Alla Shainskaya | Weizmann Institute of Science (original) (raw)

Papers by Alla Shainskaya

The Sodium Pump, 1994

Knowledge of the structure of cation binding sites and of organization of transmembrane segments ... more Knowledge of the structure of cation binding sites and of organization of transmembrane segments of cation pumps, such as Na+/K+-ATPase, is essential for an understanding of the active transport mechanism. Renal Na+/K+-ATPase extensively digested with trypsin, in the presence of Rb+ and absence of Ca2+ ions, to a Cterminal 19kDa and smaller membrane-embedded fragments (8–11kDa) of the alpha chain, and intact beta chain or beta split into two fragments, is a valuable tool for establishing structure-function relationships. In these “19kDa membranes”, cation occlusion sites are intact, and although it is evident that the sites are located within trans-membrane segments, the identity and number of segments involved is unknown. The question as to which or how many of the trans-membrane segments actually occlude the ions is unanswered, and is the major focus of the present work. The first section of the present paper analyses the nature of the antagonism between Ca2+ and Rb+ ions in “19kDa-membranes”, and explains the previous observation that the presence of Ca2+ during digestion destabilises the 19kDa fragment to trypsin and occlusion is destroyed. The second section describes the purification and identification of the limit tryptic membrane-embedded fragments, produced by digestion of “19kDa-membranes” in the presence of Ca2+.

The Sodium Pump, 1994

Organic guanidium derivatives have been characterized as competitive Na+-like antagonists in Na+/... more Organic guanidium derivatives have been characterized as competitive Na+-like antagonists in Na+/K+-ATPase (2). They compete for Na+ and Rb+ occlusion by Na+/K+-ATPase, and inhibit ATPase activity and Na+-dependent phosphorylation from ATP. Like Na+, they inhibit phosphorylation from inorganic phosphate and stabilize the E1 conformation of FITC-labeled enzyme. Two compounds m- and p- xylylene bisguanidinium, pXBG and mXBG, were found to compete for cation occlusion with the highest affinity (~8μM). This work utilizes pXBG and other Na+-like antagonists to investigate structural and functional properties of the cation binding and transport site of renal Na+/K+-ATPase. The first section summarises experiments which compare the ability of pXBG and other Na+-like antagonists with that of transported alkali metal cations to protect the enzyme against covalent modification and structural perturbations of the cation occlusion site (9). The second section describes experiments with reconstituted proteoliposomes which define the sidedness of inhibition and effects of electrical diffusion potentials on inhibition by Na+ antagonists.

Gene, 2012

In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra-and ... more In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra-and intergender communication. Blood samples taken at onset and peak of daily sexual activity from dominant and subordinate Oreochromis niloticus males and females were fractionated by native gel electrophoresis and the fast-migrating proteins were subjected to mass spectrometry. Mining the sequence data of the Cichlid Genome Consortium, we identified 11 proteins from the lipocalin super-family and characterized their genes' structures. Phylogenetic and structural analyses subdivided these genes into two classes: (I) 3-coding-exon apolipoproteins and (II) more complex 6-coding-exon sulfide-bond-containing lipocalins. Five apolipoproteins and PTGDSL1, TBTBP, and MSP proteins were modulated by gender and sexual behavior. PTGDSL1 protein was only observed in the plasma serum of dominant males. However, the cysteine residue in the position that is crucial for synthetase activity in mammalian prostaglandin D synthetases was not conserved in PTGDSL1 or PTGDSL2 proteins. In line with previous reports suggesting their involvement in male functions as pheromone transporters, TBTBP and MSP proteins were not detected in females at the onset of daily activity. Their increasing amount in males was concordant with the increase in apolipoproteins AFP4L, APOA4a, APOA4b, APO14kD and APOC2, which were detected exclusively in dominant males, indicating a possible role in mobilization of the energy required to maintain their social hierarchy.

The Environmentalist, 2012

ABSTRACT Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is... more ABSTRACT Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is well known in literature, the individual role of different isoforms of pump in determination of its radio-sensitivity is not clear yet. This is the subject of the present investigation. Using isotope, electro-physiological and enzymological methods, the effect of γ-ionizing radiation on cell membrane voltage-current characteristics, acetylcholine-induced membrane current, 22Na+ and 45Ca2+ exchange between cells and bathing solution, Na+K+-ATPase activity, dose-dependent ouabain binding with cell membrane, intracellular cAMP and membrane phosphorylation in snail neurons were studied. The changes in neurons as a result of 30-min γ-radiation exposure of snails to 5.16 Ci/kg at the end of the first 30 min of post-radiation period were as follows: the increase in membrane ionic conductance reversed the ouabain sensitivity of acetylcholine-induced currents, stimulation of 22Na+ and 45Ca2+ uptakes, inhibition of Na/K pump, activation Na/Ca exchange in reversed mode, increase in ouabain binding with high-affinity α3 and decrease with α2 middle-affinity receptors, decrease in intracellular cAMP content and membrane dephosphorylation. On the basis of the obtained data, it is suggested that both α3 and α2 catalytic subunits of Na++K+-ATPase serve primary membrane sensors through the activation of which the biological effect of γ-radiation on neurons is realized. The IR has activation effects on α3-dependent Na+/Ca2+ exchange in forward and its inactivation on α2-dependent reverse modes.

The Journal of biological chemistry, Jan 23, 2018

Proceedings of the National Academy of Sciences of the United States of America, Sep 27, 2016

It is well established that the expression profiles of multiple and possibly redundant matrix-rem... more It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochem...

Archives of Microbiology, Aug 16, 2008

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, ... more The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95°C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small non-speciWc channels with an exclusion limit of 260-300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic-strands. This research presents porins which were isolated and characterized for the Wrst time from bacteria belonging to the Desulfovibrionaceae family.

Molecular Microbiology, 2003

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like... more Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Mol Microbiol, 2002

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like... more Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Acta Physiologica Hungarica

The biological effect of ionizing radiation (IR) in lethal and sublethal doses on the sodium-pota... more The biological effect of ionizing radiation (IR) in lethal and sublethal doses on the sodium-potassium transport systems in the fractions, enriched of neuron and glial cells and in cortex slices from rat brain was investigated. It was shown that IR leads to marked disturbances in the activity of Na,K-ATPase both in neuron and in glial cells. Some phasic character of alterations may be noted, which is expressed in different degree for various cellular elements of the brain. Using the surviving brain slices we have shown that IR causes essential phasic changes in potassium ion reaccumulation in different times after exposure. The mechanisms of the disturbance of Na,K-pump function in nervous tissue after irradiation are under discussion.

The Journal of biological chemistry, Jan 22, 2013

Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of moo... more Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of mood and anxiety disorders. Here, we demonstrate that incubation (2 h) of murine islets or Min6 β cell line with the SSRIs paroxetine, fluoxetine, or sertraline inhibited insulin-induced Tyr phosphorylation of insulin receptor substrate (IRS)-2 protein and the activation of its downstream targets Akt and the ribosomal protein S6 kinase-1 (S6K1). Inhibition was dose-dependent with half-maximal effects at ∼15-20 μM. It correlated with a rapid dephosphorylation and activation of the IRS kinase GSK3β. Introduction of GSK3β siRNAs eliminated the inhibitory effects of the SSRIs. Inhibition of IRS-2 action by 30 μM SSRI was associated with a marked inhibition of glucose-stimulated insulin secretion from murine and human pancreatic islets. Secretion induced by basic secretagogues (KCl and Arg) was not affected by these drugs. Prolonged treatment (16 h) of Min6 cells with sertraline resulted in the ...

Oncogene, 2013

The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING ... more The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.

Oncogene, 2012

The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING ... more The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.

The Journal of biological chemistry, 2000

The ␥ subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryy... more The ␥ subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, ␥ runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the ␥ chains of rat kidney Na,K-ATPase shows that ␥ a (upper) has a mass of 7184.0 ؎ 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while ␥ b (lower) has a mass of 7337.9 ؎ 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of ␥ a , TELSANH, are replaced by Ac-MDRWYL in ␥ b , but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either ␥ a or ␥ b of the Na,K-ATPase, respectively. ␥ a or ␥ b cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to ␥ a or ␥ b of renal Na,K-ATPase. Additional minor bands seen after transfection, namely ␥ a in human embryonic kidney and ␥ b in HeLa, are presumably cellspecific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na,K-ATPase contains two variants of the ␥ subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.

The Journal of biological chemistry, 1993

The Journal of biological chemistry, 1996

This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the ␤ s... more This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the ␤ subunit of Na/K-ATPase leads to changes in the kinetics of occlusion of Rb ؉ ions. The experiments utilize extensively trypsinized Na/K-ATPase, "19-kDa membranes," which lack cytoplasmic loops of the ␣ subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containing transmembrane segments and extracellular loops are intact. The ␤ subunit is partially split into NH 2 -and COOH-terminal fragments of 16 and Ϸ50 kDa, respectively. Cation occlusion and ouabain binding are preserved. The 19-kDa membranes were incubated, at 37°C, with a selection of proteases, in the presence of Rb ؉ ions. In these conditions, only ␣-chymotrypsin destroyed the ability to occlude Rb ؉ ions. This process was associated with truncation of the 16-kDa fragment of the ␤ subunit in two stages. In the first stage, chymotrypsin removed 10 residues from the 16-kDa fragment to form a 15-kDa fragment (NH 2terminal Ile 15 ) and 4 or 6 residues from the NH 2 terminus of the ␣ subunit fragment beginning at Asp 68 . In these membranes Rb ؉ occlusion was still intact at 37°C. Strikingly, however, deocclusion of two Rb ؉ ions, which is characteristically biphasic in 19-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb ؉ ions compared with 19-kDa membranes; and, consistent with the lower Rb ؉ affinity, Rb ؉ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb ؉ occlusion. In the second stage, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH 2 -terminal Leu 24 ), followed by thermal destabilization of Rb ؉ occlusion even at high concentrations of Rb ؉ ions. Eventually, the thermally inactivated complex of fragments of ␣ and ␤ subunits was digested to the limit peptides. The results suggest that the cytoplasmic domain of the ␤ subunit interacts with that of the ␣ subunit, possibly with residues leading into the first transmembrane segment, and controls access of Rb ؉ ions into or out of the occlusion sites.

The Journal of biological chemistry, 1994

The Sodium Pump, 1994

Knowledge of the structure of cation binding sites and of organization of transmembrane segments ... more Knowledge of the structure of cation binding sites and of organization of transmembrane segments of cation pumps, such as Na+/K+-ATPase, is essential for an understanding of the active transport mechanism. Renal Na+/K+-ATPase extensively digested with trypsin, in the presence of Rb+ and absence of Ca2+ ions, to a Cterminal 19kDa and smaller membrane-embedded fragments (8–11kDa) of the alpha chain, and intact beta chain or beta split into two fragments, is a valuable tool for establishing structure-function relationships. In these “19kDa membranes”, cation occlusion sites are intact, and although it is evident that the sites are located within trans-membrane segments, the identity and number of segments involved is unknown. The question as to which or how many of the trans-membrane segments actually occlude the ions is unanswered, and is the major focus of the present work. The first section of the present paper analyses the nature of the antagonism between Ca2+ and Rb+ ions in “19kDa-membranes”, and explains the previous observation that the presence of Ca2+ during digestion destabilises the 19kDa fragment to trypsin and occlusion is destroyed. The second section describes the purification and identification of the limit tryptic membrane-embedded fragments, produced by digestion of “19kDa-membranes” in the presence of Ca2+.

The Sodium Pump, 1994

Organic guanidium derivatives have been characterized as competitive Na+-like antagonists in Na+/... more Organic guanidium derivatives have been characterized as competitive Na+-like antagonists in Na+/K+-ATPase (2). They compete for Na+ and Rb+ occlusion by Na+/K+-ATPase, and inhibit ATPase activity and Na+-dependent phosphorylation from ATP. Like Na+, they inhibit phosphorylation from inorganic phosphate and stabilize the E1 conformation of FITC-labeled enzyme. Two compounds m- and p- xylylene bisguanidinium, pXBG and mXBG, were found to compete for cation occlusion with the highest affinity (~8μM). This work utilizes pXBG and other Na+-like antagonists to investigate structural and functional properties of the cation binding and transport site of renal Na+/K+-ATPase. The first section summarises experiments which compare the ability of pXBG and other Na+-like antagonists with that of transported alkali metal cations to protect the enzyme against covalent modification and structural perturbations of the cation occlusion site (9). The second section describes experiments with reconstituted proteoliposomes which define the sidedness of inhibition and effects of electrical diffusion potentials on inhibition by Na+ antagonists.

Gene, 2012

In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra-and ... more In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra-and intergender communication. Blood samples taken at onset and peak of daily sexual activity from dominant and subordinate Oreochromis niloticus males and females were fractionated by native gel electrophoresis and the fast-migrating proteins were subjected to mass spectrometry. Mining the sequence data of the Cichlid Genome Consortium, we identified 11 proteins from the lipocalin super-family and characterized their genes' structures. Phylogenetic and structural analyses subdivided these genes into two classes: (I) 3-coding-exon apolipoproteins and (II) more complex 6-coding-exon sulfide-bond-containing lipocalins. Five apolipoproteins and PTGDSL1, TBTBP, and MSP proteins were modulated by gender and sexual behavior. PTGDSL1 protein was only observed in the plasma serum of dominant males. However, the cysteine residue in the position that is crucial for synthetase activity in mammalian prostaglandin D synthetases was not conserved in PTGDSL1 or PTGDSL2 proteins. In line with previous reports suggesting their involvement in male functions as pheromone transporters, TBTBP and MSP proteins were not detected in females at the onset of daily activity. Their increasing amount in males was concordant with the increase in apolipoproteins AFP4L, APOA4a, APOA4b, APO14kD and APOC2, which were detected exclusively in dominant males, indicating a possible role in mobilization of the energy required to maintain their social hierarchy.

The Environmentalist, 2012

ABSTRACT Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is... more ABSTRACT Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is well known in literature, the individual role of different isoforms of pump in determination of its radio-sensitivity is not clear yet. This is the subject of the present investigation. Using isotope, electro-physiological and enzymological methods, the effect of γ-ionizing radiation on cell membrane voltage-current characteristics, acetylcholine-induced membrane current, 22Na+ and 45Ca2+ exchange between cells and bathing solution, Na+K+-ATPase activity, dose-dependent ouabain binding with cell membrane, intracellular cAMP and membrane phosphorylation in snail neurons were studied. The changes in neurons as a result of 30-min γ-radiation exposure of snails to 5.16 Ci/kg at the end of the first 30 min of post-radiation period were as follows: the increase in membrane ionic conductance reversed the ouabain sensitivity of acetylcholine-induced currents, stimulation of 22Na+ and 45Ca2+ uptakes, inhibition of Na/K pump, activation Na/Ca exchange in reversed mode, increase in ouabain binding with high-affinity α3 and decrease with α2 middle-affinity receptors, decrease in intracellular cAMP content and membrane dephosphorylation. On the basis of the obtained data, it is suggested that both α3 and α2 catalytic subunits of Na++K+-ATPase serve primary membrane sensors through the activation of which the biological effect of γ-radiation on neurons is realized. The IR has activation effects on α3-dependent Na+/Ca2+ exchange in forward and its inactivation on α2-dependent reverse modes.

The Journal of biological chemistry, Jan 23, 2018

Proceedings of the National Academy of Sciences of the United States of America, Sep 27, 2016

It is well established that the expression profiles of multiple and possibly redundant matrix-rem... more It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochem...

Archives of Microbiology, Aug 16, 2008

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, ... more The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95°C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small non-speciWc channels with an exclusion limit of 260-300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic-strands. This research presents porins which were isolated and characterized for the Wrst time from bacteria belonging to the Desulfovibrionaceae family.

Molecular Microbiology, 2003

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like... more Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Mol Microbiol, 2002

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like... more Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Acta Physiologica Hungarica

The biological effect of ionizing radiation (IR) in lethal and sublethal doses on the sodium-pota... more The biological effect of ionizing radiation (IR) in lethal and sublethal doses on the sodium-potassium transport systems in the fractions, enriched of neuron and glial cells and in cortex slices from rat brain was investigated. It was shown that IR leads to marked disturbances in the activity of Na,K-ATPase both in neuron and in glial cells. Some phasic character of alterations may be noted, which is expressed in different degree for various cellular elements of the brain. Using the surviving brain slices we have shown that IR causes essential phasic changes in potassium ion reaccumulation in different times after exposure. The mechanisms of the disturbance of Na,K-pump function in nervous tissue after irradiation are under discussion.

The Journal of biological chemistry, Jan 22, 2013

Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of moo... more Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of mood and anxiety disorders. Here, we demonstrate that incubation (2 h) of murine islets or Min6 β cell line with the SSRIs paroxetine, fluoxetine, or sertraline inhibited insulin-induced Tyr phosphorylation of insulin receptor substrate (IRS)-2 protein and the activation of its downstream targets Akt and the ribosomal protein S6 kinase-1 (S6K1). Inhibition was dose-dependent with half-maximal effects at ∼15-20 μM. It correlated with a rapid dephosphorylation and activation of the IRS kinase GSK3β. Introduction of GSK3β siRNAs eliminated the inhibitory effects of the SSRIs. Inhibition of IRS-2 action by 30 μM SSRI was associated with a marked inhibition of glucose-stimulated insulin secretion from murine and human pancreatic islets. Secretion induced by basic secretagogues (KCl and Arg) was not affected by these drugs. Prolonged treatment (16 h) of Min6 cells with sertraline resulted in the ...

Oncogene, 2013

The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING ... more The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.

Oncogene, 2012

The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING ... more The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.

The Journal of biological chemistry, 2000

The ␥ subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryy... more The ␥ subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, ␥ runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the ␥ chains of rat kidney Na,K-ATPase shows that ␥ a (upper) has a mass of 7184.0 ؎ 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while ␥ b (lower) has a mass of 7337.9 ؎ 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of ␥ a , TELSANH, are replaced by Ac-MDRWYL in ␥ b , but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either ␥ a or ␥ b of the Na,K-ATPase, respectively. ␥ a or ␥ b cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to ␥ a or ␥ b of renal Na,K-ATPase. Additional minor bands seen after transfection, namely ␥ a in human embryonic kidney and ␥ b in HeLa, are presumably cellspecific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na,K-ATPase contains two variants of the ␥ subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.

The Journal of biological chemistry, 1993

The Journal of biological chemistry, 1996

This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the ␤ s... more This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the ␤ subunit of Na/K-ATPase leads to changes in the kinetics of occlusion of Rb ؉ ions. The experiments utilize extensively trypsinized Na/K-ATPase, "19-kDa membranes," which lack cytoplasmic loops of the ␣ subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containing transmembrane segments and extracellular loops are intact. The ␤ subunit is partially split into NH 2 -and COOH-terminal fragments of 16 and Ϸ50 kDa, respectively. Cation occlusion and ouabain binding are preserved. The 19-kDa membranes were incubated, at 37°C, with a selection of proteases, in the presence of Rb ؉ ions. In these conditions, only ␣-chymotrypsin destroyed the ability to occlude Rb ؉ ions. This process was associated with truncation of the 16-kDa fragment of the ␤ subunit in two stages. In the first stage, chymotrypsin removed 10 residues from the 16-kDa fragment to form a 15-kDa fragment (NH 2terminal Ile 15 ) and 4 or 6 residues from the NH 2 terminus of the ␣ subunit fragment beginning at Asp 68 . In these membranes Rb ؉ occlusion was still intact at 37°C. Strikingly, however, deocclusion of two Rb ؉ ions, which is characteristically biphasic in 19-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb ؉ ions compared with 19-kDa membranes; and, consistent with the lower Rb ؉ affinity, Rb ؉ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb ؉ occlusion. In the second stage, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH 2 -terminal Leu 24 ), followed by thermal destabilization of Rb ؉ occlusion even at high concentrations of Rb ؉ ions. Eventually, the thermally inactivated complex of fragments of ␣ and ␤ subunits was digested to the limit peptides. The results suggest that the cytoplasmic domain of the ␤ subunit interacts with that of the ␣ subunit, possibly with residues leading into the first transmembrane segment, and controls access of Rb ؉ ions into or out of the occlusion sites.

The Journal of biological chemistry, 1994