The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBP␣ and C/EBP) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse ␣ 1 -acid glycoprotein promoter, we detected multiple forms of C/EBP␣ and C/EBP proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBP␣s with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42-and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBP isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBP␣ forms specific complexes with the ␣ 1 -acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBP␣ and C/EBP isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBP␣ and C/EBP isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBP␣ and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBP␣ and the third AUG codon of the 20-kDa C/EBP. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.
The recently discovered repulsive guidance molecule c (RGMc or hemojuvelin) gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that is expressed in striated muscle and in liver. Mutations in this gene have been linked to the severe iron storage disease, juvenile hemochromatosis, although the mechanisms of action of RGMc in iron metabolism are unknown. As a first step toward understanding the molecular physiology of this protein, we studied its biosynthesis, processing and maturation. Production of RGMc occurs as an early and sustained event during skeletal muscle differentiation in culture and is secondary to RGMc gene activation. As assessed by pulse-chase studies and cell-surface labeling experiments, two classes of GPI-anchored and glycosylated RGMc molecules are targeted to the membrane and undergo distinct fates. Full-length RGMc is released from the cell surface and accumulates in extracellular fluid, where its half-life exceeds 24 hours. By contrast, the predominant membrane-associated isoform, a disulfide-linked heterodimer composed of N- and C-terminal fragments, is not found in the extracellular fluid, and is short-lived, as it disappears from the cell surface with a half-life of <3 hours after interruption of protein synthesis. A natural disease-associated RGMc mutant, with valine substituted for glycine at residue 320 (313 in mouse RGMc), does not undergo processing to generate the heterodimeric membrane-linked isoform of RGMc, and is found on the cell surface only as larger protein species. Our results define a series of biosynthetic steps leading to the normal production of different RGMc isoforms in cells, and provide a framework for understanding the biochemical basis of defects in the maturation of RGMc in juvenile hemochromatosis.
The CCAAT/enhancer binding protein alpha (C/EBPalpha) and CCAAT/enhancer binding protein beta (C/EBPbeta) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa and C/EBPbetas of 35, 20, and approximately 8.5 kDa. The DNA-binding activities and pool levels of p42(C/EBPalpha) and p30(C/EBPalpha) in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPalpha and C/EBPbeta isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42(C/EBPalpha) levels and binding activity, whereas those of p20(C/EBPalpha) and p20(C/EBPbeta) are increased. However, translation of 42-kDa C/EBPalpha is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPalpha- and C/EBPbeta-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPalpha and C/EBPbeta isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.
Kuns-Hashimoto R, Kuninger D, Nili M, Rotwein P. Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin.
The major human AP-endonuclease 1 (APE1) is a multifunctional protein that plays a central role in the repair of damaged DNA by acting as a dual-function nuclease in the base excision repair pathway. This enzyme was also independently identified as a redox activator of AP-1 DNA-binding activity and has subsequently been shown to activate a variety of transcription factors via a redox mechanism. In a third distinct role, APE1 was identified as a component of a trans-acting complex that acts as a repressor by binding to the negative calcium responsive elements (nCaRE)-A and nCaRE-B, which were first discovered in the promoter of the human parathyroid gene and later in the APE1 promoter itself. Here we show that the nuclear protein complex which binds to the nCaRE-B2 of the hAPE1 gene contains APE1 itself and the heterogeneous nuclear ribonucleoprotein L (hnRNP-L). The interaction between the APE1 and hnRNP-L proteins does not require the presence of nCaRE-B2. Our results support the possibility that the APE1 gene is down-regulated by its own product, which would be the first such example of the regulation of a DNA repair enzyme, and identify a novel function of hnRNP-L in transcriptional regulation.
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