Dataset Information

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin.

ABSTRACT: Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.

SUBMITTER: Takata T

PROVIDER: S-EPMC2525517 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin.

Takata Takumi T Oxford Julie T JT Demeler Borries B Lampi Kirsten J KJ

Protein science : a publication of the Protein Society 20080620 9

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of b ...[more]

PMID: 18567786

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Project description:Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. ?B2 crystallin in the lens of the eye readily forms dimers with other ?-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study.Structures of ?A3 and ?B2 crystallin homodimers and the ?A3/?B2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen-deuterium exchange with mass spectrometry. We further mimicked deamidation in ?B2 and probed the effect on the ?A3/?B2 heterodimer. Results were confirmed with chemical crosslinking and NMR.Both ?A3 and ?B2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of ?B2 was supported by the identification of chemical crosslinks between lysines in ?B2 within the heterodimer that were inconsistent with ?B2's extended homodimeric structure. The compact structure of ?A3 was supported by an overall decrease in mobility of ?A3 in the heterodimer detected by NMR. In ?B2, peptides 70-84 and 121-134 were exposed in the homodimer, but buried in the heterodimer with ?50% decreases in deuterium levels. Homologous peptides in ?A3, 97-109 and 134-149, had 25-50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between ?B2 and ?A3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in ?B2 at this predicted interface led to a less compact ?B2 in the heterodimer. The more compact structure of the ?A3/?B2 heterodimer was also more heat stable than either of the homodimers.The major structural proteins in the lens, the ?-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of ?B2, may facilitate formation of different size higher-ordered structures found in the transparent lens.Understanding complex hetero-oligomer interactions between ?-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Dataset Information

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin.

Publications

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin.

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