Prothymosin ␣ is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin ␣ contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin ␣ in vivo by pulse-labeling HeLa cells with [ 32 P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of Prothymosin ␣ is a small, highly acidic (1, 2), nuclear (3-6) protein found in virtually all mammalian tissues (7-11). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12). When cells are disrupted with detergents, the protein readily leaks out of the nucleus, suggesting that stable interactions with nucleosomes or with the nuclear matrix are not an inherent part of its activity (2, 3). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin ␣ mRNA is plentiful only in rapidly dividing cells (13)(14)(15); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12); the amount of prothymosin ␣ is directly proportional to the proliferative activity of the tissue from which it is isolated (13); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin ␣ mRNA prevents synchronized human myeloma cells from entering mitosis (16). Hence, a deficiency in prothymosin ␣ is associated with failure to complete the cell cycle.Prothymosin ␣ has several unusual features. The human protein, which is almost identical to that of all other mammals (17), has 109 amino acids, nearly 50% of which are acidic (1, 2). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3, 6), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3,18). The absence of all aromatic residues renders the protein transparent at 280 nm; it also lacks methionine, cysteine, and histidine. Based on biophysical data, it is believed to have an unfolded structure (19), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin ␣ does not bind SDS and stains anomalously with silver stain (2). The protein and the peptides derived from it exhibit poor immunogenicity. However, due to yet another aberrant property, the ability to partition quantitatively into the aqueous phase ...
Nuclear hormone receptors are a family of transcription factors regulated by small molecules derived from the endogenous metabolism or diet. There are forty-eight nuclear hormone receptors in the human genome, twenty of which are still orphans. In this review, we make a brief historical journey from the first observations by Berthold in 1849 to the era of orphan receptors that began with the sequencing of the Caenorhabditis elegans genome in 1998. We discuss the evolution of nuclear hormone receptors and the putative ancestral ligands as well as how the ligand universe has expanded over time. This leads us to define four classes of metabolites—fatty acids, terpenoids, porphyrins and amino acid derivatives—that generate all known ligands for nuclear hormone receptors. We conclude by discussing the ongoing efforts to identify new classes of ligands for orphan receptors.
Prothymosin alpha gene expression accompanies growth of all mammalian cells. The protein, which is abundant, exceedingly acidic, and localized to the nucleus, is further distinguished by the presence of clustered phosphorylated glutamic acid residues (Trumbore et al., 1997, J Biol Chem 272:26394-26404). These glutamyl phosphates are energy rich and unstable in vivo and in vitro (Wang et al., 1997, J Biol Chem 272:26405-26412). To understand the function of prothymosin alpha in greater detail, the turnover of its phosphates was examined in metabolically manipulated cells. Phosphate half-lives in growing, mock transfected, and vector-transfected COS cells were compared with the half-life in cells transfected with the prothymosin alpha gene to determine the fate of the predominantly ectopic phosphorylated protein. The values obtained--72-75 min in cells with normal levels of the protein, but 118 min in cells with surplus prothymosin alpha--led us to conclude that underutilized phosphates persist whereas functioning phosphates disperse. Cell-cycle-specific differences in the half-lives were observed in NIH3T3 cells: 72 min while cycling, 83 or 89 min during arrest in or progression through S phase, but 174 min during M-phase arrest. In the presence of actinomycin D, the value was about 145 min regardless of whether cells were quiescent or growing. In these experiments, reduced utilization of prothymosin alpha's glutamyl phosphates, signaled by an increase in their half-lives, accompanied the attenuation or abolition of transcription. Our data suggest that prothymosin alpha fuels an energy-requiring step in the production, processing, or export of RNA.
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