Isolation and Characterization of a 14.5‐kDa Trichloroacetic‐Acid‐Soluble Translational Inhibitor Protein from Human Monocytes that is Upregulated Upon Cellular Differentiation

Abstract

A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography ; five tryptic peptides were subjected to protein sequencing. The full-length cDNA of the protein was cloned and sequenced from a Agtl 1 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057dYJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation-dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.

TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-κB Activation and IL-10 Production

“…We screened a total of 5 million independent clones and obtained 78 ␤-galactosidase-positive clones. Sequence analysis of 20 clones indicated that most clones encode a poorly characterized protein p14.5 (40). Subsequent transient transfection and coimmunoprecipitation experiments indicated that BAFF-R did not interact with p14.5 in 293 cells (data not shown).…”

Redox Proteomics Uncovers Peroxynitrite-sensitive Proteins That Help Escherichia coli to Overcome Nitrosative Stress

“…RidA belongs to the YjgF/YER057c/UK114 superfamily, which is highly conserved across a wide range of organisms. This protein family has a variety of assigned functions, ranging from translational inhibition and activation of a proteinase, to nuclease and chaperone activity (33)(34)(35)(36). Recent studies identified an enamine/imine deaminase activity of RidA in Salmonella enterica (15).…”

Members of the YjgF/YER057c/UK114 Family of Proteins Inhibit Phosphoribosylamine Synthesis in Vitro

“…Plasmid pTrpD (pIR-DW) contained wild-type trpD in pSU19 under the control of the plasmid encoded lac promoter and has been described (19). UK114/hp14.5 cDNA was provided by G. Schmitz (20).…”