Purification and primary structure of a macromolecular‐translocation inhibitor II of glucocorticoid‐receptor binding to nuclei from rat liver

Abstract

The nuclear binding of the activated glucocorticoid-receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI-I, MTI-II and MTI-III). In this study, we purified the most potent of the three species (MTI-II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two-dimensional PAGE. Purified MTI-II inhibits GR binding to DNA containing glucocorticoid-response elements. To obtain the amino acid sequence of MTI-II, we digested the MTI-II with endopeptidases. The N-terminal amino acid sequences of the four digested fragments indicated that MTI-II is an 11.5-kDa Zn 2+ -binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI-II (ZnBP, parathymosin) and that GR binds to the MTI-II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.Keywords: glucocorticoid receptor; nuclear-translocation inhibitor; parathymosin; purification; zinc-binding protein.When nuclei or DNA were incubated with increasing concentrations of cytosol containing activated (transformed) glucocorticoid receptor (GR), receptor binding to nuclei or DNA reached apparent saturation [1±4]. Several laboratories have reported that this pseudo-saturation is due to the presence of an endogenous macromolecular inhibitor (or inhibitors) in the cytosol of rat liver [1±5] and rat hepatoma cells [6]. With regard to the physiological role of these inhibitors, it is speculated that the target cells may have an inherent mechanism that regulates the interaction of GR with the genomic machinery and thereby controls hormone responses at the cellular level [2,5]. Alternatively, the inhibitor may play a role in the release of the receptor from its nuclear-binding state [5]. We found that AH 130 ascites tumor cells (glucocorticoidresistant cells) have approximately five times more inhibitory activity than cells from rat liver [7], and that the inhibitory activity in regenerating rat liver following 70% hepatectomy is increased significantly during periods of DNA synthesis and subsequent mitosis and then decreases to normal level [8]. Thus, the inhibitors may be involved in cell proliferation by inhibiting GR binding to nuclei.Previously, we separated the macromolecular-translocation inhibitors (MTIs), which inhibit GR binding to nuclei in vitro, from the cytosol of rat liver [9] and other cells [7,8] into at least three components (MTI-I, MTI-II and MTI-III). Recently, we purified MTI-III to apparent homogeneity and characterized i...

Parathymosin Affects the Binding of Linker Histone H1 to Nucleosomes and Remodels Chromatin Structure

“…Early studies have identified ParaT as a zinc-binding protein, which interacts with several enzymes involved in carbohydrate metabolism (30,31). Recently, ParaT was found to inhibit the binding of the activated glucocorticoid receptor to nuclei, suggesting its involvement in the regulation of glucocorticoid steroids action (32).…”

Macromolecular Translocation Inhibitor II (Zn2+-binding Protein, Parathymosin) Interacts with the Glucocorticoid Receptor and Enhances Transcription in Vivo

“…In previous in vitro experiments (8), we found that highly purified GR binds to an MTI-II affinity matrix. In the context of the findings on PT␣, we speculated that MTI-II might be involved in regulating the transcriptional activity of GR in vivo.…”

“…Hormone-We previously showed that highly purified GR binds to an MTI-II affinity matrix in vitro and is eluted from the matrix with 200 mM NaCl (8). To elucidate whether MTI-II interacts with GR in vivo, we examined whether MTI-II could be co-immunoprecipitated with the intrinsic GR and vice versa.…”

“…We purified the most potent of these in vitro inhibitors (MTI-II) to apparent homogeneity and determined the N-terminal amino acid sequences of four enzymedigested fragments, which showed that MTI-II is identical to the 11.5-kDa Zn 2ϩ -binding protein ZnBP (also known as parathymosin) (8). MTI-II is a small and very acidic protein consisting of 101 amino acid residues, of which 11 are aspartic acid and 36 are glutamic acid residues, yielding a pI value of about 4.0.…”

Prothymosin-α and parathymosin expression predicts poor prognosis in squamous and adenosquamous carcinomas of the gallbladder

“…PTMA and PTMS, which were originally obtained by bovine thymus extraction, are members of the α-thymosin family (24). Previous studies have demonstrated that PTMA and PTMS activate the immune response to curb the viral infection in patients with human immunodeficiency virus, hepatitis B virus or hepatitis C virus (25,26).…”