rat pulmonary surfactant was studied at phospholipid concentrations of 50, 200 and 400 micrograms/ml. At the highest concentration, adsorption was rapid, reaching surface tensions below 30 mN/m within 1 s, while at the lowest concentration, approximately 3 min were required. Upon a first quasi static or dynamic compression, stable surface tensions below 1 mN/m could be obtained by a film area reduction of approximately 50%. After three to four cycles the surface tension-area relations became stationary, and the tension fell from 25-30 to approximately 1 mN/m for a film area reduction of less than 20%. Hysteresis became negligible, provided the films were not collapsed by further area reduction. Under these conditions, the films could be cycled for more than 20 min without any noticeable loss in surface activity. After only three to four consecutive cycles, surfactant films exhibited the low surface tensions, collapse rates and compressibilities characteristic of alveolar surfaces in situ. Remarkably, surface tension and area are interrelated in the captive bubble which may promote low and stable surface tensions. If the surface tension of the captive bubble suddenly increases ('click') because of mechanical vibration or unstable surfactant, the bubble shape changes from flat to more spherical. The associated isovolumetric decrease in surface area prevents the surface tension from rising as much as it would have in a constant-area situation. This feedback mechanism may also have a favorable effect in stabilizing alveolar surface tension at low lung volumes.     

Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein

The T347S polymorphism in the human apolipoprotein (apo) A-IV gene is present at high frequencies among all the world''s populations. Carriers of a 347S allele exhibit faster clearance of triglyceride-rich lipoproteins, greater adiposity, and increased risk for developing atherosclerosis, which suggests that this conservative amino acid substitution alters the structure of apo A-IV. Herein we have used spectroscopic and surface chemistry techniques to examine the structure, stability, and interfacial properties of the apo A-IV 347S isoprotein. Circular dichroism spectroscopy revealed that the 347S isoprotein has similar α-helical structure but lower thermodynamic stability than the 347T isoprotein. Fluorescence spectroscopy found that the 347S isoprotein exhibits an enhanced tyrosine emission and reduced tyrosine→tryptophan energy transfer, and second derivative UV absorption spectra noted increased tyrosine exposure, suggesting that the 347S isoprotein adopts a looser tertiary conformation. Surface chemistry studies found that although the 347S isoprotein bound rapidly to the lipid interface, it has a lower interfacial exclusion pressure and lower elastic modulus than the 347T isoprotein. Together, these observations establish that the T347S substitution alters the conformation of apo A-IV and lowers its interfacial activity—changes that could account for the effect of this polymorphism on postprandial lipid metabolism.     

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.     

Comparison of the relative biological availability and stability of commercial sources of vitamin K activity

In a test designed to examine the stability and biological availability of different sources of menadione available to the feed industry, encapsulated menadione sodium bisulphite (Kastab) and menadione dimethylpyrimidinol bisulphite appeared to be relatively equal in both stability under steam pelleting and biological availability as measured by chick prothrombin times. Menadione sodium bisulphite complex did not appear to be as biologically available but possessed equal stability under pelleting. Particle size of all supplements was similar, suggesting no probable differences in distribution into the feed. The data from this study support the current National Research Council suggestion of 400 μg of menadione activity per kg of diet.     

Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking,stability, and activity

The function of the human norepinephrine transporter (hNET) depends on its presence at the cell surface. A role for the hNET C-terminus in trafficking the transporter to the surface has been suggested by the report of a bovine NET C-terminal splice variant that accumulates within heterologous host cells, and a human variant homolog has also been reported. We examined the relevance of the C-terminus of hNET to trafficking and function using transfected LLC-PK1 cells. The intracellular and surface expression of NET proteins was evaluated by Western blots, and their functional capacities were assessed using transport assays. We found that the C-terminal residues encoded by hNET 1a enable the efficient maturation and surface expression of hNET and therefore critically impact transporter activity. Alternative splicing causes the retention of immature hNETs within the cell, whereas introduced C-terminal deletions result in significant degradation. The loss of the terminal isoleucine alone (Delta617-hNET) is sufficient to cause the degradation of hNET, an effect that can be mimicked by nonconservative point mutations at the terminal position. The phenotype of Delta617-hNET is recapitulated in neuronal SK-N-MC cells, but is significantly less severe in HEK-293 cells, suggesting a role for host cell factors in enabling the biosynthetic progression of wild-type hNET. Additional proximal residues may act at other steps to affect the expression of the fully mature protein on the cell surface (Q608A) and to more directly affect transporter activity (F609A). Together our studies document a critical contribution of the hNET C-terminus to transporter trafficking, stability, and function.     

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

The N-terminus modulates human Caf1 activity, structural stability and aggregation

Caf1 is a deadenylase component of the CCR4-Not complex. Here we found that the removal of the N-terminus resulted in a 30% decrease in human Caf1 (hCaf1) activity, but had no significant influence on main domain structure. The removal of the N-terminus led to a decrease in the thermal stability, while the existence of the N-terminus promoted hCaf1 thermal aggregation. Homology modeling indicated that the N-terminus had a potency to form a short α-helix interacted with the main domain. Thus the N-terminus played a role in modulating hCaf1 activity, stability and aggregation.