Size of Human Lens β-Crystallin Aggregates Are Distinguished by N-terminal Truncation of βB1

Ajaz, M. Saleh; Ma, Zijuan; Smith, David L.; Smith, Jean B.

doi:10.1074/jbc.272.17.11250

Cited by 63 publications

(86 citation statements)

References 27 publications

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“…Here two issues come to the fore. The first is to determine whether the N-terminal arm of B1 actually facilitates hetero-oligomerization as has been suggested (8,21,22). The second is to determine how hetero-oligomerization affects the phase behavior of the multicomponent -crystallin mixtures.…”

Section: Discussionmentioning

confidence: 99%

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Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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Human B1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native B1 and truncated B1∆N41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of B1 aqueous solutions. The oligomerization studies show that the energy of attraction between the B1∆N41 proteins is about 10% greater than that of the B1 proteins. We have found that B1∆N41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of B1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for B1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between B1 and B1∆N41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of B1 in maintaining lens transparency.

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Section: Discussionmentioning

confidence: 99%

“…It was found that H consists mainly of B1 and A4, while L2 consists mainly of B2 homodimers (10). An intermediate molecular weight fraction of hetero-oligomers, L1 (≈80 kDa), appears after 1 year of age (8)(9)(10). The -crystallin subunits of the heterooligomers assemble by noncovalent specific interactions.…”

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confidence: 99%

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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“…The described modifications to this family of proteins include: *The proteases trypsin, subtilisin, and elastase were chosen because they consistently produced peptides with different specificity resulting in high total sequence coverage by tandem mass spectrometry. truncation of N and C termini (30)(31)(32)(33)(34), deamidation (35)(36)(37), racemization (38), phosphorylation (33,35,39), oxidation (33,37,38,40), acetylation (41,42), carbamylation (43), disulfide formation (35,44), and glycation (42). Finally, because these proteins are long lived, they are prone to adventitious modification; therefore, it is necessary to have the ability to survey many possible modifications during a single experiment.…”

Section: Mapping Protein Modification Sites In Human Lens Tissue Withoutmentioning

confidence: 99%

Shotgun identification of protein modifications from protein complexes and lens tissue

MacCoss

McDonald

Saraf

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

550

493

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Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co-and posttranslational modifications of proteins.

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“…These quiescent, organelle-free cells contain high concentrations of three families of water-soluble proteins, the crystallins. Age-long accumulation of protein damage, truncations and side-chain modifications [3][4][5][6] in a low protein turn-over environment change the solubility and stability of the crystallins which can lead to formation of protein aggregates.…”

Section: Introductionmentioning

confidence: 99%

Specificity of αA‐crystallin binding to destabilized mutants of βB1‐crystallin

Mchaourab¹,

Kumar²,

Koteiche³

2007

FEBS Letters

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To elucidate the structural and energetic basis of attractive protein interactions in the aging lens, we investigated the binding of destabilized mutants of bB1-crystallin to the lens chaperones, a-crystallins. We show that the mutations enhance the binding affinity to aA-but not aB-crystallin at physiological temperatures. Complex formation disrupts the dimer interface of bB1-crystallin consistent with the binding of a monomer. Binding isotherms obtained at increasing concentrations of bB1-crystallin deviate from a classic binding equilibrium and display cooperative-like behavior. In the context of bB1-crystallin unfolding equilibrium, these characteristics are reflective of the concentration-dependent change in the population of a dimeric intermediate that has low affinity to aA-crystallin. In the lens, where a-crystallin binding sites are not regenerated, this may represent an added mechanism to maintain lens transparency.

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Size of Human Lens β-Crystallin Aggregates Are Distinguished by N-terminal Truncation of βB1

Cited by 63 publications

References 27 publications

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Shotgun identification of protein modifications from protein complexes and lens tissue

Specificity of αA‐crystallin binding to destabilized mutants of βB1‐crystallin

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