The complete sequence of human lens gamma s-crystallin.
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ABSTRACT:
SUBMITTER: Smith JB
PROVIDER: S-EPMC1136663 | biostudies-other | 1995 Apr
REPOSITORIES: biostudies-other
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Project description:Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.
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Project description:Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
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Project description:Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. betaB2 is the most abundant beta-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. betaB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among beta-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type betaB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes betaB2 and may disrupt the function of betaB2 in the lens.
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Project description:Lens cells contain high concentrations of thiol-rich proteins, gamma-crystallins and reduced glutathione. Solutions of bovine gamma-crystallin react avidly with either reduced or oxidized glutathione to form protein-glutathione mixed disulphides. A method of purification of a gamma-II crystallin-glutathione adduct containing two mixed disulphide groups is described.
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Project description:A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.
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Project description:Lens is a member of the Papilionoideae subfamily of Fabaceae and is generally used as a source of vegetable protein as part of human diets in many regions worldwide. Chloroplast (cp) genomes are highly active genetic components of plants and can be utilized as molecular markers for various purposes. As one of the wild lentil species, the Lens ervoides cp genome has been sequenced for the first time in this study using next-generation sequencing. The de novo assembly of the cp genome resulted in a single 122,722 bp sequence as two separate coexisting structural haplotypes with similar lengths. Results indicated that the cp genome of L. ervoides belongs to the inverted repeat lacking clade. Several noteworthy divergences within the coding regions were observed in ndhB, ndhF, rbcL, rpoC2, and ycf2 genes. Analysis of relative synonymous codon usage showed that certain genes, psbN, psaI, psbI, psbE, psbK, petD, and ndhC, preferred using biased codons more often and therefore might have elevated expression and translation efficiencies. Overall, this study exhibited the divergence level between the wild-type and cultured lentil cp genomes and pointed to certain regions that can be utilized as distinction markers for various goals.
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Project description:The amino acid sequence of gamma-crystallin (fraction II) from calf lens has been determined; this indicates it to be a single-chain polypeptide of 165 amino acid residues.
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Project description:Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All of the major crystallins exhibited N?-succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC-MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses (age > 60 years) relative to young lenses (age < 30 years). Immunohistochemical analyses revealed that SuccK was present in epithelium and fiber cells. Western blotting and immunoprecipitation experiments revealed that SuccK is particularly prominent in ?B-crystallin, and succinylation in vitro revealed that ?B-crystallin is more prone to succinylation than ?A-crystallin. Mass spectrometric analyses showed succinylation at K72, K90, K92, K166, K175, and potentially K174 in human lens ?B-crystallin. We detected succinylation at K72, K82, K90, K92, K103, K121, K150, K166, K175, and potentially K174 by mass spectrometry in mildly succinylated ?B-crystallin. Mild succinylation improved the chaperone activity of ?B-crystallin along with minor perturbation in tertiary and quaternary structure of the protein. These observations imply that succinylation is beneficial to ?B-crystallin by improving its chaperone activity with only mild conformational alterations.
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Project description:Cataracts are a major cause of blindness worldwide and commonly occur in individuals over 70 years old. Cataracts can also appear earlier in life due to genetic mutations. The lens proteins, αA- and αB-crystallins, are chaperone proteins that have important roles maintaining protein solubility to prevent cataract formation. Mutations in the CRYAA and CRYAB crystallin genes are associated with autosomal dominant early onset human cataracts. Although studies about the proteomic and genomic changes that occur in cataracts have been reported, metabolomics studies are very limited. Here, we directly investigated cataract metabolism using gas-chromatography-mass spectrometry (GC-MS) to analyze the metabolites in adult Cryaa-R49C and Cryab-R120G knock-in mouse lenses. The most abundant metabolites were myo-inositol, L-(+)-lactic acid, cholesterol, phosphate, glycerol phosphate, palmitic and 9-octadecenoic acids, α-D-mannopyranose, and β-D-glucopyranose. Cryaa-R49C knock-in mouse lenses had a significant decrease in the number of sugars and minor sterols, which occurred in concert with an increase in lactic acid. Cholesterol composition was unchanged. In contrast, Cryab-R120G knock-in lenses exhibited increased total amino acid content including valine, alanine, serine, leucine, isoleucine, glycine, and aspartic acid. Minor sterols, including cholest-7-en-3-ol and glycerol phosphate were decreased. These studies indicate that lenses from Cryaa-R49C and Cryab-R120G knock-in mice, which are models for human cataracts, have unique amino acid and metabolite profiles.
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Project description:Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.
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