Cataract as a protein condensation disease: the Proctor Lecture.
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Aging↗
Biomedical subjects
G B Benedek
Publications and source records attributed to G B Benedek.
On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.
We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.
Amyloid beta-Peptides↗
Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.
We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.
Animals↗
Pantethine inhibits the formation of high-Tc protein aggregates in gamma B crystallin solutions.
PURPOSE: Solutions of the bovine lens protein gamma B (or gamma II) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, gamma IIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (approximately equal to 32% of total protein by weight) and loosely associated dimers (approximately equal to 66%). gamma IIH has a phase separation temperature (Tph), at least 40 degrees C higher than that of native gamma II crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of gamma IIH. METHODS: gamma II crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tph of the solutions was monitored as a function of incubation time. Corresponding to each Tph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of gamma IIH formed. RESULTS: Incubation of gamma II crystallin with increasing amounts of pantethine lowers Tph and suppresses the formation of gamma IIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the Tph of gamma II crystallin is lowered from 8 degrees C in the native protein, to 2 degrees C, -3 degrees C respectively, at a protein concentration of approximately equal to 200 mg/ml. The amount of gamma IIH accumulated decreases from approximately 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-treated protein solutions. For complete suppression of the rise in Tph and inhibition of gamma IIH formation, a 2:1 mole ratio of pantethine to protein is required. CONCLUSIONS: We suggest that pantethine reacts with two cysteine residues of gamma IIH crystallin by forming a mixed disulfide, and effectively suppress protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.
Animals↗
Elastic free energy of anisotropic helical ribbons as metastable intermediates in the crystallization of cholesterol.
We report measurements of the geometrical structure and temporal evolution of metastable helical intermediates in the pathway for cholesterol crystallization in native and model biles. We find that the lecithin component in the bile can dramatically affect the kinetics along this pathway. We also present a theoretical description of these helical intermediates using an elastic free energy appropriate for anisotropic bilayers of tilted chiral amphiphiles, which provides a quantitative description of the observed helical ribbon geometry and insight into the relative free energies of the observed metastable intermediates.
Bile↗
Solid-liquid phase boundaries of lens protein solutions.
We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf gamma-crystallin proteins: gamma II, gamma IIIa, and gamma IIIb. We also studied the liquidus line for solutions of native gamma IV-crystallin calf lens protein, which consists of 85% gamma IVa/15% gamma IVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-liquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location of the liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures greater than 70 degrees C: gamma IIIa and gamma IV, and those in which liquidus lines flatten at temperatures less than 50 degrees C: gamma II and gamma IIIb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.
Animals↗
Binary-liquid phase separation of lens protein solutions.
We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf gamma IIIa-, gamma IIIb-, and gamma IVa-crystallin, have very similar amino acid sequences and three-dimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for gamma II-crystallin can be fit, near the critical point, to the function /(Cc-C)/Cc/ = A [(Tc - T)/Tc]beta, where beta = 0.325, Cc is the critical protein concentration in mg/ml, Tc is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A and Cc are approximately the same for all four coexistence curves (A = 2.6 +/- 0.1, Cc = 289 +/- 20 mg/ml), but that Tc is not the same. For gamma II- and gamma IIIb-crystallin, Tc approximately 5 degrees C, whereas for gamma IIIa- and gamma IVa-crystallin, Tc approximately 38 degrees C. By comparing the published protein sequences for calf, rat, and human gamma-crystallins, we postulate that a few key amino acid residues account for the division of gamma-crystallins into low-Tc and high-Tc groups.
Animals↗
Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis.
We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.
Age Factors↗
Heterogeneity of gamma-crystallins from spiny dogfish (Squalus acanthias) eye lens.
Mammalian lenses contain multiple gamma-crystallin gene products, which are differentially synthesized during lens development. We now report the isolation and characterization of multiple gamma-crystallins from lenses of adult spiny dogfish (Squalus acanthias) aged about 20-30 years. About 50% of total lens protein solubilized in 50 mM phosphate, pH 7.0; about 25% of this soluble fraction consists of gamma-crystallins as determined by gel filtration. These gamma-crystallins appear homogeneous with respect to molecular weight (approximately equal to 20,000) on SDS-polyacrylamide gels, but their isoelectric points range from below pH 6 to above 10. Preparative cation-exchange chromatography on SP-Sephadex at pH 4.8 resolves four major subfractions, while anion-exchange on DEAE-cellulose at pH 9.5 resolves seven subfractions. Although these procedures separate basic from acidic polypeptides, most of these gamma-crystallin subfractions still consist of polypeptide mixtures, as determined by ion-exchange HPLC and isoelectric focusing. Analytical cation-exchange HPLC on SynChropak CM300 at pH 6.0 resolves at least 10 different gamma-crystallin components. Amino acid compositions of all the subfractions are similar, yet distinct in the sense that three subclasses can be distinguished. Sulfhydryl residues range from three to six per chain, most of which are buried. The large heterogeneity of gamma-crystallins in adult lens may result from different gene products in combination with post-translational modification.
Amino Acids↗
Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies.
We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.
Apolipoprotein A-I↗
Formation of mixed micelles and vesicles of human apolipoproteins A-I and A-II with synthetic and natural lecithins and the bile salt sodium taurocholate: quasi-elastic light scattering studies.
We employed quasi-elastic light scattering to systematically study the interactions of human apolipoproteins A-I and A-II (apo A-I and apo A-II) with synthetic and natural lecithins, analogues of the major membrane lipids found in high-density lipoproteins (HDL). Equilibrium values of the mean hydrodynamic radius (Rh) of systems with varying concentrations of dimyristoylphosphatidylcholine (DMPC) to apolipoprotein showed that as the percentage of DMPC was increased, three distinct regions were observed. At low DMPC to apolipoprotein ratios, Rh values either increased, remained constant, or decreased, depending upon the total apolipoprotein concentration, which influenced the size of pure apolipoprotein micelles [Donovan, J. M., Benedek, G. B., & Carey, M. C. (1987) Biochemistry (preceding paper in this issue)]. When the percentage of DMPC approached the micellar phase boundary, Rh values uniformly diverged (50 to approximately 150 A); with the percentage of DMPC in excess of the micellar phase boundary, large Rh values (200-300 A) were observed that were consistent with unilamellar apo A-I/DMPC or apo A-II/DMPC vesicles. Decreases in total solute concentration (1.0-0.25 mg/mL) and/or elevations in temperature (25-37 degrees C) shifted micellar phase limits to lower percentages of DMPC in the case of both apolipoproteins. Although apo A-I interacted spontaneously with DMPC at 25 degrees C, it was necessary to dilute mixed micellar solutions of sodium taurocholate (TC) and egg yolk phosphatidylcholine (EYPC) with apo A-I solutions to form apo A-I/EYPC mixed micelles. Despite the presence of submicellar concentrations of TC (below 3 mM, the lower limit of its critical micellar concentration), Rh values of apo A-I/EYPC mixed micelles were similar to those observed for the apo A-I/DMPC system. Dilution of micellar TC/EYPC solutions with low concentrations of apo A-I (0.001-0.10 mg/mL) influenced the width of the mixed micellar zone, the kinetics of micelle-to-vesicle transitions, and the size of metastable vesicles. Dilution with higher apo A-I concentrations (0.05 and 0.1 mg/mL) resulted in the transformation of bile salt rich EYPC micelles into apo A-I rich EYPC micelles with an intervening zone of metastable vesicles. The micelle-to-vesicle transition was abolished by dilution with an apo A-I concentration of 0.5 mg/mL, suggesting that bile salts and apo A-I can directly interchange in micellar solubilization of EYPC.(ABSTRACT TRUNCATED AT 400 WORDS)
Apolipoprotein A-I↗
Human lens gamma-crystallins: isolation, identification, and characterization of the expressed gene products.
We have isolated the individual gamma-crystallins expressed in young human lenses and identified with which of the six known human gamma-crystallin genes they each correspond. We find that at least 90% of the gamma-crystallins synthesized in the young human lens are the products of genes gamma G3 and gamma G4. We demonstrate that gamma G4-crystallin undergoes a temperature-dependent phase separation, and we have measured the low-concentration branch of its coexistence curve (phase separation temperature vs. concentration) up to about 40 mg/ml. By comparison, we found no evidence of gamma G3-crystallin phase separating, even at lower temperatures and higher concentrations. This is consistent with predictions based on sequence homology between human and rat gamma-crystallins. The implications of these findings for human inherited and senile cataracts are considered.
Aging↗
Binary liquid phase separation and critical phenomena in a protein/water solution.
We have investigated the phase diagram of aqueous solutions of the bovine lens protein gamma II-crystallin. For temperatures T less than Tc = 278.5 K, we find that these solutions exhibit a reversible coexistence between two isotropic liquid phases differing in protein concentration. The dilute and concentrated branches of the coexistence curve were characterized, consistently, both by measurements of the two coexisting concentrations, c(T), and by measuring the cloud temperatures for various initial concentrations. We estimate that the critical concentration, cc, is 244 mg of protein per ml solution. The coexistence curve is well represented by the absolute value of (c - cc)/cc = 5.2 square root (Tc - T)/Tc. Using the temperature dependence of the scattered light intensity along isochores parallel to the critical isochore, we estimated the location of the spinodal line and found it to have the form (c - cc)/cc = 3.0 square root (Tc - T)/Tc. The ratio of the widths of the coexistence curve and the spinodal line, (5.2/3.0), is close to the mean-field value square root 3. We have also observed the growth of large crystals of gamma II-crystallin in some of these aqueous solutions and have made preliminary observations as to the factors that promote or delay the onset of crystallization. These findings suggest that selected protein/water systems can serve as excellent model systems for the study of phase transitions and critical phenomena.
Animals↗
Quantitative detection of the molecular changes associated with early cataractogenesis in the living human lens using quasielastic light scattering.
We have used quasielastic light scattering to detect and quantitatively characterize the molecular changes associated with the early stages of cataractogenesis in the living human lens. The autocorrelation function of the fluctuations in the light scattered by the lens shows the presence of two major species responsible for the scattering. The first, fast diffusing species (f), has a diffusivity of approximately 3 x 10(-7) cm2/sec and corresponds to the alpha crystallin proteins. The second, slow diffusing species (s), has a diffusivity of approximately 10(-9) cm2/sec and corresponds to the diffusivity of a large aggregate. The intensity of light If and Is scattered into the collection optics by each of these species was also measured. We studied a group of 49 individuals ranging in age from 21 years to 82 years. In this group 40 presented with preoperative cataract development. In this patient population we found that regardless of age, or position in the lens that a plot of Itot = If+Is versus Is could be well fitted by a straight line with a slope less than unity and a positive intercept Ifo. It has been possible to explain this finding using a two state model for the molecular changes associated with early cataractogenesis. In this model the proteins in the slow diffusing species are aggregates each containing a definite number of rapidly diffusing proteins. The early development of cataract is represented by the redistribution of protein between the unaggregated form (f) and the aggregated form (s). The prediction for the relationship between Itot and Is based on this two state model is in very good agreement with our experimental data. Indeed the measured position of the point (Itot, Is) along this line provides a sensitive, and quantitative measure of the degree of cataract development at any selected location in the lens.
Adult↗
In vivo measurement of the aging rabbit lens using quasielastic light scattering.
We have been able to analyze the autocorrelation function of the light scattered from the rabbit lens in vivo in terms of a two component, exponential fit. We have measured the intensity and the decay rate associated with each of these two components as a function of age and position in the lens. As far as is possible we have interpreted these results in terms of molecular changes in the state of association of the protein constituents in the aging rabbit lens. These studies suggest that the method of quasielastic light scattering spectroscopy can provide a useful probe of the protein modifications that occur in both normal and cataractous human lenses.
Aging↗
Permanent suppression of phase separation cataract in calf lens using amine modification agents.
Low temperature induced opacification (cold cataract) of the nucleus of young mammalian lenses is associated with a phase separation of proteins in the lens cell cytoplasm. Calf lenses were treated with a variety of imido-esters and N-hydroxysuccinimide-esters, which react specifically with amino groups. Many potent inhibitors of phase separation cataract were identified which lower the opacification temperature by 6 degrees C or more. Lenses generally remain clear, colorless and soft. Furthermore, suppression of the cold cataract temperature is permanent upon removal of excess reagent.
Animals↗
A quantitative microprobe analysis of elements in cortical and nuclear cells of the calf lens.
The outer cortical cells in the calf lens remain transparent under conditions that produce opacity in central nuclear cells. The nuclear cells opacify by a mechanism of cellular restructuring that is associated with a cytoplasmic phase separation while cortical cells do not opacify by this mechanism. In this study the differences in elemental composition of nuclear and cortical cells were analyzed using X-ray emission spectroscopy (XES) of tissue that was prepared for scanning electron microscopy. It was necessary to develop special methods of fixation and dehydration to prevent significant distortion of lens tissue and minimize solubilization and redistribution of elements during the histological processing of the tissue. We calibrated the microprobe for the quantitative analysis using gelatin standards which contained known concentrations of sulfur, potassium, phosphorus, chlorine, and cesium. The standard curves were used to determine proportionality constants, which related the intensity of X-ray emission to the molar concentration of each element, and to determine the minimum detectable levels of each element. An important finding is that the intensity of the X-ray emission is dependent on sample density only at low protein concentration. At the high protein concentrations that exist in lens, the intensity is not affected by sample density.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Opacification of gamma-crystallin solutions from calf lens in relation to cold cataract formation.
To determine the molecular mechanisms for cold cataract formation in the nucleus of the young mammalian lens, we have investigated the thermally reversible opacification of gamma-crystallin solutions isolated from calf lens. Coexistence curves (plots of opacification temperature Tc versus protein concentration) were determined for the individual gamma-crystallin fractions II, III, and IV as well as for the unfractionated gamma-crystallin mixtures isolated from the nucleus and cortex. The coexistence curve of gamma IV-crystallin is remarkably elevated above those of gamma II- and gamma III-crystallin and the gamma-crystallin mixtures. The gamma IV-crystallin fraction is the major determinant of the opacification temperature within the whole lens or isolated cytoplasm. Quasielastic light-scattering spectroscopy of gamma IV-crystallin solutions indicates that above Tc there are two populations of protein aggregates of distinctly different mean size. As the temperature is lowered towards Tc, both populations increase in size. Opacification occurs when the population of large scatterers, which is composed of less than 0.1% protein by weight, reaches an average radius of about 20,000 A.
Animals↗