Dataset Information

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

ABSTRACT: AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

SUBMITTER: Ecroyd H

PROVIDER: S-EPMC1698675 | biostudies-other | 2007 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets