Interactome of Paraoxonase PON2 Reveals New Pathways for Tumor Growth Regulation.

The interactome of paraoxonase-2 encoded by the PON2 gene was investigated. A cDNA library was screened using a yeast two-hybrid system to search for new proteins interacting with human PON2. Analysis of the identified candidates, along with previously published data on interactors obtained by other methods, indicates the presence of a significant number of indirect interactions between PON2 and EGFR and, consequently, possible regulation of tumor growth with mutant EGFR involving PON2.

[P4-ATP-ase Atp8b1/FIC1: structural properties and (patho)physiological functions].

P4-ATP-ases comprise an interesting family among P-type ATP-ases, since they are thought to play a major role in the transfer of phospholipids such as phosphatydylserine from the outer leaflet to the inner leaflet. Isoforms of P4-ATP-ases are partially interchangeable but peculiarities of tissue-specific expression of their genes, intracellular localization of proteins, as well as regulatory pathways lead to the fact that, on the organismal level, serious pathologies may develop in the presence of structural abnormalities in certain isoforms. Among P4-ATP-ases a special place is occupied by ATP8B1, for which several mutations are known that lead to serious hereditary diseases: two forms of congenital cholestasis (PFIC1 or Byler disease and benign recurrent intrahepatic cholestasis) with extraliver symptoms such as sensorineural hearing loss. The physiological function of the Atp8b1/FIC1 protein is known in general outline: it is responsible for transport of certain phospholipids (phosphatydylserine, cardiolipin) for the outer monolayer of the plasma membrane to the inner one. It is well known that perturbation of membrane asymmetry, caused by the lack of Atp8B1 activity, leads to death of hairy cells of the inner ear, dysfunction of bile acid transport in liver-cells that causes cirrhosis. It is also probable that insufficient activity of Atp8b1/FIC1 increases susceptibility to bacterial pneumonia.Regulatory pathways of Atp8b1/FIC1 activity in vivo remain to be insufficiently studied and this opens novel perspectives for research in this field that may allow better understanding of molecular processes behind the development of certain pathologies and to reveal novel therapeutical targets.

[Tissue specificity of alternative splicing products of mouse mRNA encoding new protein hampin homologous to the Drosophila MSL-1 protein].

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.

[Role of nitric oxide in tumor growth]. / Biologicheskaia aktivnost' oksida azota v mekhanizmakh opukholevogo rosta.

This review article will analyze the role of nitric oxide in antiblastomous organism resistance, in particular some mechanisms of NO-mediated apoptosis in different cells and NO involvement in etiological mechanisms as well as tumor growth promotion. The possible mechanisms of nitric oxide dual effect are discussed. The data about NO as a mediator in different methods of cancer treatment are given. In conclusion, we have determined some principles of application of NO-modulating agents in cancer therapy.

The betam protein, a member of the X,K-ATPase beta-subunits family, is located intracellularly in pig skeletal muscle.

The sequence of the pig cDNA encoding the muscle-specific betam-protein, a member of the X,K-ATPase beta-subunits family, was determined. Two alternatively spliced transcripts encoding polypeptide chains of 355 and 351 residues were identified. The tissue specificity of expression of betam and other X,K-ATPase beta-subunit genes was studied by RT-PCR performed on 24 tissues from newborn pigs. The betam expression was shown to be highly tissue-specific, being detected at the highest level in skeletal muscle, at a lower level in heart, and at much lower level in skin. The betam transcripts are more abundant in the tissues from the newborn than adult. Immunoblotting and deglycosylation shift assay indicated that skeletal muscle membranes of newborn pigs contain betam protein with an electrophoretic mobility and carbohydrate content very similar to that of human betam. Fractionation of membranes from both newborn and adult pig skeletal muscles by isopycnic centrifugation revealed that the majority of the betam protein is concentrated in the sarcoplasmic reticulum-containing fractions. This intracellular location is a unique property that distinguishes the betam protein from other members of the X,K-ATPase beta-subunit family.

Intersubunit interactions in human X,K-ATPases: role of membrane domains M9 and M10 in the assembly process and association efficiency of human, nongastric H,K-ATPase alpha subunits (ATP1al1) with known beta subunits.

Na,K- and H,K-ATPase (X,K-ATPase) alpha subunits need association with a beta subunit for their maturation, but the authentic beta subunit of nongastric H,K-ATPase alpha subunits has not been identified. To better define alpha-beta interactions in these ATPases, we coexpressed human, nongastric H,K-ATPase alpha (AL1) and Na,K-ATPase alpha1 (alpha1NK) as well as AL1-alpha1 and alpha1-AL1 chimeras, which contain exchanged M9 and M10 membrane domains, together with each of the known beta subunits in Xenopus oocytes and followed their resistance to cellular and proteolytic degradation and their ER exit. We show that all beta subunits (gastric betaHK, beta1NK, beta2NK, beta3NK, or Bufo bladder beta) can associate efficiently with alpha1NK, but only gastric betaHK, beta2NK, and Bufo bladder beta can form stably expressed AL1-beta complexes that can leave the ER. The trypsin resistance and the forces of subunit interaction, probed by detergent resistance, are lower for AL1-beta complexes than for alpha1NK-beta complexes. Furthermore, chimeric alpha1-AL1 can be stabilized by beta subunits, but alpha1-AL1-gastric betaHK complexes are retained in the ER. On the other hand, chimeric AL1-alpha1 cannot be stabilized by any beta subunit. In conclusion, these results indicate that (1) none of the known beta subunits is the real partner subunit of AL1 but an as yet unidentified, authentic beta should have structural features resembling gastric betaHK, beta2NK, or Bufo bladder beta and (2) beta-mediated maturation of alpha subunits is a multistep process which depends on the membrane insertion properties of alpha subunits as well as on several discrete events of intersubunit interactions.

[Identification of Escherichia coli nitrate reductase as an antigen for a monoclonal antibody with previously unknown specificity]. / Identifikatsiia nitratreduktazy Escherichia coli kak antigena monoklonal'nogo antitela neizvestnoi ranee spetsifichnosti.

The immunoaffinity chromatography of total membrane proteins from Escherichia coli helped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknown E. coli antigen. Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified by N-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from the E. coli cells grown upon induction of nitrate reductase. It was shown that the 3A6 antibody specifically recognizes the alpha subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.

Immunochemical demonstration of a novel beta-subunit isoform of X, K-ATPase in human skeletal muscle.

Recently we have identified mRNA encoding a hitherto unknown mammalian X,K-ATPase beta-subunit expressed predominantly in muscle tissue (Pestov, N. B. et al. (1999) FEBS Lett. 456, 243-248). Here we demonstrate the existence of the predicted protein, designated as beta(m) (beta(muscle)), in human adult skeletal muscle membranes using immunoblotting with beta(m)-specific antibodies generated against recombinant polypeptide formed by extramembrane beta(m) domains. The electrophoretic mobility of beta(m) was shown to be abnormally low due to the presence of Glu-rich sequences. In contrast to mature forms of other known X,K-ATPase beta-subunits, carbohydrate moiety of beta(m) is sensitive to endoglycosidase H and appears to be composed of short high-mannose or hybrid N-glycans. This finding argues in favor of an intracellular location of beta(m) in human skeletal muscle.

Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli. Effects on catalytic and H+-pumping activities.

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the alpha and beta subunits remained tightly associated. A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.

[Epitope mapping of monoclonal antibodies to human plasma membrane Ca2-ATPase]. / Kartirovanie épitopov monoklonal'nykh antitel k Ca2-ATPase plazmati cheskikh membran cheloveka.

Overlapping fragments of the fourth isoform of human plasma membrane Ca(2+)-ATPase (hPMCA4) and several fragments of hPMCA1 were expressed in bacterial cells and purified by metal affinity chromatography. Enzyme immunoassays of the fragments helped map epitopes for 4 monoclonal antibodies (2D8, 8B8, 7C8 and 5E6). The epitope for 2D8 was localized within the 222-249 site (i.e., in the putative transduction domain), the epitopes for 8B8 and 7C8 were localized within the 330-353 site, in which phospholipids are presumably bound, and the 5E6 epitope was found within the 791-843 site, where the putative hinge region is situated. 2D8 recognizes hPMCA1 and hPMCA4 isoforms, while 8B8 and 7C8 are specific for hPMCA4. The amino acid sequences of these epitopes and phage-displayed mimotopes were compared.

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic alpha subunit.

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic alpha subunit of a non-gastric ouabain-sensitive H,K-ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT-PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue-specific expression patterns indicates that animal and human non-gastric H,K-ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non-gastric H,K-ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

[Monoclonal antibodies to the alpha-subunit of the putative human H+,K+-ATPase encoded by the atp1al1 gene]. / Monoklonal'nye antitela k alpha-sub"edinitse gipoteticheskoi H+,K+-ATP-azy cheloveka, kodiruemoi genom atp1aq11.

The N-terminal fragment of ATP1AL1, the possible catalytic subunit of human ouabain-sensitive H+,K(+)-ATPase, was expressed in Escherichia coli cells as two recombinant proteins: the Ser14-Ile104 fragment or the same fragment containing His6 sequence at its N-end. The second protein was purified by metal-affinity chromatography and used as an antigen to construct two hybridoma lines producing antibodies of the IgM class. These monoclonal antibodies were shown to recognize not only the starting antigen but also the full-size recombinant ATP1AL1 protein and do not react with Na+,K(+)-ATPase.