Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins.

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.

Prion protein expression in mammalian lenses.

PURPOSE: Aging and oxidative stress resulting from over-expression of Alzheimer precursor protein (betaAPP) have been studied as important factors contributing to the major age-related (sporadic), and minor (hereditary) forms of Alzheimer's disease (AD), and muscle inclusion body myositis, (IBM). AD and prion proteins accumulate in plaques linked with AD and scrapie diseases, and in rimmed vacuoles of IBM. Soluble beta-amyloid (Abeta) fibrillar forms are now thought to play a critical role in and outside of cells by producing oxidative stress. In lens, betaAPP and Abeta increase in cultured lenses exposed to oxidative stress, and in areas of lens fiber cell degeneration in thiamine (vitamin B1) deprived mice, a classic model of systemic oxidative stress. The purpose of the present study is to extend our studies of amyloid disease-related protein expression in mammalian lenses. METHODS: Western blot, immunohistochemical detection, and RT-PCR methods were used to identify and quantitate prion protein expression in human, monkey, and guinea pig lenses. RESULTS: We demonstrate for the first time that prion protein gene expression increases with oxidative stress in cultured human lens epithelial cells. In addition, we detected greater prion protein gene expression in fiber cells than epithelial cells in vivo. This is consistent with increases in prion protein expression demonstrated in myoblasts and neuronal cells induced to differentiate. Our initial investigations of prion protein in human lens cataracts identified increased prion protein immunoreactivity in regions of lens fiber cell degeneration. CONCLUSIONS: The present data indicate that prion protein expression increases during lens development, and is substantially increased in cultured human lens epithelial cells exposed to oxidative stress. We also provide evidence that prion protein immunoreactivity can be increased in regions of fiber cell disorganization. These data suggest a potential role for prion protein as a marker for some types of lens pathology, and in the mechanism of oxidative stress-related lens degeneration.

Refinement of 3D structure of bovine lens alpha A-crystallin.

In absence of 3D structures for alpha-crystallin subunits, alpha A and alpha B, we utilized a number of experimental and molecular modeling techniques to generate working 3D models of these polypeptides (Farnsworth et al., 1994. In Molecular Modeling: From Virtual Tools to Real Problems (Eds. Kumosinski, T.F. and Liebman, M.N.) ACS Symposium Series 576, Ch. 9:123-134, 1994, ACS Books, Washington DC). The refinement of the initial bovine alpha A model was achieved using a more accurate estimation of secondary structure by new/updated methods for analyzing the far UV-CD spectra and by neural network secondary structure predictions in combination with database searches. The spectroscopic study reveals that alpha-crystallin is not an all beta-sheet protein but contains approximately 17% alpha-helices, approximately 33% beta-structures and approximately 50% turns and coils. The refinement of the alpha A structure results in an elongate, asymmetric amphipathic molecule. The hydrophobic N-terminal domain imparts the driving force for subunit aggregation while the more flexible, polar C-terminal domain imparts aggregate solubility. In our quaternary structure of the aggregate, the monomer is the minimal cooperative subunit. In bovine alpha A, the highly negatively charged C-terminal domain has three small positive areas which may participate in dimer or tetramer formation of independently expressed C-terminal domains. The electrostatic potential of positive areas is modulated and become more negative with phosphorylation and ATP binding. The refined bovine alpha A model was used to construct alpha A models for the human, chick and dogfish shark. A high degree of conservation of the three dimensional structure and the electrostatic potential was observed. Our proposed open micellar quaternary structure correlates well with experimental data accumulated over the past several decades. The structure is also predictive of the more recent data.

Interaction of DNA with bovine lens alpha-crystallin: its functional implications.

Under normal conditions, lens aggregates of alpha-crystallin subunits, alpha A and alpha B, are found in the cytoplasm. However, during stress in nonlenticular tissues, alpha B translocates to the nucleus. A sequence study revealed that both subunits share a consensus sequence with other DNA binding proteins. These observations prompted us to investigate DNA binding with alpha-crystallin by UV-mediated photo-crosslinking. The data show that both single and double stranded DNA crosslink mainly with tetramers of alpha-crystallin subunits. The formation of tetramers appears to modify alpha-crystallin interactive properties and, therefore, its induction may have functional significance. These observations suggest that alpha-crystallin may have a nuclear function which includes DNA binding.

Localization of B-DNA and Z-DNA in terminally differentiating fiber cells in the adult lens.

We examined histochemically and immunohistochemically the distribution of B- and Z-DNA in the epithelium and terminally differentiating dog lens fiber cells. On the basis of anti-DNA antibody reactivity, qualitative and quantitative data on B- and Z-DNA in cells were determined. Anti-B-DNA immunoreactivity gradually declined throughout nucleated fibers, with a precipitous decrease at approximately 90 microns. Anti-Z-DNA antibody binding decreased with a sudden loss of immunoreactivity at approximately 90 microns. The pattern of anti-B- and Z-DNA staining correlates with the loss of alpha-crystallin immunoreactivity, the major lens crystallin, and decreased eosin staining of proteins. Germinative zone cell nuclei showed the highest DNA probe binding values, followed by the superficial fibers, central zone, middle fibers, and deep fibers. The presence of single-stranded (ss)DNA in deeper fibers was detected by anti-ss-DNA antibodies. This is indicative of DNA degradation. These observations suggest that a dramatic reorganization of lens fiber cells' supramolecular order occurs at approximately 90 microns, the phase transition zone.

Effects of temperature and concentration on bovine lens alpha-crystallin secondary structure: a circular dichroism spectroscopic study.

Elucidation of the structure of alpha-crystallin, the major protein in all vertebrate lenses, is important for understanding its role in maintaining transparency and its function in other tissues under both normal and pathological conditions. Progress toward a unified consensus concerning the tertiary and quaternary structures of alpha-crystallin depends, in part, on an accurate estimation of its secondary structure. For the first time, three algorithms, SELCON, K2D and CONTIN were used to analyze far ultra-violet circular dichroism (UV-CD) spectra of bovine lens alpha-crystallin to estimate the secondary structure and to determine the effects of temperature and concentration. Under all experimental conditions tested, the analyses show that alpha-crystallin contains 14% alpha-helix, 35% beta-sheet and the remainder, random coil and turns. The results suggest that alpha-crystallin is best classified as a mixed protein. In addition, increased temperature and concentration of alpha-crystallin result in increased alpha-helices with a compensatory decrease in beta-sheets. Such structural alterations in alpha-crystallin may be functionally important during terminal differentiation of the lens fiber cells that is accompanied by increased protein concentrations and its role as a chaperone-like protein.

A comparison of structural relationships among alpha-crystallin, human Hsp27, gamma-crystallins and beta B2-crystallin.

The 3D structures of alpha-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that alpha-crystallin is primarily a beta-sheet globular protein similar to alpha-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56-67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1-6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of beta/gamma-crystallins. Similar analyses of alpha-crystallin subunits, alpha A and alpha B, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike beta/gamma-crystallins, both Hsp27 and alpha-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in alpha-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in alpha-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that alpha-crystallin and members of beta/gamma superfamily are structurally dissimilar.

Effect of thiobase incorporation into duplex DNA during the polymerization reaction.

6-Thioguanine, a thioderivative of the nucleic acid base guanine, is a metabolic inhibitor and possesses antitumor activity. In addition, it inhibits the synthesis of nucleotides and is incorporated readily into the nucleic acid during the polymerization reaction by substituting for guanine. The biological activity of 6-thioguanine has been related to the alterations in the dimensions and energetics of a duplex DNA as a result its incorporation in place of guanine (Bugg and Thewalt 1975). With the advancement in the computer hardware technology and emergence of sophisticated molecular modeling packages, it is now possible to simulate the biological activities of different drugs. An effort has been made to explain the biological activities of thoiderivatives of nucleic acid bases. For this, semiempirical MNDO quantum chemical calculations were performed on some non-complementary (thiosubstituted) base pairs to understand the effects of substitution of sulfur for oxygen in nucleic acid bases. The base pairs considered in this study are thioguanine-cytosine (TG-C), guanine-thiocytosine (G-TC) and thioguanine-thiocytosine (TG-TC). The results obtained suggest that the replacement of guanine by thioguanine and/or replacement of cytosine by thiocytosine weakens the stability of the double helical DNA by reducing the strength of the hydrogen bonds.

Electrostatic parameters of the theoretical quaternary structure of bovine alpha-crystallin.

By changing the ionic strength, the effects of charge modification on the electrostatic properties of our predicted "open' micellar quaternary structure composed of bovine alpha A subunits were determined. The electrostatic potential values (phi) at 6 arbitrary points surrounding the protein and at all atomic sites of the protein were calculated using the non-linear Poisson-Boltzmann equation. The effective charge (q) of our predicted aggregate ranged from 16 at 0.0022 M to 45 at 0.1472 M ionic strengths. The variation of potential (phi), as well as charge, is a hyperbolic function of ionic strength (R2, 0.901). Plotting the inverse charge (l/q) against inverse ionic strength (1/l) it is possible to calculate maximum charge (q(max)) (approximately 48) at saturation. This value is close to previously reported experimental (50 +/- 5), and our theoretical charge (45), values at physiological ionic strength (0.145 M). These data indicate that maximal repulsion among the alpha-crystallin aggregates occurs at or near physiological ionic strength. Also, half-maximal charge (q(max)/2) at 0.003-0.004 M indicates a transition state at very low ionic strength. The calculated volume available for the mobile solvent in our quaternary structure is approximately 70%. These data are in good agreement with experimental values for bovine alpha-crystallin in solution reported by Xia et al. (Biophys. J., 1994; 66: 861-872). This agreement provides support for our predicted models of alpha-crystallin and a level of confidence in the reliability of the theoretical calculations. Since an ionic gradient exists between the lens cortical and nuclear regions, the modification of charge on alpha-crystallin by varying ionic strength could contribute to the function of alpha-crystallin as a modulator of lens supermolecular order during fiber cell maturation.

alpha-Crystallin quaternary structure: molecular basis for its chaperone activity.

alpha-Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an 'open' micellar structure composed of alpha A subunits is used to simulate chaperoning of partially heat denatured soluble gamma-crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 alpha/gamma molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601-13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311-21]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509-16]; i.e. the bound gamma-crystallin monomers are not in a central cavity, but are separated by alpha A subunits.

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy.

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37 degrees C: binding sites per 400 kDa aggregate = 10 and Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 10(3) M-1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum of 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P(i), AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.

Advances in computer technology: impact on the practice of medicine.

Advances in computer technology provide a wide range of applications which are revolutionizing the practice of medicine. The development of new software for the office creates a web of communication among physicians, staff members, health care facilities and associated agencies. This provides the physician with the prospect of a paperless office. At the other end of the spectrum, the development of 3D work stations and software based on computational chemistry permits visualization of protein molecules involved in disease. Computer assisted molecular modeling has been used to construct working 3D models of lens alpha-crystallin. The 3D structure of alpha-crystallin is basic to our understanding of the molecular mechanisms involved in lens fiber cell maturation, stabilization of the inner nuclear region, the maintenance of lens transparency and cataractogenesis. The major component of the high molecular weight aggregates that occur during cataractogenesis is alpha-crystallin subunits. Subunits of alpha-crystallin occur in other tissues of the body. In the central nervous system accumulation of these subunits in the form of dense inclusion bodies occurs in pathological conditions such as Alzheimer's disease, Huntington's disease, multiple sclerosis and toxoplasmosis (Iwaki, Wisniewski et al., 1992), as well as neoplasms of astrocyte origin (Iwaki, Iwaki, et al., 1991). Also cardiac ischemia is associated with an increased alpha B synthesis (Chiesi, Longoni et al., 1990). On a more global level, the molecular structure of alpha-crystallin may provide information pertaining to the function of small heat shock proteins, hsp, in maintaining cell stability under the stress of disease.

Effect of catalytically active recruited crystallins on lens metabolism.

The impact on duck lens metabolism of recruited epsilon-crystallin/LDH-B4 and tau-crystallin/enolase was investigated by NMR spectroscopy. A comparison of the duck lens metabolite profile with that of the calf in which recruited crystallins are absent revealed significant increases in ATP, alpha-glycerophosphate (alpha-GP) and pyridine dinucleotide concentrations. The alterations in the concentrations of ATP and alpha-GP appear to be related to both the high concentration of NAD and the elevated reduced to oxidized NADH/NAD+ ratio.

Methylisocyanate and actin polymerization: the in vitro effects of carbamylation.

Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4 degrees C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.

Modulation of actin polymerization by an exogenous protein, lysozyme.

Several methods (fluorescence, high- and low-shear viscosity, and electron microscopy) have been applied to measure the effects of lysozyme on actin polymerization. Under our conditions, at pH 8.0 and 20 degrees C, lysozyme is predominantly dimeric and its major effect is to inhibit the steady-state polymerization of actin. Those actin filaments formed in the presence of lysozyme are significantly shortened with recurrent amorphous densities along the filament length. However, at pH 6.4 and 37 degrees C, lysozyme is monomeric and actin filament cross-linking is observed. We reasoned that in hen egg white lysozyme the tripeptide L-arginyl-glycyl-aspartate (RGD), a sequence capable of mimicking a portion of the receptor sites of extracellular matrix proteins, might be important in lysozyme self-association and, therefore, actin-lysozyme interaction. The presence of RGD in the lysozyme-actin polymerizing solutions at pH 8.0 and 20 degrees C caused an inhibition of the dimeric lysozyme effects, while RGD alone had no effects on actin polymerization. Therefore, RGD most likely binds to a complementary RGD sequence on lysozyme and alters its ability to interact with actin and modify polymerization.

31P NMR studies of the ATP/alpha-crystallin complex: functional implications.

Evidence is presented for the binding of ATP to alpha-crystallin in the lens by 31P NMR spectroscopic measurements. The chemical shift data as well as the T1 and T2 values indicate that P beta and P gamma of ATP are of prime importance in binding. In addition, it is demonstrated that the association of alpha-crystallin with purified fiber cell membranes is significantly enhanced by the addition of ATP. These results suggest that ATP modulates the functional behavior of alpha-crystallin.

Regional variations in electrolytes related to resistivity during bovine lens maturation.

Little is known regarding the behavior of ions in protein-rich cytoplasm characteristic of lens fiber cells. Resistivity is dependent upon the electrolyte concentration available to conduct an applied current and the mobility of these electrolytes. In the present study, the relative importance of these factors in the increasing cortico-nuclear resistivity gradient reported for both calf and bovine lens homogenates was analysed. Relative ion mobility for regions of the lens was determined by the calculation of the ratio of resistivity of lens homogenates to resistivity of aqueous solutions of freely mobile KCl at the same molarity. The increasing resistivity ratios in the calf cortex, transition zone and nucleus suggest an increasingly impaired ion mobility from the outer to the inner lens regions.

Investigation of lens glycolytic enzymes: species distribution and interaction with supramolecular order.

Distribution of several glycolytic enzymes in the lenses of different vertebrate species and their organization in the calf lenses were studied. Though no general pattern of enzyme activities in different species is discernible, high activities of TPI followed, in decreasing order, by GAPDH, enolase, PK, LDH and aldolase appear to be more common. Our observation on the unusually high activities of aldolase in the pig, enolase in the sheep and LDH in the duck lens are interesting in view of the already known dual function of LDH as an enzyme and a structural protein (epsilon-crystallin) in duck. Controlled treatment with detergents Brij-58 and Triton X-100 caused distinctly differential purturbations in the lens cells. In spite of fiber membrane disruption and partial actin dissolution by Brij-58, no significant increase in the release of glycolytic enzymes compared to control was observed. This suggests that none of the enzymes existed as a completely soluble and freely diffusible fraction. But treatment with a strong detergent (Triton X-100) caused the release of higher amounts of enzymes suggesting either a direct or indirect interaction with the cytomatrix components. Aldolase appears to be maximally bound in the cytosol followed by TPI, GAPDH, LDH and PK in decreasing order. Although thin lens slices were incubated with the detergents for a total period of 40 min and the loss of fiber architecture and organization confirmed by light microscopy, in the Triton X-100 treated tissues less than 25% of the total activity of any enzyme except TPI appeared in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)