Dataset Information

Succinylation Is a Gain-of-Function Modification in Human Lens ?B-Crystallin.

ABSTRACT: Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All of the major crystallins exhibited N?-succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC-MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses (age > 60 years) relative to young lenses (age < 30 years). Immunohistochemical analyses revealed that SuccK was present in epithelium and fiber cells. Western blotting and immunoprecipitation experiments revealed that SuccK is particularly prominent in ?B-crystallin, and succinylation in vitro revealed that ?B-crystallin is more prone to succinylation than ?A-crystallin. Mass spectrometric analyses showed succinylation at K72, K90, K92, K166, K175, and potentially K174 in human lens ?B-crystallin. We detected succinylation at K72, K82, K90, K92, K103, K121, K150, K166, K175, and potentially K174 by mass spectrometry in mildly succinylated ?B-crystallin. Mild succinylation improved the chaperone activity of ?B-crystallin along with minor perturbation in tertiary and quaternary structure of the protein. These observations imply that succinylation is beneficial to ?B-crystallin by improving its chaperone activity with only mild conformational alterations.

SUBMITTER: Nandi SK

PROVIDER: S-EPMC6662615 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Succinylation Is a Gain-of-Function Modification in Human Lens αB-Crystallin.

Nandi Sandip K SK Rakete Stefan S Nahomi Rooban B RB Michel Cole C Dunbar Alexandra A Fritz Kristofer S KS Nagaraj Ram H RH

Biochemistry 20190220 9

Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All of the major crystallins exhibited N<sup>ε</sup>-succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC-MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses ...[more]

PMID: 30758948

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Project description:AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alphaA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alphaA-crystallin by MGO (with 20-500 microM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of beta(L)- and gamma-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured beta(L)- and gamma-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of alphaA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alphaA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of alphaA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alphaA-crystallin responsible for the chaperone function and refolding are the same in the native alphaA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alphaA-crystallin's ability to refold denatured proteins.

Dataset Information

Succinylation Is a Gain-of-Function Modification in Human Lens ?B-Crystallin.

Publications

Succinylation Is a Gain-of-Function Modification in Human Lens αB-Crystallin.

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