Mammalian prothymosin alpha links to tRNA in Escherichia coli cells.

Prothymosin alpha, a small and highly acidic nuclear protein related to cell proliferation, is known to be covalently attached to a small unidentified cytoplasmic RNA in mammalian cells. Here we demonstrate that recombinant rat prothymosin a links covalently to an RNA when overproduced in Escherichia coli cells. The RNA species of this complex is represented by a wide range of bacterial tRNAs. tRNA(Lys), tRNA(3Ser), tRNA(2Ile), and tRNA(mMet) were identified by sequencing. Prothymosin alpha appears to be linked to the 5' terminus of tRNA. tRNA attachment site lies close to the carboxy-terminus of prothymosin alpha. Furthermore, the carboxy-terminal peptide of prothymosin alpha is also competent for tRNA binding. The site of tRNA attachment coincides with the nuclear localization signal of prothymosin alpha, and tRNA binding might be expected to affect subcellular localization of this protein.     

Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha

Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.     

Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.     

Nuclear targeting of prothymosin alpha

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.     

Functional discontinuities in prothymosin alpha caused by caspase cleavage in apoptotic cells

Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.     

Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha.

Prothymosin alpha is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin alpha appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin alpha molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin alpha shared domains with mobile proteins such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin alpha. The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin alpha is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.     

Prothymosin alpha fragmentation in apoptosis

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.     

Does prothymosin alpha affect the phosphorylation of elongation factor 2?

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.     

Segments of Escherichia coli genome similar to the exons of human prothymosin alpha gene.

Identification of the putative prothymosin alpha homolog in Escherichia coli cells prompted the search for a prothymosin alpha-coding gene in the E. coli genome. A set of interspersed DNA segments was identified, which match various parts of the human prothymosin alpha molecule. Their location in the E. coli genome and high degree of similarity with the appropriate regions of the human prothymosin alpha gene suggest that some kind of trans-splicing should exist in E. coli, which could be responsible for bringing these putative bacterial prothymosin alpha-coding exons together.     

Identification of a low-Mr acidic nuclear protein as prothymosin alpha

We have purified to homogeneity a 15-kDa perchloric acid (PCA)-soluble protein from rat thymus nuclei. This highly acidic protein showed a Mr of ca. 30 kDa in acetic acid/urea gels, probably due to oligomer formation. Sequence analysis of internal tryptic and thermolytic peptides revealed that the purified protein is, in fact, prothymosin alpha, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin alpha is a rather abundant nuclear protein in rat thymus; its concentration is comparable to that of a well-characterized nonhistone protein HMG-14. The subcellular localization and physicochemical properties of prothymosin alpha suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.     

Divalent metal cation binding properties of human prothymosin alpha.

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.     

Novel functions of the well-known protein--prothymosin alpha is involved in protection of cells against apoptosis and oxidative stress

Several novel functions of the well-known and intensively studied protein prothymosin alpha have recently been revealed. In addition to such traditional functions of this protein as immunomodulatory activity and stimulation of cellular proliferation, prothymosin alpha was shown to be involved in protection of cells against apoptosis and in regulation of expression of the oxidative stress-protective genes. Methods and approaches used for revelation of prothymosin alpha novel functions are described in this review.     

Identification of N-terminal acetylation of recombinant human prothymosin alpha in Escherichia coli

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin alpha is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin alpha expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin alpha, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin alpha produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin alpha and thymosin alpha1.     

Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA

A 13 kDa protein, covalently linked to a small RNA from the cytoplasm of mouse cells, was studied. Sequence analysis of its tryptic peptides revealed that the RNA-linked protein is identical to prothymosin alpha. Very similar RNA-protein complexes were identified in human, bovine and yeast cells. Tryptic peptide maps of 125I-labelled RNA-linked proteins of diverse origin demonstrated their marked similarity, thus indicating high evolutionary conservation of prothymosin alpha from yeast to man.