Alexandra G. Evstafieva

Nuclear oncoprotein prothymosin alpha is a partner of Keap1: implications for expression of oxidative stress-protecting genes.

ABSTRACT Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin α. The in vivo and in vitro data indicated that the prothymosin α-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin α was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin α by using prothymosin α overproduction and mRNA interference approaches. Our data attribute to prothymosin α the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin α and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway

While many functions of the p53 tumor suppressor affect mitochondrial processes, the role of altered mitochondrial physiology in a modulation of p53 response remains unclear. As mitochondrial respiration is affected in many pathologic conditions such as hypoxia and intoxications, the impaired electron transport chain could emit additional p53-inducing signals and thereby contribute to tissue damage. Here we show that a shutdown of mitochondrial respiration per se does not trigger p53 response, because inhibitors acting in the proximal and distal segments of the respiratory chain do not activate p53. However, strong p53 response is induced specifically after an inhibition of the mitochondrial cytochrome bc1 (the electron transport chain complex III). The p53 response is triggered by the deficiency in pyrimidines that is developed due to a suppression of the functionally coupled mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). In epithelial carcinoma cells the activation of p53 in response to mitochondrial electron transport chain complex III inhibitors does not require phosphorylation of p53 at Serine 15 or up-regulation of p14ARF. Instead, our data suggest a contribution of NQO1 and NQO2 in stabilization of p53 in the nuclei. The results establish the deficiency in pyrimidine biosynthesis as the cause of p53 response in the cells with impaired mitochondrial respiration.

Prothymosin α fragmentation in apoptosis

We observed fragmentation of an essential proliferation‐related human nuclear protein prothymosin α in the course of apoptosis induced by various stimuli. Prothymosin α cleavage occurred at the DDVD99 motif. In vitro, prothymosin α could be cleaved at D99 by caspase‐3 and ‐7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin α and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin α fragmentation may therefore be proposed to disable intranuclear proliferation‐related function of prothymosin α in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated 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.

Mutational analysis of human prothymosin α reveals a bipartite nuclear localization signal

Mutants of human prothymosin α with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin α‐inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin α by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP‐prothymosin α fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin α through an unknown mechanism, without influencing the nuclear import of the protein.

Topography of RNA in the ribosome: location of the 3′-end of 5 S RNA on the central protuberance of the 50 S subunit

The 5 S RNA is an integral part of the prokaryotic ribosome and plays an important role in polypeptide synthesis. However, the specific function of 5 S RNA and/or associated proteins is unknown” The removal of 5 S RNA from 50 S ribosomal subunits strongly impairs various functional activities of ribosomes [ 1,2]. The o&y exception is the EF-Gdependent GTP hydrolysis which is not influenced by the presence of 5 S RNA f2]. Therefore, the direct focalization of the 5 S RNA-protein domain with respect to other ribosomai components with known functions is of great interest. Here we report the localization of the 3’,

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin α

‘-terminal stem af the Escherichiu coli 5 S RNA on the surface of the 50 S subunit. This was done using the immunealectron microscopy approach applied to localize the 3’ends of the f 6 S [3] and 23 S RNA [4] on the 30 S and 50 S subunits, respectively. The 3 ‘end nucleotide residue of 5 S RNA was found to be located on the outward surface of the central protuberance of the 50 S subunit. These data together with the known secondary structure of the 3’,5’-terminal stem of the 5 S RNA allow one to conclude that its S’end is also located in this region of the 50 S subunit.

TOPOGRAPHY OF RNA IN THE RIBOSOME: LOCALIZATION OF THE 3'-END OF THE 23 S RNA ON THE SURFACE OF THE 50 S RIBOSOMAL SUBUNIT BY IMMUNE ELECTRON MICROSCOPY

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.

A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4

The macromolecular structure of the major components of ribosomes (their RNAs) is now under intensive investigation. In spite of the substantial progress in the elucidation of primary and secondary structures of Escherichia coli 5 S, 16 S, and 23 S RNA, our knowledge of the spatial organization of rRNA in both intact ribosomal subunits and 70 S ribosomes is still very limited (for references, see [ 1,2]). In [3] we proposed a general approach to the identification of external RNA regions in ribosomal subunits. It is based on the chemical modification of selected points in RNA by reagents containing a hapten phenyl

Human prothymosin α inhibits division of yeast Saccharomyces cerevisiae cells, while its mutant lacking nuclear localization signal does not

D-lactoside residue. This approach has been applied to the location of the 3’-end on the 16 S RNA on 30 S subunits [3]. A similar method has been used in [4] utilizing the dinitrophenyl hapten. Here we have located the 3’-end of Escherichia coli 23 S RNA on the lateral surface of the 50 S subunit under its side elongated protuberance (rod-like appendage).