Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

Osmoregulation of dimer resolution at the plasmid pJHCMW1 mwr locus by Escherichia coli XerCD recombination

Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E. coli ARG box consensus. The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.     

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.     

A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo-archaeal nucleoside diphosphate kinase

Nucleoside diphosphate kinase from extremely halophilic archaeon (HsNDK) requires above 2M NaCl concentration for in vitro refolding. Here an attempt was made to isolate mutations that allow HsNDK to refold in low salt media. Such a screening resulted in isolation of an HsNDK mutant, Gly114Arg, which efficiently refolded in the presence of 1M NaCl. This mutant, unlike the wild type enzyme, was expressed in Escherichia coli as an active form. The residue 114 is in close proximity to Glu155 of the neighboring subunit in the three dimensional hexameric structure of the HsNDK. It is thus possible that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK, stabilizing the hexameric subunit assembly.     

Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

Varietal differences in uptake and utilization of nitrogen and other macro-elements in seedlings of barley, Hordeum vulgare

Growth and uptake of N, P, S, K, Ca and Mg in barley ( Hordeum vulgare L.) were studied in water culture using young plants of 17 cultivars. Large varietal differences were obtained in dry weight production and mineral accumulation. The differences were not the same for plants grown in high- and low-salt media. For plants grown under both conditions there was a good correlation between dry weight production and total N content. Total shoot contents of K and Ca were closely correlated with shoot dry weight. Utilization of P and S in high- and low-salt plants and Mg in low-salt plants was variable in relation to dry weight production in both types of nutrient conditions. The correlation between dry weight and total content of Mg in high-salt plants was good. These differences in mineral economy between young barley plants were partly caused by varietal differences in relative growth rate, and in high-salt seedlings also by differences in seed content of N. The significance of root size, and of uptake, root-shoot partitioning and use-efficiency of specific elements differed; all four factors were important for P and S, but had varying impact on K, Mg and Ca. For N, differences in root size and ion accumulation were the most important factors causing varietal variation in mineral nutrition. – In a special experiment seedlings of barley were transferred to N-free nutrient solution after six days of adequate N supply. There was no significant varietal differences in use-efficiency ratio of N. Root/shoot partitioning of N was unaffected.     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes

The efficacy of PNA vs DNA oligomers for the recovery of femtomolar concentrations of 16S rDNA targets was determined with solution- and mixed-phase hybridization formats and limiting dilution quantitative PCR. Several results contradict existing perceptions of expected PNA behavior deduced from hybridization studies with oligonucleotide targets at high concentration. For example, DNA probes in the solution hybridization format performed as well as or better than PNA probes under high- or low-salt conditions, regardless of hybridization time or target size. In the mixed-phase hybridization format, however, PNA probes showed certain advantages, with more rapid and efficient binding/recovery of target nucleic acids regardless of target size. Recovery of target DNA with PNA probes was always more efficient in low-salt (20 mM in Na(+)) than high-salt (400 mM in Na(+-)) phosphate buffer. Recovery of target DNA by PNA probes was enhanced in the presence of excess, nontarget DNA, and differences in PNA efficacy under low- or high-salt conditions vanquished. In contrast, DNA probe performance was unaffected by the presence or absence of exogenous DNA in both solution- and mixed-phase hybridization formats. The absolute recovery and detection limit of the affinity purification method with either DNA or PNA probes was approximately 10(2) input target molecules at zeptamolar concentrations.     

Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension

Vascular tissues express arginase that metabolizes L-arginine to L-ornithine and urea and thus reduces substrate availability for nitric oxide formation. Dahl salt-sensitive (Dahl-S) rats with salt-induced hypertension show endothelial dysfunction, including decreased vascular nitric oxide formation. This study tests the hypothesis that increased vascular arginase activity contributes to endothelial dysfunction in hypertensive Dahl-S rats. Male Dahl-S rats (5-6 wk) were placed on high (8%) or low (0.3%) NaCl diets for 4 wk. With respect to the low-salt group, mean arterial blood pressure was increased in the high-salt animals. Immunohistochemical stainings for arginase I and II were enhanced in arterioles isolated from high-salt Dahl-S rats. Experiments used isolated Krebs buffer-superfused first-order gracilis muscle arterioles with constant pressure (80 mmHg) and no luminal flow or constant midpoint but altered endpoint pressures to establish graded levels of luminal flow (0-50 microl/min). In high-salt arterioles, responses to an endothelium-dependent vasodilator acetylcholine (1 nmol/l to 3 micromol/l) and flow-induced dilation were decreased. Acute in vitro treatment with an inhibitor of arginase, 100 micromol/l (S)-(2-boronoethyl)-L-cystine, or the nitric oxide precursor, 1 mmol/l L-arginine, similarly enhanced acetylcholine and flow-induced maximal dilations and abolished the differences between high- and low-salt arterioles. These data show that arteriolar arginase expression is increased and that endothelium-dependent vasodilation is decreased in high-salt Dahl-S rats. Acute pretreatment with an arginase inhibitor or with L-arginine restores endothelium-dependent vasodilation and abolishes the differences between high- and low-salt groups. These results suggest that enhanced vascular arginase activity contributes to endothelial dysfunction in Dahl-S rats with salt-induced hypertension and identifies arginase as a potential therapeutic target to prevent endothelial dysfunction.     

Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins

Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.     

Efficient expression of haloarchaeal nucleoside diphosphate kinase via strong porin promoter in moderately halophilic bacteria

Enzymes from extremely halophilic archaea require high concentration of salts for their proper folding and consequently are expressed as an unfolded and inactive form in Escherichia coli. Moderate halophile, which accumulates protein stabilizers, i.e., compatible solutes, is an attractive host cell for the recombinant production of heterologous proteins, since such protein stabilizers may help folding of expressed proteins. Here, we succeeded in efficient expression and purification to homogeneity of recombinant haloarchaeal nucleoside diphosphate kinase (HsNDK) in moderate halophile using newly isolated strong porin promoter.     

Characterisation of a highly stable α-amylase from the halophilic archaeon Haloarcula hispanica

Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted -amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.     

Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system

The role of betaine as a factor influencing the salt resistance of the respiratory system in resting cells of the moderately halophilic halotolerant bacterium Ba(1) was studied. Betaine accelerated succinate oxidation in cells obtained from low-salt medium, and stimulation of the respiratory rate was stronger the higher the sodium chloride concentration in the assay medium. The stimulatory effect also depended on the ratio of betaine concentration to the amount of bacteria present. Accumulation of labelled betaine by the bacterial cells was demonstrated; like the respiratory stimulation, it was favourably influenced by an increase in the sodium chloride concentration of the medium. In cells harvested from a high-salt medium and washed with 2.0m-sodium chloride, betaine caused no increase in the respiratory rate, nor was the already high salt resistance of the respiratory system further improved by the addition of betaine. When, however, these cells lost their salt resistance as a result of washing in the absence of sodium chloride, betaine was able to restore it to its original level. In contrast with respiration in low-salt-grown bacteria, that in high-salt-grown cells was not affected by betaine, even after they were washed in the absence of sodium chloride, when the sodium chloride concentration was optimum.     

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.     

Passive Proton Conductance Is the Major Reason for Membrane Depolarization and Conductance Increase in Chara buckellii in High-Salt Conditions

Chara buckellii G.O.A., a salt-tolerant alga, has a less negative membrane potential (Em) when cultured in saline medium (artificial Waldsea water) than when cultured in freshwater. The cell hyperpolarizes and membrane conductance (Gm) decreases when the external medium is changed from Waldsea control solution (WCS), a high-salt medium, to low-salt medium containing sufficient sorbitol to generate the same osmotic potential as WCS. Banding pattern and proton flux experiments show that C. buckellii has higher passive proton influx in the alkaline band in high-salt medium than in low-salt medium. Decrease of the passive proton influx by darkness or low external pH dramatically hyperpolarizes the membrane and decreases the conductance. The pH dependence curves of Em and Gm also indicate the existence of high passive proton conductance (GH) in C. buckellii. Ion substitution experiments show that Em and Gm of saltwater cells are not dependent on K+, Na+, Cl-, or SO42+. Mg2+ also affects Em and Gm, but its effect is probably on GH. We conclude that GH is the most important cause of the membrane depolarization and conductance increase in the saltwater alga C. buckellii.     

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.     

Divalent Metal Ion-Induced Folding Mechanism of RNase H1 from Extreme Halophilic Archaeon Halobacterium sp. NRC-1

RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.