ABSTRACT
Two Puralpha-binding proteins (PurBPs) were found in nuclear extract from mouse brain during P4-P10 by the overlay assay. At P14, they were decreased significantly in nuclear extract and increased in the S3 fraction, indicating their dynamic translocation during development. Western blot analysis also demonstrated concomitant translocation of Puralpha with the PurBPs during P7-P14, when neuronal circuit proceeds. Immunocytochemical study with cultured hippocampal neurons from rat E18 confirmed that nuclear Puralpha was translocated to cytoplasm after plating for 7-14 days. These results suggest that spatiotemporal translocation of Puralpha with the PurBPs from nuclei to cytoplasm has a crucial role in neuronal development.
Subject(s)
Cell Nucleus/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytoplasm/metabolism , Hippocampus/cytology , Neurons/metabolism , Age Factors , Animals , Animals, Newborn , Blotting, Western/methods , Cells, Cultured , DNA-Binding Proteins , Embryo, Mammalian , Hippocampus/growth & development , Immunohistochemistry/methods , Indoles/metabolism , Nerve Tissue Proteins , Rats , Subcellular Fractions/metabolism , Time Factors , Transcription FactorsABSTRACT
Pur alpha is an abundant protein in the brain and binds to a (GGN)n sequence, PUR element. It has been shown that Pur alpha not only interacts with single stranded DNA and RNA, but also with various proteins. In the present study, we tried to search for Pur alpha-binding proteins (PurBPs) in mouse brain by the overlay assay with GST-Pur alpha as a ligand. Three PurBPs of 35, 38 and 40 kDa were found mostly in the nuclear extract (N.Ext.) and they were not detected by the pretreatment of N.Ext. with trypsin, but not with RNase or DNase. The three PurBPs disappeared by the addition of ssCRE (single stranded cAMP response element) containing a PUR element, but not by DeltaGGN ssCRE (deletion of the PUR element from the ssCRE). The PurBPs were abundantly expressed in the brain as Pur alpha. We also determined a region in Pur alpha which is required for the association with the PurBPs by using deletion mutants of Pur alpha. These biochemical properties of the PurBPs are different from the reported nuclear Pur alpha-binding proteins such as Sp1 and pRb.
Subject(s)
Brain Chemistry/physiology , DNA-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Brain/cytology , Cell Nucleus/chemistry , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Ligands , Male , Mice , Nerve Tissue Proteins/genetics , Neurons/metabolism , Protein Binding , Subcellular Fractions/chemistryABSTRACT
Reticulon3 (RTN3), which belongs to a reticulon family, is first isolated from the retina, but little is known about its function. We investigated the distribution of RTN3 in rat retina and olfactory bulb by immunohistochemistry. In the retina, Müller cells highly expressed RTN3. The expression level of RTN3 in the optic nerve was high in the embryo, but low in the adult. In the olfactory system, RTN3 was highly expressed in the olfactory nerve both in developmental and adult stages. Further, RTN3 was co-localized with synaptophysin in tubulovesicular structures in the developing axon of cultured cortical neurons. These results suggest that RTN3 may play an important role in the developing axons and also in some glial cells such as Müller cells.
Subject(s)
Gene Expression Regulation/physiology , Nerve Tissue Proteins/biosynthesis , Olfactory Bulb/metabolism , Retina/metabolism , Animals , Female , Immunohistochemistry , Mice , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Olfactory Bulb/chemistry , Pregnancy , Rats , Rats, Sprague-Dawley , Retina/chemistryABSTRACT
The Ku autoantigen/KARP-1 (Ku86 autoantigen related protein-1) plays an important role in the double-strand break repair of mammalian DNA as a DNA-binding component of DNA-dependent protein kinase (DNA-PK) complex. KARP-1 is differently transcribed from the human Ku86 autoantigen gene locus and it is implicated in the control of DNA-dependent protein kinase activity. We cloned rKAB1, a rat homolog of KAB1 (KARP-1 binding protein 1 of human) from a rat hippocampal cDNA library. rKAB1 mRNA was specifically expressed in the brain and the thymus. EGFP-tagged rKAB1 protein localized in cell nucleus and in the condensed chromosome during the mitotic cell division. We found that rKAB1 works as a protective protein against cell damage by oxidative stress.