New Insights on Respiratory Syncytial Virus Prevention.

Respiratory syncytial virus (RSV) is a well-known infant pathogen transmitted mainly by droplets. It is a leading cause of upper respiratory tract infections in children, usually with a mild course of illness. RSV has also been a threat to older people, especially those with underlying medical conditions. For a long time, prevention was limited to passive immunoprophylaxis with palivizumab for high-risk infants. There was a strong need to find other treatment or prevention methods against RSV infections. In addition, after the coronavirus disease 2019 (COVID-19) pandemic, some significant changes in RSV epidemiology have been observed. Researchers noticed the shift in RSV seasonality and age distribution and the increased number of cases in older infants and adults. All of these made the need to find other medical options even stronger. Fortunately, two protein-based vaccines against RSV have successfully passed all phases of clinical trials and have been approved for use by adults and older people. One of them is also approved for infants from birth to 6 months of age (after maternal immunisation during pregnancy) and for pregnant women between 24 and 36 weeks of pregnancy. Also, a new passive immunisation option named nirsevimab (a highly potent monoclonal antibody with a long half-life) is now available for the paediatric group. In this review, we will discuss the previous and current RSV prevention methods in the light of structural discoveries of RSV antigens.

Effects of Co-Ingestion of ß-Hydroxy-ß-Methylbutyrate and L-Arginine α-Ketoglutarate on Jump Performance in Young Track and Field Athletes.

The aim of the study was to determine the effect of simultaneous supplementation of ß-hydroxy-ß-methylbutyrate and L-Arginine α-ketoglutarate on lower limb power and muscle damage in medium distance runners aged 15.3 (±0.9) years old. METHODS: The study group consisted of 40 volunteers aged 14-17 years practicing medium distance running for at least two years. The study lasted 12 days and followed a randomized, double-blind, placebo-controlled, parallel design. All subjects attended a familiarization session on day 0 before the test. The subjects were randomly divided into two groups: supplements and placebo group. The same training cycle protocol was used in both groups during the 12-day training period. Morning warm-up involved 10 min jogging at 60-75% of maximal heart rate and countermovement jump height measurement. Main training units were carried out for both groups with the same volume. Training load assessment (the daily session Rating of Perceived Exertion (s-RPE) method) method takes into consideration the intensity and the duration of the training session to calculate the "training load" (TL). RESULTS: At the end of the training cycle, a significant (p = 0.002) decrease in the countermovement jump (CMJ) height was found in the placebo group when compared to the baseline. In the supplement group, there was no decrease in the countermovement jump height. Creatine kinase and lactate dehydrogenase concentration increased during the training days similarly in both groups and decreased on rest days. There were no differences between groups in enzymes concentration. The research results indicate that the supplement combination used in the supplements group prevented a reduction in the CMJ values. In contrast to the supplements group, in the placebo group, the CMJ changes were statistically significant: a noticeable (p = 0.002) decrease in CMJ was noted between the baseline measurement and the 6th measurement. The well-being of the subjects from both groups changed significantly during the training period, and the intergroup differences in the mood level were similar and not statistically significant. CONCLUSIONS: The results of this study indicate that the daily co-supplementation with calcium salt of ß-hydroxy-ß-methylbutyrate (7.5 g) and L-Arginine α-ketoglutarate (10 g) during training might help to prevent decline in jump performance. No influence on muscle damage markers or mood was shown.

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate.

H1N1 influenza virus is still regarded as a serious pandemic threat. The most effective method of protection against influenza virus and the way to reduce the risk of epidemic or pandemic spread is vaccination. Influenza vaccine manufactured in a traditional way, though well developed, has some drawbacks and limitations which have stimulated interest in developing alternative approaches. In this study, we demonstrate that the recombinant H1 vaccine based on the hydrophilic haemagglutinin (HA) domain and produced in the yeast system elicited high titres of serum haemagglutination-inhibiting antibodies in mice. Transmission electron microscopy showed that H1 antigen oligomerizes into functional higher molecular forms similar to rosette-like structures. Analysis of the N-linked glycans using mass spectrometry revealed that the H1 protein is glycosylated at the same sites as the native HA. The recombinant antigen was secreted into a culture medium reaching approximately 10 mg/l. These results suggest that H1 produced in Pichia pastoris can be considered as the vaccine candidate against H1N1 virus.

Highly sensitive electrochemical biosensor based on redox - active monolayer for detection of anti-hemagglutinin antibodies against swine-origin influenza virus H1N1 in sera of vaccinated mice.

BACKGROUND: In this work, we report an electrochemical biosensor for the detection of anti-hemagglutinin antibodies against the swine virus H1N1 present in mice sera immunized with mixture of His6-H1 HA in monomeric and oligomeric form. The oriented immobilization of the recombinant His-tagged hemagglutinin (His6-H1 HA) consists of: (i) formation of a mixed layer of 4-mercaptobutanol (MBT) and the thiol derivative of dipyrromethene (DPM); (ii) complexation of Cu (II) by DPM; (iii) immobilization of His6-H1 HA via coordination bonds between Cu (II) sites from DPM-Cu (II) complex and imidazole nitrogen atoms of a histidine tag; (iv) filling free spaces with bovine serum albumin. The interactions between recombinant His6- H1 HA covalently attached to the electrode surface and the anti-hemagglutinin H1 antibodies present in mice sera were explored with Osteryoung square-wave voltammetry. RESULTS: This analytical device was able to detect the antibodies present in vaccinated mice sera diluted from 1 × 109 to 1 × 108 fold. CONCLUSIONS: The unprecedented sensitivity of described biosensor is much better than widely use ELISA test and other analytical methods for determination of antibodies against the influenza A viruses. It has been proved that redox active DPM-Cu (II) monolayer is a universal platform suitable for stable and oriented immobilization of any His-tagged sensing elements. Thus, this universal layer could be a base of numerous analytical devices suitable for detection of antibodies against different viruses.

Transcriptional response to a prime/boost vaccination of chickens with three vaccine variants based on HA DNA and Pichia-produced HA protein.

Highly pathogenic avian influenza causes severe economic losses and is a potential threat to public health. Better knowledge of the mechanisms of chicken response to the novel types of vaccines against avian influenza might be helpful in their successful implementation into poultry vaccination programs in different countries. This work presents a comprehensive analysis of gene expression response elicited in chicken spleens by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens. All groups of vaccinated chickens displayed changes in spleen transcriptomes in comparison to the control group with 423, 375 and 212 identified differentially expressed genes in protein/protein, DNA/DNA and DNA/protein group, respectively. Genes with most significantly changed expression belong to immune-related categories. Depending on a group, a fraction of 15-34% of up-regulated and a fraction of 15-42% of down-regulated immune-related genes are shared by all groups. Interestingly, the most upregulated genes encode ß-defensins, short peptides with antimicrobial activity and immunomodulatory functions. Microarray results were validated with RT-qPCR method, which confirmed differential regulation of the selected immune-related genes. Immune-related differentially expressed genes and metabolic pathways identified in this work are compared to the available literature data on gene expression changes in vaccinated and non-vaccinated chickens after influenza infection.

The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen.

Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

A prime/boost vaccination with HA DNA and Pichia-produced HA protein elicits a strong humoral response in chickens against H5N1.

Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris.

An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge.

Highly pathogenic avian influenza is an on-going problem in poultry and a potential human pandemic threat. Pandemics occur suddenly and vaccine production must be fast and effective to be of value in controlling the spread of the virus. In this study we evaluated the potential of a recombinant protein from the extracellular domain of an H5 hemagglutinin protein produced in a yeast expression system to act as an effective vaccine. Protein production was efficient, with up to 200 mg purified from 1 L of culture medium. We showed that the deletion of the multibasic cleavage site from the protein improves oligomerization and, consequentially, its immunogenicity. We also showed that immunization with this deleted protein protected chickens from challenge with a highly pathogenic avian influenza H5N1 virus. Our results suggest that this recombinant protein produced in yeast may be an effective vaccine against H5N1 virus in poultry.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.

Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.

Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same ß2αß fold characteristic for Kazal-family serine proteinase inhibitors.

Application of Ni(II)-assisted peptide bond hydrolysis to non-enzymatic affinity tag removal.

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His(6). This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions.

Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a general sequence R1-(Ser/Thr)-Xaa-His-Zaa-R, which occurs in the presence of Ni(II) ions [A. Krezel, E. Kopera, A. M. Protas, A. Wyslouch-Cieszynska, J. Poznanski, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355-3366]. In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV-vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in practical applications of Ni(II)-dependent peptide bond hydrolysis.

Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: reaction conditions and molecular mechanism.

Recently we screened a combinatorial library of R(1)-(Ser/Thr)-Xaa-His-Zaa-R(2) peptides (Xaa = 17 common alpha-amino acids, except Asp, Glu, and Cys; Zaa =19 common alpha-amino acids, except Cys; R(1) = CH(3)CO-Gly-Ala, R(2) = Lys-Phe-Leu-NH(2)) and established criteria for selecting Ser/Thr, Xaa, and Zaa substitutions optimal for specific R(1)-Ser/Thr peptide bond hydrolysis in the presence of Ni(II) ions (Krezel, A.; Kopera, E.; Protas, A. M.; Poznanski, J.; Wyslouch-Cieszynska, A.; Bal, W. J. Am. Chem. Soc. 2010, 132, 3355-3366). The screening results were confirmed by kinetic studies of hydrolysis of seven peptides: R(1)-Ser-Arg-His-Trp-R(2), R(1)-Ser-Lys-His-Trp-R(2), R(1)-Ser-Ala-His-Trp-R(2), R(1)-Ser-Arg-His-Ala-R(2), R(1)-Ser-Gly-His-Ala-R(2), R(1)-Thr-Arg-His-Trp-R(2), and R(1)-Thr-His-His-Trp-R(2). In this paper, we used the same seven peptides to investigate the molecular mechanism of the hydrolysis reaction. We studied temperature dependence of the reaction rate at temperatures between 24 and 75 degrees C, measured stability constants of Ni(II) complexes with hydrolysis substrates and products, and studied the course of R(1)-Ser-Arg-His-Trp-R(2) peptide hydrolysis under a broad range of conditions. We established that the specific square planar complex containing the Ni(II) ion bonded to the His imidazole nitrogen and three preceding peptide bond nitrogens (4N complex) is required for the reaction to occur. The reaction mechanism includes the N-O acyl shift, yielding an intermediate ester of R(1) with the Ser/Thr hydroxyl group. This ester hydrolyzes spontaneously, yielding final products. The Ni(II) ion activates the R(1)-Ser peptide bond by destabilizing it directly through peptide nitrogen coordination and, indirectly, by imposing a strain in the peptide chain.

Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.

Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological applications of Ni(II)-dependent peptide bond hydrolysis.

Overexpression of phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium.

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 muM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of gamma-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in gamma-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.

Reaction of the XPA zinc finger with S-nitrosoglutathione.

S-Nitrosoglutathione (GSNO) is an intracellular redox signaling molecule, also implicated in nitrosative stress. GSNO actions include modifications of Cys thiols in proteins. In this study, we focused on a GSNO reaction with a Cys4 zinc finger (ZF) sequence of human protein XPA, crucial to the nucleotide excision repair pathway of DNA repair. By using a corresponding synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) and combining the detection of noncovalent and covalent complexes by ESI-MS with zinc release monitored by the zinc-sensitive chromophore 4-(2-pyridylazo)resorcinol (PAR), we demonstrated that the reaction of XPAzf with GSNO yielded S-nitrosylated intermediates, intrapeptide disulfides, and mixed glutathione disulfides. The reaction started with the formation of a complex of GSNO with ZnXPAzf followed by thiol transnitrosylation reactions and the final formation of disulfides. The results obtained suggest that at low levels/transient exposures, GSNO may act as a reversible regulator of Cys4 ZF activity, whereas transnitrosylation by GSNO, occurring at prolonged exposures, may cause deleterious effects to the functions of Cys 4 ZF proteins. In the case of XPA, this may lead to DNA repair inhibition.

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2).

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.

A zinc-finger like metal binding site in the nucleosome.

UV spectroscopy demonstrated that chicken mononucleosomes bind Co(II) and Zn(II) ions at submicromolar concentrations in a tetrahedral mode, at a conserved zinc finger-like site, composed of Cys110 and His113 residues of both H3 molecules. Neither of these metal ions substituted for another, indicating a limited binding reversibility. Molecular modeling indicated that the tetrahedral site is formed by unhindered rotations around Calpha-Cbeta bonds in the side chains of the zinc binding residues. The resulting local rearrangement of the protein structure shields the bound metal ion from the solvent, explaining the observed lack of reversibility of the binding. Consequences of these findings for zinc homeostasis, metal toxicology and nucleosomal regulation are discussed.

Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a peptide containing threonine and histidine residues.

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH2, its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E(a) = 92.0 kJ mol(-1) and activation entropy DeltaS# = 208 J mol(-1) K(-1). The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH2. These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.

Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.