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The absence of SIRT3 and SIRT5 promotes the acetylation of lens proteins and improves the chaperone activity of ?-crystallin in mouse lenses.

ABSTRACT: Acetylation of lysine residues occurs in lens proteins. Previous studies have shown an improvement in the chaperone activity of ?A-crystallin upon acetylation. Sirtuins are NAD+-dependent enzymes that can deacylate proteins. The roles of sirtuins in regulating the acetylation of lens proteins and their impacts on the function of ?-crystallin are not known. Here, we detected sirtuin activity in mouse lenses, and SIRT3 and SIRT5 were present primarily in the mitochondria of cultured primary mouse lens epithelial cells. Western blotting showed higher levels of protein acetylation in the lenses of SIRT3 KO and SIRT5 KO mice than in lenses of WT mice. Mass spectrometry analyses revealed a greater number of acetylated lysine residues in ?-crystallin isolated from the SIRT3 and SIRT5 KO lenses than from WT lenses. ?-Crystallin isolated from SIRT3 and SIRT5 KO lenses displayed a higher surface hydrophobicity and higher chaperone activity than the protein isolated from WT lenses. Thus, SIRTs regulate the acetylation levels of crystallins in mouse lenses, and acetylation in lenses enhances the chaperone activity of ?-crystallin.

SUBMITTER: Nandi SK

PROVIDER: S-EPMC6504558 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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The absence of SIRT3 and SIRT5 promotes the acetylation of lens proteins and improves the chaperone activity of α-crystallin in mouse lenses.

Nandi Sandip K SK Nahomi Rooban B RB Harris Peter S PS Michel Cole R CR Fritz Kristofer S KS Nagaraj Ram H RH

Experimental eye research 20190305

Acetylation of lysine residues occurs in lens proteins. Previous studies have shown an improvement in the chaperone activity of αA-crystallin upon acetylation. Sirtuins are NAD<sup>+</sup>-dependent enzymes that can deacylate proteins. The roles of sirtuins in regulating the acetylation of lens proteins and their impacts on the function of α-crystallin are not known. Here, we detected sirtuin activity in mouse lenses, and SIRT3 and SIRT5 were present primarily in the mitochondria of cultured pri ...[more]

PMID: 30849386

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Project description:Mouse Cx50D47A and human Cx50D47N are non-functional connexin mutants that cause dominantly-inherited cataracts. In tissue culture expression experiments, they both exhibit impaired cellular trafficking and gap junction plaque formation. Lenses of mice expressing Cx50D47A have cataracts, reduced size, drastically decreased levels of connexin50, and less severely reduced levels of connexin46. The PERK-dependent pathway of the ER response to misfolded proteins is activated, and they have impaired differentiation with retained cellular organelles. Since treatments that enhance protein folding improve trafficking and plaque formation by Cx50D47N and other mutant connexins in vitro, and they are successful therapeutics for some other diseases caused by misfolded proteins, we tested the efficacy of the chemical chaperone, 4-phenylbutyrate (4-PBA) in cultured cells and mice expressing Cx50D47A. 4-PBA treatment increased the formation of Cx50D47A-containing plaques at appositional membranes of transiently transfected HeLa cells. Heterozygous Cx50D47A mice were treated with 4-PBA by addition to the drinking water and parenteral injection of pregnant mice (starting 10 days after pairing of males and females) and their pups. Lenses from 1-month-old mice were examined by darkfield illumination and immunofluorescence microscopy. Protein levels were determined by immunoblotting. Cataract size and density were not detectably different between the control and the 4-PBA-treated groups. Lens size was not increased following treatment. Levels of connexin46 and connexin50 were significantly increased in lenses of 4-PBA-treated mice compared with saline-treated animals. Immunofluorescence showed an increased abundance of connexin46 immunoreactivity and puncta. The ratio of phosphorylated to total EIF2? was not altered, and levels of organellar proteins were not significantly reduced, suggesting that the ER response to misfolded proteins and differentiation were not changed. Thus, treatment with 4-PBA improved critical pathological issues in these mice (low connexin and gap junction abundance), but the magnitude of this recovery (especially for Cx50) was inadequate to impact the reduced size or the opacification of Cx50D47A lenses.

Dataset Information

The absence of SIRT3 and SIRT5 promotes the acetylation of lens proteins and improves the chaperone activity of ?-crystallin in mouse lenses.

Publications

The absence of SIRT3 and SIRT5 promotes the acetylation of lens proteins and improves the chaperone activity of α-crystallin in mouse lenses.

Similar Datasets

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