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Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone.

ABSTRACT: Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-crystallin oligomers formed long-lived stable complexes with their gammaD-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with alpha-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

SUBMITTER: Acosta-Sampson L

PROVIDER: S-EPMC3072757 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone.

Acosta-Sampson Ligia L King Jonathan J

Journal of molecular biology 20100601 1

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insolubl ...[more]

PMID: 20621668

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Project description:Multiple interactive domains are involved in the activity of the stress protein, alphaB crystallin that protects against the unfolding, aggregation, and toxicity of amyloidogenic proteins. Six peptides corresponding to the interactive sequences 41STSLSPFYLRPPSFLRAP58, 73DRFSVNLDVKHFS85, 101HGKHEERQDE110, 113FISREFHR120, 131LTITSSLSSDGV142, and 156ERTIPITRE164 in human alphaB crystallin were synthesized and evaluated in Thioflavin T fluorescence assays for their effects on the modulation of fibrillation of four disease-related amyloidogenic proteins: amyloid-beta, alpha-synuclein, transthyretin, and beta2-microglobulin. The 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110 peptides in the conserved alpha crystallin core domain of alphaB crystallin were the most effective fibril inhibitors. 73DRFSVNLDVKHFS85 completely inhibited alpha-synuclein fibrillation and reduced the fibrillation of amyloid-beta, transthyretin, and beta2-microglobulin by >50%. 101HGKHEERQDE110 completely inhibited amyloid-beta fibrillation and reduced the fibrillation of alpha-synuclein, transthyretin, and beta2-microglobulin by >50%. The peptides FSVN, NLDV, HGKH, and HEER, which are synthetic fragments of 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110, inhibited fibrillation of all four amyloidogenic proteins by >75%. In contrast, the peptides FISREFHR, ERTIPITRE, DRFS, KHFS, and EERQ were the strongest promoters of fibrillation. Molecular modeling of the interactions between transthyretin and beta2-microglobulin and the synthetic bioactive peptides determined that residues Phe-75, Ser-76, Val-77, Asn-78, Leu-79, and Asp-80 in 73DRFSVNLDVKHFS85 and residues His-101, Lys-103, His-104, Glu-105, and Arg-107 in 101HGKHEERQDE110 interact with exposed residues in the beta strands, F and D of transthyretin and beta2-microglobulin, respectively, to modulate fibrillation. This is the first characterization of specific bioactive peptides synthesized on the basis of interactive domains in the small heat shock protein, alphaB crystallin that protect against the fibrillation of amyloidogenic proteins.

Project description:Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, betaA3/A1(102-117) peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by beta- and gamma-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of alphaB-crystallin. The present study was performed to identify the interacting sites of the betaA3/A1(102-117) peptide in alphaB-crystallin.betaA3/A1(102-117) peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotin-containing cross-linker. The biotin-derivatized peptide was then incubated with alphaB-crystallin at 37 degrees C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to alphaB-crystallin. Label transfer was confirmed by western blot, and the labeled alphaB-crystallin was digested with trypsin. Tryptic peptides from alphaB-crystallin carrying the biotin label were purified by avidin affinity chromatography, and betaA3/A1(102-117) peptide interacting sites in alphaB-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS).We found that the betaA3/A1(102-117) peptide interacted with alphaB-crystallin regions (70)LEKDR(74), (83)HFSPEELKVK(92), (91)VKVLGDVIEVHGK(103), (93)VLGDVIEVHGKHEER(107), and (121)KYR(123), which are part of the alpha-crystallin domain, and were previously shown to be part of the functional chaperone site in alphaB-crystallin. The betaA3/A1(102-117) peptide also interacted with regions at the COOH-terminal extension of alphaB-crystallin, (150)KQVSGPER(157), (164)EEKPAVTAAPK(174), and (164)EEKPAVTAAPKK(175). When two of the hydrophobic residues of betaA3/A1(102-117) peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property.This study confirmed the interactions between a low molecular weight peptide derived from betaA3/A1-crystallin found in aged and cataract lenses and alphaB-crystallin. The binding of betaA3/A1(102-117) peptide to the chaperone site and the COOH-terminal extension of alphaB-crystallin may explain its anti-chaperone property.

Project description:Molecular chaperones including the small heat shock proteins, alphaB crystallin and sHSP27 participate in the assembly, disassembly, and reorganization of the cytoskeleton during cell development and differentiation. While alphaB crystallin and sHSP27 stabilize and modulate filament assembly and re-organization, the sequences and structural domains mediating interactions between these proteins and filaments are unknown. It is important to define these interactive domains in order to understand differential interactions between chaperones and stable or unfolding filaments and their function in the cellular stress response. Protein pin arrays identified sequences in human alphaB crystallin that selectively interacted with native or partially unfolded filament proteins desmin, glial-fibrillary acidic protein, and actin. Circular dichroism spectroscopy determined differences in the structure of these filaments at 23 and 45 degrees C. Seven alphaB crystallin sequences had stronger interactions with desmin and six sequences had stronger interactions with glial-fibrillary acidic protein at 23 degrees C than at 45 degrees C. The alphaB crystallin sequences (33)LESDLFPTSTSLSPFYLRPPSFLR(56) and (129)DPLTITSSLSSDGV(145) had the strongest interactions with actin at 23 degrees C, while (57)APSWFDTG(64), (111)HGFISREF(118), (145)VNGPRKQVSG(154), and (155)PERTIPITREEK(165) had the strongest interactions with actin at 45 degrees C. The actin interactive sequences of alphaB crystallin overlapped with previously identified alphaB crystallin chaperone sequences and were synthesized to evaluate their effect on the assembly and aggregation of actin. Full-length alphaB crystallin and the core domain chaperone sequence (131)LTITSSLSSDGV(143) promoted actin polymerization at 37 degrees C and inhibited depolymerization and aggregation at 50 degrees C. The results support the hypothesis that interactive domains in alphaB crystallin have multiple functions in stabilizing the cytoskeleton and protecting cytosolic proteins from unfolding.

Dataset Information

Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone.

Publications

Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone.

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