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Joseph Horwitz - One of the best experts on this subject based on the ideXlab platform.
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Alpha Crystallin the quest for a homogeneous quaternary structure
Experimental Eye Research, 2009Co-Authors: Joseph HorwitzAbstract:Alpha A and Alpha B Crystallins are key members of the small heat-shock protein family. In addition to being a major structural protein of the lens, they are constitutively found in many other cells, where their function is not completely understood. Alpha B Crystallin is also known to be over-expressed in many neurological diseases. To date, all efforts to crystallize Alpha A or Alpha B have failed. Thus, high-resolution data on the tertiary and quaternary structures of Alpha Crystallin is not available. The main reason for this failure seems to be the polydisperse nature of Alpha Crystallin. This review deals mainly with the polydisperse properties of Alpha Crystallin and the influence of post-translational modification, chemical modifications, truncations and mutation on its quaternary structure.
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the function of Alpha Crystallin in vision
Seminars in Cell & Developmental Biology, 2000Co-Authors: Joseph HorwitzAbstract:Abstract The Alpha-Crystallins account for approximately one-third of the total soluble protein in the lens, contributing to its refractive power. In addition, Alpha-Crystallin also has a chaperone-like function and thus can bind unfolding lens proteins. Alpha B-Crystallin is also found outside the lens, having an extensive tissue distribution. It is over-expressed in response to stresses of all kinds, where it is thought to serve a general protective function. Recently, it has been shown in humans that naturally occurring point mutations in the Alpha-Crystallins result in a deficit in chaperone-like function, and cause cataracts as well as a desmin-related myopathy. This review summarizes much of the past and current knowledge concerning the structure and functions of Alpha-Crystallin.
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Interaction of .Alpha.-Crystallin with Spin-Labeled Peptides
Biochemistry, 1995Co-Authors: Zohreh Toossi Farahbakhsh, Qingling Huang, Linlin Ding, Christian Altenbach, Heinz-juergen Steinhoff, Joseph Horwitz, Wayne L. HubbellAbstract:Alpha-Crystallin is a major protein of the vertebrate lens once thought to be highly specialized for conferring transparency. However, recent work has revealed a wide tissue distribution and a sequence homology to small heat shock proteins, suggesting a more general role for the protein. Like other molecular chaperons, Alpha-Crystallin is known to bind to unfolded proteins and suppress nonspecific aggregation in vitro. In the present work, spin-labeled derivatives of the insulin B chain and melittin were used to investigate the state of these proteins bound to Alpha-Crystallin. Insulin was selected since unfolding can be triggered by reduction of the interchain disulfide bonds, a treatment that does not affect Alpha-Crystallin. Upon reduction of insulin, the separated B chains aggregate. In the presence of Alpha-Crystallin, the B chains bind to Alpha-Crystallin and aggregation is suppressed. Melittin, a 26 amino acid peptide from bee venom, was selected for study since it is a random coil under physiological conditions, and its interaction with Alpha-Crystallin can be directly studied. EPR analysis of the spin-labeled peptides shows that the nitroxide side chains are immobilized in a polar environment on Alpha-Crystallin and that they are separated by 25 A or more in the complex, indicating that the bound proteins are not clustered. The bound B chains of insulin are not in a fully extended conformation, and melittin does not appear to bind to a hydrophobic surface in Alpha-Crystallin as an amphipathic helix, as it does to membranes and some other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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Chaperone-like activity of Alpha-Crystallin. The effect of NADPH on its interaction with zeta-Crystallin.
Journal of Biological Chemistry, 1994Co-Authors: Joseph Horwitz, J. S. ZiglerAbstract:Abstract Alpha-Crystallin, a major structural protein of the ocular lens of vertebrates, has been characterized recently as a molecular chaperone (Horwitz, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10449-10453 and Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520). While Alpha-Crystallins prevent the aggregation of various proteins denatured by heat or chaotropic agents, neither the mode of interaction between target proteins and Alpha-Crystallin nor the specific conformational requirements, if any, of the target protein are known. Here, we demonstrate that the ability of Alpha-Crystallin to prevent thermally induced aggregation of zeta-Crystallin/NADPH:quinone oxidoreductase, an abundant Crystallin of guinea pigs and camelids, is strongly dependent on the presence of the obligate cofactor (NADPH) of the target enzyme. zeta-Crystallin in the absence of NADPH is readily aggregated at 41 degrees C, and Alpha-Crystallin added at a 1:1 (w/w) ratio offers very little protection. In contrast, in the presence of NADPH zeta-Crystallin remains stable to 45 degrees C and with the addition of Alpha-Crystallin (1:1 (w/w)) is protected from aggregation even at 55 degrees C. Cibacron blue 3GA, a nonmetabolized pyridine nucleotide analog, which has very high binding affinity to zeta-Crystallin had similar effects. NADH and NAD+, which are not bound by zeta-Crystallin, had no such effect. Complex formation between Alpha-Crystallin and non-native zeta-Crystallin was demonstrated in the presence of either cibacron blue 3GA or NADPH. Circular dichroism spectroscopy of zeta-Crystallin in the presence and absence of NADPH or cibacron blue indicated that nucleotide binding was accompanied by a change in the protein's aromatic amino acid environment but that the secondary structure was unaffected. The data suggest that subtle change in the conformation of denaturing proteins can markedly affect the ability of Alpha-Crystallin to protect them from aggregation.
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structural and functional similarities of bovine Alpha Crystallin and mouse small heat shock protein a family of chaperones
Journal of Biological Chemistry, 1993Co-Authors: Karin B Merck, Joseph Horwitz, Patricia J T A Groenen, Christina E M Voorter, W A De Haardhoekman, H Bloemendal, W W De JongAbstract:Abstract Alpha-Crystallin, composed of the subunits Alpha A and Alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine Alpha-Crystallin, with respect to several known properties of the latter. We show that Alpha-Crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of Alpha A-Crystallin, Alpha B-Crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like Alpha-Crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like Alpha B-Crystallin. Our results thus corroborate that Alpha-Crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.
Roger J.w. Truscott - One of the best experts on this subject based on the ideXlab platform.
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Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, Alpha-Crystallin, in maintaining lens flexibility.
Aging cell, 2007Co-Authors: Karl R Heys, Michael G Friedrich, Roger J.w. TruscottAbstract:Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, Alpha-Crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble Alpha-Crystallin from the centre of the lens. These alterations in Alpha-Crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 degrees C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for Alpha-Crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of Alpha-Crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.
W W De Jong - One of the best experts on this subject based on the ideXlab platform.
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Mutations and modifications support a 'pitted-flexiball' model for Alpha-Crystallin.
International journal of biological macromolecules, 1998Co-Authors: R H Smulders, M A Van Boekel, W W De JongAbstract:Alpha-Crystallin is renown for resisting crystallization and electron microscopic image analysis. The spatial conformation thus remaining elusive, the authors explored the structure and chaperone functioning by analyzing the effects of site-directed mutagenesis, the properties of naturally occurring aberrant forms of Alpha-Crystallin and the influence of chemical modifications. The authors observed that the globular multimeric structure, as well as the chaperoning capacity are remarkably tolerant towards changes and modifications in the primary structure. The essential features of the quaternary structure--globular shape, flexibility, highly polar exterior and accessible hydrophobic surface pockets--support a 'pitted-flexiball' model, which combines tetrameric subunit building blocks in an open micelle-like arrangement.
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The influence of some post-translational modifications on the chaperone-like activity of Alpha-Crystallin.
Ophthalmic research, 1996Co-Authors: M A Van Boekel, J J Harding, S E Hoogakker, W W De JongAbstract:We investigated the influence of phosphorylation, glycation, carbamylation and oxidative modification on the capacity of Alpha-Crystallin to protect beta-Crystallins against heat denaturation. Simple modification of lysine residues by early glycation or carbamylation had no effect. However, late (cross-linking) glycation products and oxidative modifications decreased the chaperone-like activity of Alpha-Crystallin. Homopolymers of Alpha A-Crystallin had a higher protecting capacity compared with those of Alpha B-Crystallin. The in vivo phosphorylated forms of especially Alpha A- but also Alpha B-Crystallin revealed a somewhat better protecting ability than the respective non-phosphorylated forms.
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Evolution of the Alpha-Crystallin/small heat-shock protein family.
Molecular Biology and Evolution, 1993Co-Authors: W W De Jong, J A M Leunissen, C. E. M. VoorterAbstract:Abstract 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.
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structural and functional similarities of bovine Alpha Crystallin and mouse small heat shock protein a family of chaperones
Journal of Biological Chemistry, 1993Co-Authors: Karin B Merck, Joseph Horwitz, Patricia J T A Groenen, Christina E M Voorter, W A De Haardhoekman, H Bloemendal, W W De JongAbstract:Abstract Alpha-Crystallin, composed of the subunits Alpha A and Alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine Alpha-Crystallin, with respect to several known properties of the latter. We show that Alpha-Crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of Alpha A-Crystallin, Alpha B-Crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like Alpha-Crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like Alpha B-Crystallin. Our results thus corroborate that Alpha-Crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.
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evolution of the Alpha Crystallin small heat shock protein family
Molecular Biology and Evolution, 1993Co-Authors: W W De Jong, J A M Leunissen, Christina E M VoorterAbstract:Abstract 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.
C Slingsby - One of the best experts on this subject based on the ideXlab platform.
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wrapping the Alpha Crystallin domain fold in a chaperone assembly
Journal of Molecular Biology, 2005Co-Authors: Robin Stamler, Guido Kappe, Wilbert C Boelens, C SlingsbyAbstract:Small heat shock proteins (sHsps) are oligomers that perform a protective function by binding denatured proteins. Although ubiquitous, they are of variable sequence except for a C-terminal similar to 90-residue "Alpha-Crystallin domain". Unlike larger stress response chaperones, sHsps are ATP-independent and generally form polydisperse assemblies. One proposed mechanism of action involves these assemblies breaking into smaller subunits in response to stress, before binding unfolding substrate and reforming into larger complexes. Two previously solved non-metazoan sHsp multimers are built from dimers formed by domain swapping between the Alpha-Crystallin domains,. adding to evidence that the smaller subunits are dimers. Here, the 2.5 angstrom resolution structure of an sHsp from the parasitic flatworm Taenia saginata Tsp36, the first metazoan crystal structure, shows a new mode of dimerization involving N-terminal regions, which differs from that seen for non-metazoan sHsps. Sequence differences in the a-Crystallin domains between metazoans and nonmetazoans are critical to the different mechanism of dimerization, suggesting that some structural features seen for Tsp36 may be generalized to other metazoan sHsps. The structure also indicates scope for flexible assembly of subunits, supporting the proposed process of oligomer breakdown, substrate binding and reassembly as the chaperone mechanism. It further shows how sHsps can bind coil and secondary structural elements by wrapping them around the Alpha-Crystallin domain. The structure also illustrates possible roles for conserved residues associated with disease, and suggests a mechanism for the sHsp-related pathogenicity of some flatworm infections. Tsp36, like other flatworm sHsps, possesses two divergent sHsp repeats per monomer. Together with the two previously solved structures, a total of four Alpha-Crystallin domain structures are now available, giving a better definition of domain boundaries for sHsps.
Larry J. Takemoto - One of the best experts on this subject based on the ideXlab platform.
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Preferential interaction of Alpha Crystallin with denatured forms of gamma Crystallin
Investigative Ophthalmology & Visual Science, 1994Co-Authors: S. Gopalakrishnan, Daniel L. Boyle, Larry J. TakemotoAbstract:Purpose. To characterize the possible interaction of Alpha Crystallin with partially denatured forms of gamma Crystallin. Methods. Gamma Crystallin was denaturated in the presence of guanidine hydrochloride, then dialyzed in the presence or absence of Alpha Crystallin. The high-molecular-weight complex formed in the presence of Alpha was characterized by gel filtration chromatography, electron microscopy, and quantitative Western blot analysis. Results. Relative to native Alpha or reconstituted aggregated of purified Alpha, the higher molecular weight complex possessed a greater mean diameter and contained increased amounts of gamma Crystallin
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An assay for intermolecular exchange of Alpha Crystallin.
Investigative Ophthalmology & Visual Science, 1992Co-Authors: S. Gopalakrishnan, Larry J. TakemotoAbstract:An affinity column of Alpha Crystallin linked to cyanogen bromide-activated Sepharose was developed to study the exchange of Alpha subunits. Alpha Crystallin bound to the Sepharose-Alpha complex was dissociated with 8 mol/l urea, followed by quantitation using high-performance reverse-phase liquid chromatography. The time course of binding at 37 degrees C showed a hyperbolic binding pattern reaching equilibrium between 6-18 hr. Under these conditions, binding of beta and gamma Crystallins to the same matrix was less than 10% of the Alpha values, as was binding of Alpha to glycine-coupled Sepharose. This assay was used to demonstrate changes in the subunit exchange of Alpha Crystallins present in high molecular weight versus lower molecular weight aggregates of the human lens. These results show that this binding procedure was a specific reproducible assay that might be used to study intermolecular interactions of the Alpha Crystallins.
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The ability of lens Alpha Crystallin to protect against heat-induced aggregation is age-dependent.
Current Eye Research, 1992Co-Authors: Joseph Horwitz, T. Emmons, Larry J. TakemotoAbstract:Alpha Crystallin was prepared from newborn and aged bovine lenses. SDS-PAGE and tryptic peptide napping demonstrated that both preparations contained only the Alpha-A and Alpha-B chains, with no significant contamination of other Crystallins. Compared with Alpha Crystallin from the aged lens, Alpha Crystallin from the newborn lens was much more effective in the inhibition of betaL Crystallin denaturetion and precipitation induced in vitro by heat. Together, these results demonstrate that during the aging process, the Alpha Crystallins lose their ability to protect against protein denaturation, consistent with the hypothesis that the Alpha Crystallins play an important role in the maintenance of protein native structure in the intact lens.
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Specificity of Alpha Crystallin binding to the lens membrane
Current Eye Research, 1990Co-Authors: F. Ifeanyi, Larry J. TakemotoAbstract:The A1, A2, B1, and B2 species of bovine Alpha Crystallin have been purified and renatured to form high molecular weight aggregates comprised of only one species, and the aggregated forms of each of these species have been tested for their ability to bind to lens membrane in vitro. The aggregated forms of Alpha-A1 and Alpha-A2 bound to membrane in a saturable manner while those of Alpha-B1 and Alpha-B2 bound in much lower amounts, in a manner inconsistent with saturable binding. Together, these results demonstrate specific and saturable binding of aggregated Alpha-A1 and Alpha-A2 to the lens membrane, suggesting that these species are responsible for the previously observed interaction between Alpha Crystallin and the lens fiber cell membrane.
