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In vivo substrates of the lens molecular chaperones ?A-crystallin and ?B-crystallin.

ABSTRACT: ?A-crystallin and ?B-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in ?A-R49C and ?B-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old ?A-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of ?A-crystallin; increased association between ?A-crystallin and filensin, actin, or creatine kinase B; increased acidification of ?B1-crystallin; increased levels of grifin; and an association between ?A3/A1-crystallin and ?A-crystallin. Homozygous ?A-R49C mutant lenses exhibited increased associations between ?A-crystallin and ?B3-, ?A4-, ?A2-crystallins, and grifin, whereas levels of ?B1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, ?-enolase, and cytochrome c increased more than 50-fold in homozygous ?A-R49C mutant lenses. In ?B-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several ?- and ?-crystallins, and degradation of ?A- and ?B-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal ?-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of ?A- and ?B-crystallin.

SUBMITTER: Andley UP

PROVIDER: S-EPMC3997384 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.

Andley Usha P UP Malone James P JP Townsend R Reid RR

PloS one 20140423 4

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated wi ...[more]

PMID: 24760011

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Project description:The demonstration of chaperone-like activity in peptides (mini-chaperones) derived from ?-crystallin's chaperone region has generated significant interest in exploring the therapeutic potential of peptide chaperones in diseases of protein aggregation. Recent studies in experimental animals show that mini-chaperones could reach intended targets and alter the disease phenotype. Although mini-chaperones show potential benefits against protein aggregation diseases, they do tend to form aggregates on storage. There is thus a need to fine-tune peptide chaperones to increase their solubility, pharmacokinetics, and biological efficacy.This review summarizes the properties and the potential therapeutic roles of mini-chaperones in protein aggregation diseases and highlights some of the refinements needed to increase the stability and biological efficacy of mini-chaperones while maintaining or enhancing their chaperone-like activity against precipitation of unfolding proteins.Mini-chaperones suppress the aggregation of proteins, block amyloid fibril formation, stabilize mutant proteins, sequester metal ions, and exhibit antiapoptotic properties. Much work must be done to fine-tune mini-chaperones and increase their stability and biological efficacy. Peptide chaperones could have a great therapeutic value in diseases associated with protein aggregation and apoptosis.Accumulation of misfolded proteins is a primary cause for many age-related diseases, including cataract, macular degeneration, and various neurological diseases. Stabilization of native proteins is a logical therapeutic approach for such diseases. Mini-chaperones, with their inherent antiaggregation and antiapoptotic properties, may represent an effective therapeutic molecule to prevent the cascade of protein conformational disorders. Future studies will further uncover the therapeutic potential of mini-chaperones. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Project description:S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.

Dataset Information

In vivo substrates of the lens molecular chaperones ?A-crystallin and ?B-crystallin.

Publications

In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.

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