Biochemistry (Moscow) (v.82, #1)

Extracellular actin in health and disease by N. P. Sudakov; I. V. Klimenkov; V. A. Byvaltsev; S. B. Nikiforov; Yu. M. Konstantinov (1-12).
This review considers the functions of extracellular actin–cell surface bound, associated with extracellular matrix, or freely circulating. The role of this protein in different pathological processes is analyzed: its toxic effects and involvement in autoimmune diseases as an autoantigen. The extracellular actin clearance system and its role in protection against the negative effects of actin are characterized. Levels of free-circulating actin, anti-actin immunoglobulins, and components of the actin clearance system as prognostic biomarkers for different diseases are reviewed. Experimental approaches to protection against excessive amounts of free-circulating F-actin are discussed.
Keywords: actin; danger associated molecular patterns; gelsolin; Gc-globulin; anti-actin antibodies; pathologies; biomarkers

Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathionedependent formaldehyde dehydrogenase.
Keywords: Bacillus subtilis ; formaldehyde dehydrogenase; alcohol dehydrogenase; metal dependent; protein folding; circular dichroism

Apoptotic endonuclease EndoG inhibits telomerase activity and induces malignant transformation of human CD4+ T cells by D. A. Vasina; D. D. Zhdanov; E. V. Orlova; V. S. Orlova; M. V. Pokrovskaya; S. S. Aleksandrova; N. N. Sokolov (24-37).
Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active fulllength hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.
Keywords: EndoG; telomerase; hTERT; alternative splicing; malignant transformation

Cells resistant to toxic concentrations of manganese have increased ability to repair DNA by K. A. Zakharcheva; L. V. Gening; K. Yu. Kazachenko; V. Z. Tarantul (38-45).
Manganese (Mn) is crucially important for vital activity of cells and has many biological functions. Nevertheless, high doses of Mn taken up by an organism over a long period may cause neurodegenerative diseases such as manganism and Parkinsonism. The molecular mechanisms of this Mn toxicity are still poorly studied. It is now believed that Mn-induced pathophysiological neural processes are multifaceted and affect several metabolic pathways. In particular, Mn ions might affect the processes of DNA replication and repair. To test this possibility, we obtained an SKOV-3 cell line resistant to the toxic action of Mn ions. We found that these cells are characterized by the activation of poly(ADP-ribose)polymerase, which leads to increased ability to repair DNA. Thus, the model used here supports the suggestion that at least one cause of Mn cytotoxicity might be disorders of the processes involved in DNA replication and repair.
Keywords: Mn toxicity; manganism; Parkinsonism; DNA repair

The plasma membrane Pma1 H+-ATPase of the yeast Saccharomyces cerevisiae contains conserved residue Asp739 located at the interface of transmembrane segment M6 and the cytosol. Its replacement by Asn or Val (Petrov et al. (2000) J. Biol. Chem., 275, 15709-15716) or by Ala (Miranda et al. (2011) Biochim. Biophys. Acta, 1808, 1781-1789) caused complete blockage of biogenesis of the enzyme, which did not reach secretory vesicles. It was proposed that a strong ionic bond (salt bridge) could be formed between this residue and positively charged residue(s) in close proximity, and the replacement D739A disrupted this bond. Based on a 3D homology model of the enzyme, it was suggested that the conserved Arg811 located in close proximity to Asp739 could be such stabilizing residue. To test this suggestion, single mutants with substituted Asp739 (D739V, D739N, D739A, and D739R) and Arg811 (R811L, R811M, R811A, and R811D) as well as double mutants carrying charge-neutralizing (D739A/R811A) or charge-swapping (D739R/R811D) substitutions were used. Expression of ATPases with single substitutions R811A and R811D were 38-63%, and their activities were 29-30% of the wild type level; ATP hydrolysis and H+ transport in these enzymes were essentially uncoupled. For the other substitutions including the double mutations, the biogenesis of the enzyme was practically blocked. These data confirm the important role of Asp739 and Arg811 residues for the biogenesis and function of the enzyme, suggesting their importance for defining H+ transport determinants but ruling out, however, the existence of a strong ionic bond (salt bridge) between these two residues and/or importance of such bridge for structure–function relationships in Pma1 H+-ATPase.
Keywords: yeast; plasma membrane; secretory vesicles; Pma1 H+-ATPase; transmembrane segments; H+ transport; sitedirected mutagenesis

Subcellular localization and detection of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy by T. N. Erokhina; E. A. Lazareva; K. R. Richert-Pöggeler; E. V. Sheval; A. G. Solovyev; S. Y. Morozov (60-66).
Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.
Keywords: Tobacco mosaic virus ; ORF6 protein; overlapping genes; subcellular localization; monoclonal antibodies; immunoelectron microscopy

The kinetics of irradiation-induced changes in leaf optical transparence (ΔT) and non-photochemical quenching (NPQ) of chlorophyll fluorescence in Tradescantia fluminensis and T. sillamontana leaves adapted to different irradiance in nature was analyzed. Characteristic times of a photoinduced increase and a dark decline of ΔT in these species were 12 and 20 min, respectively. The ΔT was not confirmed to be the main contributor to the observed middle phase of NPQ relaxation kinetics (τ = 10-28 min). Comparison of rate of photoinduced increase in ΔT and photosystem II quantum yield recovery showed that the former did not affect the tolerance of the photosynthetic apparatus (PSA) to irradiances up to 150 μmol PAR·m–2·s–1. Irradiance tolerance correlated with the rate of “apparent NPQ” induction. Considering that the induction of apparent NPQ involves processes significantly faster than ΔT, we suggest that the photoprotective mechanism induction rate is crucial for tolerance of the PSA to moderate irradiance during the initial stage of light acclimation (first several minutes upon the onset of illumination).
Keywords: Tradescantia ; excessive light stress; leaf optical transparence; chlorophyll fluorescence

Cancer: Bad luck or punishment? by A. V. Lichtenstein (75-80).
Contrasting opinions on the role of extrinsic and intrinsic factors in cancer etiology (Tomasetti, C., and Vogelstein, B. (2015) Science, 347, 78-81; Wu, S., et al. (2016) Nature, 529, 43-47) variously define priorities in the war on cancer. The correlation between the lifetime risk of several types of cancer and the total number of divisions of normal selfrenewing cells revealed by the authors has given them grounds to put forward the “bad luck” hypothesis. It assumes that ~70% of cancer variability is attributed to random errors arising during DNA replication in normal, noncancerous stem cells, i.e. to internal factors, which is impossible either to expect or to prevent. This assumption caused many critical responses that emphasize, on the contrary, the defining role of extrinsic factors in cancer etiology. The analysis of epidemio-logical and genetic data presented in this work testifies in favor of the “bad luck” hypothesis.
Keywords: cancer; bad luck hypothesis; carcinogenesis; mutagenesis; extrinsic and intrinsic causes of cancer; cancer prevention

Arguments supporting the “bad luck” hypothesis presented by C. Tomasetti and B. Vogelstein ((2015) Science, 347, 78–81) and A. V. Lichtenstein ((2017) Biochemistry (Moscow), 82, 75–80) are critically discussed. Those arguments are not sufficient for recognition of the “bad luck” hypothesis and the leading role of internal factors in spontaneous tumor development.
Keywords: “bad luck” hypothesis; causes of tumor development; external and internal factors of carcinogenesis

The opinion is presented according to which the “bad luck” hypothesis (Tomasetti, C., and Vogelstein, B. (2015) Science, 347, 78–81), which has recently received experimental confirmation, has the right to exist, and its criticisms are largely unfounded.
Keywords: cancer; “bad luck” hypothesis; carcinogenesis; mutagenesis; external and internal factors; cancer prevention

Remark to response of A. V. Lichtenstein by V. N. Manskikh (88-88).
The arguments against the “bad luck” hypothesis suggested by C. Tomasetti and B. Vogelstein ((2015) Science, 347, 78–81) are significant in any case, and new experimental studies are necessary.
Keywords: cancer; “bad luck” hypothesis; carcinogenesis; mutagenesis; extrinsic and intrinsic factors; cancer prevention