Tatyana V. Korneenko

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic α subunit1

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic α 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).

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase.

Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Nongastric H,K-ATPase: structure and functional properties.

Abstract: Nongastric H,K‐ATPases whose catalytic subunits (AL1) encoded by human ATP1AL1 and homologous animal genes comprise the third distinct group within the X,K‐ATPase family. No unique nongastric β has been identified. Precise in situ colocalization and strong association of AL1 with β1 of Na,K‐ATPase was detected in apical membranes of rodent prostate epithelium. In this tissue, β1NK serves as an authentic subunit of both the Na,K‐ and nongastric H,K‐pumps. Upon expression in Xenopus oocytes the human AL1 can assemble with β1NK, and more efficiently with gastric βHK, into functional H,K‐pumps. Both AL1/β complexes exhibit a similar K‐affinity, and their K‐transport depends on intra‐ and extracellular Na. These data provide new evidence that nongastric H,K‐ATPase can perform Na/K‐exchange, and indicate that β does not significantly affect this ion‐pump function. Analysis of human nongastric H,K‐ATPase expressed in Sf‐21 insect cells revealed that AL1/βHK exhibits substantial enzymatic activities in K‐free medium and K stimulates, but Na has inhibitory effect on ATP hydrolysis. Thus, although the nongastric H,K‐ATPase can function as Na/K exchanger, its reaction mechanism is different from that of the Na,K‐ATPase. Human nongastric H,K‐ATPase is highly sensitive to bufalin, digoxin, and digitoxin, but almost resistant to digoxigenin and ouabagenin.

Nuclear translocation of lysyl oxidase is promoted by interaction with transcription repressor p66β

Lysyl oxidase (LOX) is an amine oxidase involved in protein cross-linking of the extracellular matrix. Less well characterized is the role that LOX plays among nuclear proteins, and molecular mechanisms of its transport to the nucleus are currently unknown. Here, we have employed yeast two-hybrid library screening and found that the LOX catalytic domain interacts with the transcription repressor p66β. This interaction has been confirmed in vitro and has been found to be accomplished through the CR2-containing domain of p66β. Moreover, co-expression of p66β and LOX in living tumor cells leads to the nuclear accumulation of LOX. Thus, p66β might be important for the regulation of LOX in the nucleus.

Intracellular location of hampin isoforms.

130 The male-specific lethal (MSL) protein complex is a well studied structure involved in chromatin rearrangement. In Drosophila , this complex is referred to as a “compensasome”, and the main function of this complex consists in alignment of gene expression between males and females. That is reached by a selective increase in the expression of X-chromosome genes due to activity of compensasome histonacetyltransferase with respect to Lys16 of H4 histon [1–3]. Although histone acetyltransferase activity is also characteristic of the human MSL complex, the latter plays a different physiological role. First, the mechanisms of X-chromosome quantitative compensation in mammals differ from those in Drosophila [4]. Second, the human MSL complex is associated with all chromosomes in the cell [1]. Third, the proteins constituting the human MSL are more diverse than those in Drosophila . For example, mammalian hampin (a homologue of the Drosophila MSL-1) has at least five various isoforms encoded by the same gene [5]. Hampin differs significantly from MSL-1. First, the amino acid sequences of these two proteins share only 27% of homology, and they nearly lack the higlyconserved regions. In addition, Drosophila MSL-1 consists of 1000 amino acids, whereas hampin is significantly shorter. On the other hand, the quaternary structure of the MSL complex seems to be rather conserved in all true protostomes, including all insects and mammals [1]. The mouse hampin has five different isoforms consisting of 616, 600, 463, 370, and 233 amino acids (isoforms A–E, respectively, Fig. 1) [5]. This diversity is the result of alternative splicing at several sites. The longest product of alternative splicing, hampin A, is encoded by nearly all exons of the gene (except exon 2) and contains all four domains characteristic of hampin isoforms (Fig. 1): the highly variable proline-rich domain I with the amino acid sequence of low complexity; domain IIcc that contains the coiled-coil region; the poorly conserved domain III; and the conserved domain IVpehe containing several highly conserved residues (Pro, Glu, His, and Glu), which are present even in the Drosophila MSL-1.

P4-ATPase Atp8b1/FIC1: Structural features and physiological functions in health and disease

The P4 ATPase family of P-type ATPases is of especial interest, since the main function of P4 ATPases is the translocation of phospholipids, phosphatidylserine in particular, from the outer monolayer of the plasma membrane to the inner one. P4 ATPase isoforms are redundant to some extent, but structural defects of certain isoforms can still lead to rather severe pathologies at the whole-organism level due to tissue specificity of expression of the corresponding genes and variability of the intracellular localization of the proteins and regulatory pathways. The product of the gene ATP8B1 occupies a special place among P4 ATPases, since a number of point mutations in this gene are known to cause severe hereditary diseases, namely, two forms of hereditary cholestasis (Byler disease and benign recurrent intrahepatic cholestasis) with extrahepatic symptoms including sensorineural hearing loss, pneumonia, impaired function of the sweat glands, and growth retardation. The physiological functions of the protein Atp8b1/FIC1 were characterized to a certain extent; they consist in the translocation of certain phospholipids (phosphatidylserine and cardiolipin) from the outer monolayer of the plasma membrane to the inner one. Disturbance of membrane asymmetry due to insufficient activity of Atp8b1/FIC1 is known to result in loss of hair cells in the inner ear, disruption of transport of bile acids in hepatocytes, and liver cirrhosis. Insufficient activity of Atp8b1/FIC1 is likely to increase the susceptibility of the organism to bacterial infections. It should be noted that in vivo regulation pathways for Atp8b1/FIC1 activity have not yet been characterized in sufficient detail. Therefore, investigation of this protein holds promise for better understanding of molecular mechanisms underpinning the development of pathologies, as well as for identification of potential therapeutic targets.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.

Postnatal regulation of X,K-ATPases in rat skin and conserved lateroapical polarization of Na,K-ATPase in vertebrate epidermis.

Development of epidermis creates stratified epithelium with different sets of ion‐transporting enzymes in its layers. We have characterized expression of Na,K‐ and H,K‐ATPase α and β subunits and FXYD isoforms in rat skin. Maturation of rat skin from newborn to adult is associated with an increase in FXYD4 and a decrease of Na,K‐ATPase α1‐isoform, ATP1B4 and FXYD6 transcripts. Na,K‐ATPase of rat epidermis is represented predominantly by α1 and β3 isoforms. Keratinization is associated with the loss of the Na,K‐ATPase α‐subunit and an enrichment of αng. Na,K‐ATPase α1 is abundant in the innermost layer, stratum basale, where it is lacking in basal membranes, thus indicating lateroapical polarization of Na,K‐ATPase. Immunocytochemical detection of Na,K‐ATPase in Xenopus laevis skin shows that cellular and subcellular localization of the enzyme has a pattern highly similar to that of mammals: basolateral in glandular epithelium and lateroapical in epidermis.

Matricide in Caenorhabditis elegans as an example of programmed death of an animal organism: The role of mitochondrial oxidative stress

Nematode Caenorhabditis elegans is a widely used model for studying genetic and molecular mechanisms of lifespan regulation. The choice between two life strategies—normal aging and matricide (programmed death)-made by an adult hermaphroditic C. elegans organism is based on food availability and is also affected by different kinds of stress. We have tested a hypothesis concerning an increase in matricide probability as a result of oxidative stress; this hypothesis is based on the phenoptosis theory. High concentrations of paraquat, a strong mitochondrial stressor, were shown to increase matricide propensity. Mutants with a decreased antioxidant potential of mitochondria (nnt) were more sensitive to the reagent. On the other hand, paraquat concentrations required for this effect to be observed are toxic for the progeny, whereas at low paraquat concentration matricide frequency is the same in wild-type worms and nnt mutants. Therefore, one can assume that the molecular mechanisms of matricide initiation under normal conditions are not necessarily connected to mitochondrial oxidative stress.

Tissue Specificity of Alternative Splicing of Transcripts Encoding Hampin, a New Mouse Protein 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.

Evolutionary diversification of the BetaM interactome acquired through co-option of the ATP1B4 gene in placental mammals

ATP1B4 genes represent a rare instance of orthologous vertebrate gene co-option that radically changed properties of the encoded BetaM proteins, which function as Na,K-ATPase subunits in lower vertebrates and birds. Eutherian BetaM has lost its ancestral function and became a muscle-specific resident of the inner nuclear membrane. Our earlier work implicated BetaM in regulation of gene expression through direct interaction with the transcriptional co-regulator SKIP. To gain insight into evolution of BetaM interactome we performed expanded screening of eutherian and avian cDNA libraries using yeast-two-hybrid and split-ubiquitin systems. The inventory of identified BetaM interactors includes lamina-associated protein LAP-1, myocyte nuclear envelope protein Syne1, BetaM itself, heme oxidases HMOX1 and HMOX2; transcription factor LZIP/CREB3, ERGIC3, PHF3, reticulocalbin-3, and β-sarcoglycan. No new interactions were found for chicken BetaM and human Na,K-ATPase β1, β2 and β3 isoforms, indicating the uniqueness of eutherian BetaM interactome. Analysis of truncated forms of BetaM indicates that residues 72-98 adjacent to the membrane in nucleoplasmic domain are important for the interaction with SKIP. These findings demonstrate that evolutionary alterations in structural and functional properties of eutherian BetaM proteins are associated with the increase in its interactome complexity.