Structural alterations of alpha-crystallin during its chaperone action

The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.     

Effect of the chaperone-like alpha-crystallin on the refolding of lysozyme and ribonuclease A

This article is a critical review of the evolution of diagnostic criteria for anorexia nervosa, bulimia nervosa, and binge eating disorder. The shortcomings of the current diagnostic criteria for all three disorders are addressed, as are the primary nosological challenges facing the field.     

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.     

Mutation of alpha B-crystallin: effects on chaperone-like activity

A recent paper by Plater et al. [20], showed that the mutation of a single phenylalanine residue F27R in mouse alpha B completely abolished the chaperone-like property of alpha-crystallin when assayed with insulin at 25 degrees C or with gamma-crystallin at 66 degrees C. We have produced the same mutation as well as some additional mutations in human alpha B-crystallin. Our data suggest that the F27R mutation effected the thermal stability of alpha B-crystallin making it unstable at temperatures > or = 60 degrees C. In agreement with the published work, at these temperatures the F27R human recombinant alpha B-crystallin does not protect the target protein from aggregation. When assayed with insulin or alpha-lactalbumin at 25 or 37 degrees C, however, there were no differences in the protective abilities between the native alpha B-crystallin or the F27R mutated human alpha B-crystallin. Several other multiple mutations involving proline residues were also produced. These mutations did not effect the chaperone-like properties of human alpha B-crystallin, but some of them did effect the native molecular weight size as judged by gel filtration chromatography.     

Thermodynamic stability of bovine alpha-crystallin in its interactions with other bovine crystallins

Light scattering measurements were performed on dilute solutions of alpha-crystallin mixed with different combinations of beta H, beta L and gamma-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of alpha-crystallin is progressively enhanced by its interactions with gamma [symbol: see text] (beta H + gamma) [symbol: see text] (beta H + beta L + gamma) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.     

Identification of photooxidation sites in bovine alpha-crystallin

Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine alpha-crystallin was irradiated with UV light (> 293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-22 and 57-69 of alpha B-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22. The specific sites of photooxidation indicate that the N-terminal regions of alpha A- and alpha B-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.     

Conformational change of human lens insoluble alpha-crystallin

BACKGROUND: It is useful to measure the luminal concentration of drugs which act in the gut. Dialysis of the rectum has not previously been used or validated for this purpose. AIM: To determine the precision of rectal dialysis for measuring rectal drug concentrations. METHODS: To establish the duration of dialysis required to approach equilibrium, the rate of methotrexate diffusion into dialysis bags was first determined in vitro. The precision of rectal dialysis for sampling the methotrexate concentration of colonic lumen extracellular fluid was determined in seven subjects who underwent two consecutive dialysis procedures. Subjects treated with subcutaneous methotrexate for refractory inflammatory bowel disease were studied. RESULTS: Methotrexate crossed the dialysis membrane by a first-order process, and after a 2 h in vitro dialysis, equilibration was 74 +/- 2% (mean +/- s.d.) complete. Rectal dialysis was well tolerated by all subjects. The mean +/- s.e. methotrexate concentration of 3.6 +/- 1.1 nmol/L in the first dialysate was not significantly different from 3.6 +/- 0.9 nmol/L in the second dialysate. P = 0.99 (paired two-tailed t-test). Similar precision was obtained for an endogenous molecule, potassium, secreted by the rectal mucosa. CONCLUSIONS: Dialysis of the rectum is a well tolerated and precise technique for sampling the colonic lumen extracellular fluid for quantitative analyses of exogenous and endogenous substances.     

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.     

Structural requirements for alpha-mating factor activity

The sexual hormone of S. cerevisiae, alpha-mating factor (alpha-MF, WHWLQLKPGQPMY) has structural homology with mammalian luteinizing hormone releasing hormone (LHRH, pEHWSYGLRPG-NH2) and has been shown to exhibit LHRH activity [Loumaye et al. (1982) Science 218, 1323-1325]. We have tested whether LHRH has alpha-MF activity in yeast and found that it does not. We therefore synthesized a series of hybrid peptides of alpha-MF and LHRH to study the structural features which determine alpha-MF and LHRH activities. A hybrid peptide consisting of the LHRH sequence with the C-terminal tetrapeptide (QPMY) of alpha-MF did not exhibit alpha-MF activity. Thus, the lack of alpha-MF activity of LHRH is not due solely to the absence of the C-terminal residues. Substitution of Lys7 in alpha-MF with Arg, as is found in LHRH, did not affect the alpha-MF activity, nor did an additional substitution of Trp1 with pGlu. However, the C-terminal four amino acids of alpha-MF were necessary for alpha-MF activity. Our results indicate that insertion of a Ser residue in position 4 as found in LHRH abolishes alpha-MF activity. These results suggest that, in addition to an intact C-terminus, correct spacing of the N-terminal His2 and the C-terminus is required for alpha-MF activity. The hybrid peptides all exhibited less LHRH activity than either LHRH or alpha-MF. These structure-function studies indicate that the structural homology between these two reproductive hormones may not reflect an evolutionary relationship between them.     

Quaternary structure of bovine alpha-crystallin: influence of temperature

The tertiary and quaternary structure of alpha-crystallin is still a matter of controversy. We have characterized the native alpha-crystallin quaternary structure by isolating it at the in vivo temperature and solvent conditions. It can be represented by a distribution of expanded particles with a weight average molar mass of 550,000 g/mol. On decreasing (to 4 degrees C) or increasing (up to 50 degrees C) the temperature, the size distribution increases to larger particles. Only at lower temperatures (4 degrees C), a stable population of particles is obtained with weight average molar mass of 700,000 g/mol. In all conditions, alpha-crystallin behaves as a very expanded particle with a maximum hydrodynamic volume of 3.15 ml/g. The transitions in quaternary structure are rather slow: it takes several hours to evolve from a population of aggregates, characteristic for given solvent conditions, to another distribution in size and quaternary structure on changing the environment. The quaternary structure of alpha-crystallin is an uncharacteristic parameter of the particle: a broad distribution of values can be obtained on changing the environment. Any realistic model should include this property. Our studies favor an open loose structure, where peptides can be added or removed without drastic changes of secondary and tertiary structure of the peptides.     

Evolution of the alpha-crystallin/small heat-shock protein family

The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.     

Chemical modifications and dissociation characteristics of tyrosine and tryptophan residues in alpha-crystallin

A quantitative estimation of surface accessibility of aromatic residues in alpha-crystallin from goat lens has been accomplished by chemical modifications using different specific reagents having varying sizes. Results of modification of tyrosine residues with N-acetylimidazole and tetranitromethane when combined with those of ionization studies carried out with hydroxyl ions having the smallest size reveal different classes of tyrosine residues in the native protein: 78 +/- 2 residues have been found to be easily available for modification; among the rest, 94 +/- 2 residues appear to be comparatively less exposed to the reagents while 28 +/- 2 residues are found to be completely unavailable for modification in the native protein and are modified only when the protein is denatured. Modification of tryptophan residues with H2O2 also indicates different classes of these residues available for oxidation at different concentrations of the oxidant. 34 +/- 2 residues of tryptophan are found to be easily oxidized at a lower concentration of H2O2 during the first phase of the reaction. The remaining tryptophan residues appear to be less exposed to the reagent. This is also corroborated from the studies of reactivities of these residues towards another specific but bulkier reagent, 2-hydroxy-5-nitrobenzyl bromide. These surface exposed aromatic residues in alpha-crystallin may be considered to be vulnerable to in vivo oxidative modifications forming insoluble aggregates which may finally contribute to the formation of cataract.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of alpha A- and alpha B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to alpha B-crystallin, but not to alpha A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to alpha A- and alpha B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to alpha A- and alpha B-crystallin. In rat embryos, alpha A-crystallin appeared in the lens pit at E12, and alpha B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to alpha A-crystallin. The lens fiber cells were also immunoreactive to alpha B-crystallin, but the epithelial cells were not. These findings suggest that alpha B-crystallin appears earlier than alpha A-crystallin in the human lens, but at a later period than alpha A-crystallin in the rat lens. alpha B-Crystallin was not detected in the epithelial cells of the rat lens, but was persistently present in the epithelial cells of the human lens.     

Structural features and protective activity of dexamethasone and pregnenolone-16 alpha-carbonitrile

Pretreatment with dexamethasone acetate or pregnenolone-16 alpha-carbonitrile markedly diminished the pharmacological effect of zoxazolamine in rats. This prophylactic action was associated with significantly decreased plasma drug levels, which, in turn, were correlated with enhanced hepatic drug biotransformation, induced by the steroids. Dexamethasone proved to be more active than pregnenolone-16 alpha-carbonitrile in this respect. The A-ring conformation as well as the distances of O-3-O-11, O-11-O-17, and the O-3-mean plane C-5-C-17 may be key factors in glucocorticoid activity, and the longer the distances, the greater the potency. These characteristics have no bearing on catatoxic activity for which the 16 alpha-substituent appears to be a structural prerequisite.     

Modal Perturbation Method and its Applications in Structural Systems

A new perturbation method is developed to solve any eigenvalue equation of the form (A0+ΔA)X? = (B0+ΔB)X?Λ? based on the solution of an original system described by A0X = B0XΛ. The eigenvectors of the modified system are expanded in a subspace spanned with a small number of vibration modes of the original system. In doing so, the former eigenvalue equation of the modified system is transformed into a set of algebraic equations, which require a significantly less computational effort to solve for the eigensolutions of complex structural systems. Four numerical examples show that the developed technique gives rise to the eigensolution of high accuracy and it is an effective approach for dynamic reanalysis of the structures with numerous degrees of freedom. In comparison with the conventional small parameter perturbation, the developed technique is applicable to a wider range of problems, and only m mode shapes are used based on the Ritz expansion so that the final solution can be derived efficiently. The technique also extends laboratory model tests for complex structures with the concept of dynamic hybrid tests numerically and experimentally.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.     

alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner

alpha-crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced damage and to share sequence similarity with small heat-shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by alpha-crystallin, alpha A-crystallin or alpha B-crystallin. However, phosphorylation of alpha-crystallin markedly decreased its protective effect. It is suggested that phosphorylation of alpha-crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain alpha-crystallin species. This communication presents biochemical evidence clearly demonstrating that alpha-crystallin is involved in actin polymerization-depolymerization dynamics. It is also shown that alpha-crystallin prevented heat-induced aggregation of actin filaments. alpha-crystallin was found to stabilize actin polymers decreasing dilution-induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 microM to 0.18 microM. Similar results were found with either alpha-crystallin or its purified subunits alpha A-crystallin and alpha B-crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between alpha-crystallin and actin monomers since the effect of alpha-crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the alpha-crystallin effect indicates that 2-3 alpha-crystallin monomers/actin monomer give maximum actin polymer stabilization.