A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3' untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression.
An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.
Earlier studies have shown that the chaperone activity of alpha-crystallin is significantly affected in diabetic rat and human lenses. Subsequently, mass spectrometric analysis showed diabetic lenses having high levels of the alphaA-crystallins in which different numbers of C-terminal residues were deleted. The present study was aimed to show whether cleavage of these residues influences protein structure, oligomerization, and chaperone function. For generation of various mutants, a stop codon was introduced at the positions of interest, proteins were expressed in BL21(DE3)pLys S E. coli, and the truncated alphaA-crystallins were purified by size-exclusion chromatography. The molecular masses, as determined by molecular sieve HPLC, of mutants with deletions of 1, 5, and 10 C-terminal residues (group-1) were 519-602 kDa, and those of mutants with deletions of 11, 16, and 22 C-terminal residues (group-2) were 148-152 kDa, as compared to 607 kDa for alphaA-wild type. On the basis of circular dichroism measurements, the alpha helix content was 2-fold higher and the tertiary structure was significantly altered in the group-2 mutants. Chaperoning abilities, as determined by the ADH assay and the betaL-crystallin heat denaturation assay, of the group-1 mutants, with the exception of alphaA(1-163), were slightly improved or unchanged, that of alphaA(1-163) was moderately affected, and those of the group-2 mutants were severely affected. Most strikingly, cleavage of 11 C-terminal residues including Arg-163 showed a substantial decrease in oligomeric size and chaperone function and significant changes in protein structure whereas cleavage of 10 residues had either a small effect or no effect at all. This points to an important role for the C-terminal extension, Arg-163 in particular, and no significant role for the C-terminal flexible tail in the oligomer assembly of alphaA-crystallin.
Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H2O2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H2O2-treated cells compared to controls and protected against H2O2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT1A agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT1A receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.