المؤلفون: Fang Bai, Timothy P. Fleming, Jing Hua Xi, Eric F. Wawrousek, Usha P. Andley

المصدر: The Journal of biological chemistry. 278(38)

مصطلحات موضوعية: Genome instability, DNA, Complementary, Time Factors, Cell division, Binucleated cells, Blotting, Western, Centromere, Immunoblotting, Mitosis, Apoptosis, Mice, Transgenic, Biology, Biochemistry, Retinoblastoma Protein, Mice, Lens, Crystalline, medicine, Animals, Muscle, Skeletal, Molecular Biology, Cells, Cultured, In Situ Hybridization, Fluorescence, Mice, Knockout, medicine.diagnostic_test, Cell Death, Cell Cycle, Wild type, alpha-Crystallin B Chain, Epithelial Cells, Cell Biology, DNA, Cell biology, Phenotype, Microscopy, Fluorescence, Gamma Rays, biology.protein, Antibody, Tumor Suppressor Protein p53, Cytokinesis, Cell Division, Fluorescence in situ hybridization

الوصف: alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::df13770a63f82da1f446d44fdccc77eaTest
https://pubmed.ncbi.nlm.nih.gov/12826669Test

المؤلفون: Shashank Mathur, J. Mark Petrash, Terry A. Griest, Usha P. Andley

المصدر: The Journal of biological chemistry. 271(50)

مصطلحات موضوعية: Base Sequence, Chaperonins, Protein Conformation, Protein subunit, Circular Dichroism, Mutant, Molecular Sequence Data, Wild type, Alpha-Crystallin A Chain, Cell Biology, Biology, Biochemistry, Crystallins, Lens protein, Gene Expression Regulation, Crystallin, Aldehyde Reductase, Chaperone (protein), biology.protein, Humans, Amino Acid Sequence, Cloning, Molecular, Molecular Biology, Protein secondary structure

الوصف: One of the major protein components of the ocular lens, alpha-crystallin, is composed of alphaA and alphaB chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, alpha-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the alphaA chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of alpha-crystallin modification, two mutant forms of alphaA subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of approximately 540 kDa were formed from a tryptophan-free alphaA mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the C-terminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of gamma-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of alphaA subunits, and that deletion of C-terminal sequences from the alphaA subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5379428cbdd7f41dbb1f4d6d344e1406Test
https://pubmed.ncbi.nlm.nih.gov/8943244Test