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In vitro interactions of histones and ?-crystallin.

ABSTRACT: The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether ?-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone-?-crystallin binding with a K_d of 4?×?10^-7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone-?-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone-?-crystallin mixtures showed a decrease in the oligomeric molecular weight of ?-crystallin, indicating that histones alter the oligomerization of ?-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of ?-crystallin, indicating that the interaction between ?-crystallin and histones in the lens is functionally important.

SUBMITTER: Hamilton PD

PROVIDER: S-EPMC6047474 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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<i>In vitro</i> interactions of histones and α-crystallin.

Hamilton Paul D PD Andley Usha P UP

Biochemistry and biophysics reports 20180601

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed <i>in vitro</i> analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone-α-cryst ...[more]

PMID: 30023439

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Project description:Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant ?S-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asn143 deamidated (2N-HGS) and the other with two additional Gln residues (92 and 120) deamidated (2N2Q-HGS). Isoelectric focusing measurements showed the expected lowering of the pI from 6.9 in HGS to ?6.5 in 2N-HGS and to ?6.1 in 2N2Q-HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q-HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein-protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2Q-HGS of 8.90 × 10(-4), 7.10 × 10(-4), and 6.65 × 10(-4) mL mol g(-2), respectively). Further substantiation is provided by estimates of the excess binding energy of protein-protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid-liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein-protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time.

Dataset Information

In vitro interactions of histones and ?-crystallin.

Publications

<i>In vitro</i> interactions of histones and α-crystallin.

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