RESUMO
Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic ß-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic ß-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.
Assuntos
Catarata , Cristalino/metabolismo , Dobramento de Proteína , Cadeia A de beta-Cristalina/genética , beta-Cristalinas/química , Catarata/congênito , Catarata/genética , Catarata/metabolismo , Humanos , MutaçãoRESUMO
HIRA is one of the chaperones of histone H3.3. Mutation of Hira results in embryonic lethality in mice, suggesting a critical role in embryogenesis. However, Hira-mutated Drosophila may survive to adults, indicating that it is dispensable in Drosophila development. The role of Hira in fish development is unknown. In this study we first investigated the expression of Hira during embryogenesis of gibel carp (Carassius auratus gibelio) by whole-mount in situ hybridization. We found that Hira signal appeared ubiquitously in the early embryos. After gastrulation, it appeared mainly along the anterior-posterior axis, including the tail bud. In hatching period, the signal was detected in head, heart, and the endoderm region on the back of yolk. Then by microinjection with morpholino-HIRA at the beginning of development, we observed delayed gastrulation and abnormal somitogenesis in gibel carp embryos. The HIRA morphants exhibited short trunk, limited yolk extension, and twisted tail. Most of the mutants died during embryogenesis or shortly after hatching. The rest of the HIRA morphants could survive to larvae but with severe defects in organogenesis. These data suggest that HIRA may be essential for the development of gibel carp, and this function is conserved in vertebrates.
Assuntos
Desenvolvimento Embrionário , Carpa Dourada/embriologia , Chaperonas de Histonas/fisiologia , Animais , Blastodisco/metabolismo , Carpas/genética , Feminino , Carpa Dourada/genética , Hibridização In Situ , Masculino , MutaçãoRESUMO
Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.
Assuntos
Citocinas/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Carpas , Citocinas/química , DNA Complementar/metabolismo , Hibridização In Situ , Microscopia de Fluorescência , Midkina , Dados de Sequência Molecular , Sistema Nervoso/embriologia , Neurônios/metabolismo , Filogenia , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-ZebraRESUMO
HIR/HIRA, one of the histone chaperones, encoded by Hir/Hira (histone regulation) genes, were initially identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA contains a conserved family of proteins found in various species including low eukaryotes, invertebrates and vertebrates. It is essential for proper development. Mutations of Hir/Hira genes result in very serious defects in normal development not only in yeast but also in advanced eukaryotes. Combined with the work in our group, the roles of Hir/Hira genes in the development in different species are reviewed, and the recent advances concerning the mechanisms of HIRA functioning are also summarized in an attempt to promote the research further on the exact roles of hira genes in development of eukaryotes and on their deeply operating mechanisms.
