[Phytaspases: aspartate-specific proteases involved in plant cell death].

Structure and properties of the recently discovered aspartate-specific cell death-related plant proteases named phytaspases are reviewed and compared to those of animal apoptotic proteases, caspases. Caspases (cysteine-dependent proteases) and phytaspases (serine-dependent proteases) are structurally very different, yet they share cleavage specificity and a role in programmed cell death. We demonstrate here that the distinctions in structural organization of animal and plant death proteases define differences in the strategies to regulate functioning of these proteolytic enzymes in the two kingdoms.

Programmed cell death in plants.

The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.

[Influence of prothymosin alpha and its mutants on activity of the p53 tumor suppressor].

Overexpression of the nuclear oncoprotein prothymosin a enhanced and, in a reciprocal experiment, down-regulation of endogenous prothymosin alpha by RNA interference approach inhibited transcriptional activity of the p53 tumor suppressor in the reporter gene assay. Ectopic expression of prothymosin alpha enhanced not only p53-dependent transcription, but also intracellular level of p53 in HeLa (but not HCT116) cells. Ability to stimulate p53-dependent transcription was lost by C-terminal mutants of prothymosin alpha with impaired nuclear accumulation, but not by N-terminal deletion mutants and by the double mutant of prothymosin alpha with impaired ability to bind Keap1, suggesting that prothymosinalpha-Keap1 interaction is dispensable for p53 response. Our data suggest that the central "acidic" region of prothymosin alpha together with intact nuclear localization signal is responsible for stimulation of p53-dependent transcription. This conclusion was confirmed by the fact that another protein containing long "acidic" region and nuclear localization signal, parathymosin, was able to stimulate transcription of p53-responsive reporter gene.

[Prothyomosin alpha interaction with KEAP1 doesn't lead to prothymosin alpha ubiquination and degradation].

Prothymosin alpha (ProTalpha) is a highly conserved protein in vertebrates that possesses a number of biological functions. One of these functions of ProTalpha is the ability to enhance antioxidant defence system of a cell via its interaction with Keap1 protein. Keap1 is a repressor of Nrf2, a transcription factor responsible for activation of genes that code for defensive proteins. While bound to Nrf2, Keap1 exports Nrf2 from the nucleus to the cytoplasm and, being adaptor protein for ubiquitin ligase, promotes ubiquitination of Nrf2 and its subsequent degradation by 26S proteasome. ProTalpha and Nrf2 compete for interaction with Keap1, therefore ProTalpha is able to liberate Nrf2 from complex with Keap1 and hence contribute to Nrf2-dependent transcription. Here we were interested in elucidating possible consequences for ProTalpha of its interaction with Keap1. We have shown that, despite ProTalpha interaction with Keap1, ProTalpha is a stable protein. In contrast to Nrf2 ProTalpha was not subjected to Keap-dependent ubiquitination, degradation and export from the nucleus. Furthermore, ubiquitination of ProTalpha was undetectable even when Keap1 and ubiquitin were overexpressed. It appears that ProTalpha contribution to Nrf2-dependent transcription is accomplished via the increase of free Nrf2 rather then the increase of total intracellular amount of Nrf2.

[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.

The primary and secondary structure of the 5'-end region of encephalomyocarditis virus RNA. A novel approach to sequencing long RNA molecules.

The primary structure of a 149-nucleotide fragment of encephalomyocarditis (EMC) virus RNA from the 5'-terminus of the genome up to the poly(C) tract (S fragment) has been determined. For isolation of the S fragment, site-directed fragmentation of the viral RNA with RNase H and poly(dG) was employed. For sequencing the S fragment, a novel approach has been developed, which can be used for primary structure determination of long RNA molecules. A model of the secondary structure of the S fragment is proposed, according to which this region of RNA is highly structured. The role of complementary oligonucleotide stretches near both termini of the RNA molecule is discussed.

Multiple tRNA attachment sites in prothymosin alpha.

A covalent complex formed by bacterial tRNAs and prothymosin alpha, an abundant acidic nuclear protein involved in proliferation of mammalian cells, upon production of the recombinant rat protein in Escherichia coli cells was studied. Several tRNA attachment sites were identified in the prothymosin alpha molecule using a combination of deletion analysis of prothymosin alpha and site-specific fragmentation of the protein moiety of the prothymosin alpha-tRNA complex. The electrophoretic mobilities of the tRNA-linked prothymosin alpha and its derivatives are consistent with one tRNA molecule attached to one prothymosin alpha molecule, thus suggesting that alternative tRNA linking to one of several available attachment sites occurs. The possible effect of tRNA attachment on the nuclear uptake of prothymosin alpha is discussed.

Small cytoplasmic RNA from mouse cells covalently linked to a protein.

A low-molecular-mass RNA from the cytoplasm of mouse Krebs II cells was found to be covalently linked to a protein with an apparent molecular mass of 13 kDa. The protein appears to be attached to the 5'-terminus of the RNA molecule, which is approx. 20 nucleotides long.

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.

Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal.

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha 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 alpha through an unknown mechanism, without influencing the nuclear import of the protein.

[Polymerase chain reaction]. / Polimeraznaia tsepnaia reaktsiia.

Principles of the polymerase chain reaction, its modifications and applications are discussed. Guidelines for performance of the polymerase chain reaction are briefly summarized.

[Genome-linked proteins in DNA and RNA viruses]. / Belki, kovalentno sviazannye s genomami DNK- i RNK-soderzhashchikh virusov.

General principles of the organization of viral nucleic acid--protein covalent complexes are formulated. Participation of the genome-linked proteins in the initiation of viral nucleic acid replication is discussed.

[Covalent bond between RNA and protein in encephalomyocarditis virus]. / Kovalentnoe soedinenie RNK i belka v viruse zntsefalomnokardita.

Deproteinized encephalomyocarditis [32P]RNA, after digestion with a mixture of RNases A, T1 and T2, yields mononucleotides and a labelled compound, which is positively charged at pH 3.5. This product can be digested with pronase and has a close electrophoretic mobility to a protein with a molecular weight of 7000-8000. Covalently bound nucleotide-peptides were isolated from this compound after treatment with RNases and pronase. It was shown that the 5'-terminal uridylic acid is covalently linked with peptides. The phosphodiester bond between uridylic acid and peptides is discussed.

Biotechnological approaches to creation of hypoxia and anoxia tolerant plants.

The present work provides results of a number of biotechnological studies aimed at creating cell lines and entire plants resistant to anaerobic stress. Developed biotechnological approaches were based on earlier fundamental researches into anaerobic stress in plants, so "Introduction" briefly covers the importance of the problem and focuses on works considering two main strategies of plants adaptation to anaerobic stress. Those are adaptation at molecular level where key factor is anaerobic metabolism of energy (true tolerance) and adaptation of the entire plant via formation of aerenchyma and facilitated transportation of oxygen (apparent tolerance). Thus, sugarcane and wheat cells resistant to anaerobic stress were obtained through consecutive in vitro selection under conditions of anoxia and absence of exogenous carbohydrates. Tolerant wheat cells were used to regenerate entire plants of higher resistance to root anaerobiosis. It has been demonstrated that cells tolerance to anoxia is significantly supported by their ability to utilize exogenous nitrate. Cells tolerance established itself at the genetic level and was inherited by further generations. Apart from that, other successful attempts to increase tolerance of plants to anaerobic stress by means of stimulation of glycolysis and overexpression of genes responsible for cytokinin synthesis and programmed cell death are also discussed. The presented data proved the notion of two main strategies of plants adaptation to anaerobic stress proposed earlier on the base of fundamental studies.

A plant alternative to animal caspases: subtilisin-like proteases.

Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases.

The structure of the covalent linkage between proteins and RNA in encephalomyocarditis virus.

Two protein, VPgA and VPgB, are covalently bound to the virion RNA of encephalomyocarditis (EMC) virus. Their molecular weightrs, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate are 10,000 and 8,000, respectively. A study of nucleotide-peptides isolated from VPg-RNA compound has shown that VPgA is bound to the 5'-terminal nucleotide of RNA by a phosphodiester bond. The 5'-terminal nucleotide of RNA is uridylic acid. It is the hydroxy group of the Tyr residue of VPgA that is involved in the formation of the linkage with RNA. VPgB-RNA seems to be similar to VPgA-RNA both in the structure of the RNA-protein linkage and localization of VPgB on RNA.

Encephalomyocarditis virus RNA synthesis in vitro is protein-primed.

A crude membrane-bound replication complex isolated from encephalomyocarditis virus-infected cells is able to initiate the synthesis of viral RNA. Both the formation of the primer, VPg-pU, and its utilization for the initiation of RNA chains take place in this system. A significant amount of the synthesized VPg-pU is found in the free form. The predominant product of the de novo initiation is represented by short phenol-soluble VPg oligonucleotide species, and only a small percentage of the latter appear to be elongated into longer RNA chains.

Early alteration of nucleocytoplasmic traffic induced by some RNA viruses.

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine. HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins.