Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.     

Characterization of biological activities of Brucella melitensis lipopolysaccharide

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin-specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor-alpha and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti-complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.     

A Mutation in the LPAT1 Gene Suppresses the Sensitivity of fab1 Plants to Low Temperature

Various fluorophore-based microscopic methods, comprising Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.Many biological processes rely on the dynamic assembly and disassembly of multicomponent protein complexes. Experimental methods based on genetically encoded fluorophores are widely used to study the subcellular localization and binary protein-protein interactions in living cells. The subcellular localization of proteins can be visualized by translationally fusing them to fluorophores, which are now available across a wide spectral range (Shaner et al., 2005). Fluorescence microscopy allows imaging and tracing of these proteins in real time in living cells. However, the resolution of a conventional epifluorescence microscope or a confocal laser scanning microscope is insufficient to resolve distances smaller than approximately 200 nm between biological macromolecules and to demonstrate their vicinity at the molecular scale (Bhat et al., 2006; Held et al., 2008).Two fluorophore-based methods are commonly employed to study protein-protein interactions on the stage of a light microscope and overcome its limitations: bimolecular fluorescence complementation (BiFC; Hu et al., 2002; Bracha-Drori et al., 2004; Walter et al., 2004) and Förster resonance energy transfer (FRET; Hink et al., 2002; Chen et al., 2003; Russinova et al., 2004). The BiFC approach is based on the restoration of an intact fluorophore from its nonfluorescent N- and C-terminal domains. When the two complementing fragments are fused to potentially interacting proteins, close proximity of the proteins can bring the N- and C-terminal fluorophore domains into a favorable position and orientation and thereby facilitate their association into a functional fluorophore (Hu et al., 2002; Kerppola, 2008). Since only standard fluorescence microscopic equipment is required for this technique, it is a popular method to analyze protein-protein interactions (Weinthal and Tzfira, 2009).An alternative procedure exploits a physical phenomenon referred to as FRET to obtain information about the molecular vicinity of fluorophore-labeled proteins. FRET occurs when a donor fluorophore is brought into close proximity (less than 10 nm) of a suitable acceptor fluorophore. In this case, the donor can transmit its excitation energy to the acceptor. The extent of FRET depends on various parameters, such as the distance between donor and acceptor, their spectral properties, the relative orientation of the donor and acceptor transition dipoles, and the refractive index of the medium (for a more detailed introduction to the theory of FRET, see “Materials and Methods”; Clegg, 1996; Periasamy and Day, 2005; Biskup et al., 2007). The extent of FRET can be estimated by several means, such as by assessing the sensitized emission (Shah et al., 2001, 2002), by determining the donor fluorescence before and after acceptor photobleaching (APB; Bhat et al., 2005), or by measuring the fluorescence lifetime of the donor (Russinova et al., 2004; Bhat et al., 2005; Tonaco et al., 2006; Shen et al., 2007; Osterrieder et al., 2009). Depending on the method, FRET measurements require more or less sophisticated microscopic equipment.Many biological processes involve protein complexes composed of more than two proteins, which are difficult to study with the experimental approaches outlined above. For example, biologically active N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complexes are composed of three distinct protein partners. SNAREs, together with accessory proteins and cytosolic calcium, catalyze membrane fusion events in eukaryotic cells. A binary or ternary t-SNARE complex composed of a Qa-SNARE, also called syntaxin, and Qb- and Qc-SNARE domains present in either one protein (synaptosome-associated protein [SNAP-25]) or as two separate units is located at the target membrane. This t-SNARE complex interacts with an R-SNARE (vesicle-associated membrane protein [VAMP]) present on the vesicle, and the formation of the fusogenic SNARE complex brings the opposing membranes into close proximity (Jahn and Scheller, 2006; Leabu, 2006). Besides their role in cellular homoeostasis, SNAREs play a major role in various plant biological processes, such as cytokinesis, gravitropism, and defense against pathogens (Lipka et al., 2007).The orthologous Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare) syntaxins, AtPEN1 and HvROR2, respectively, are essential in restricting the invasion of nonadapted powdery mildew species or in broad-spectrum powdery mildew resistance conferred by loss-of-function mlo mutants, respectively (Collins et al., 2003; Assaad et al., 2004). Both syntaxins form a binary t-SNARE complex with the orthologous SNAP-25-like proteins AtSNAP33 and HvSNAP34 (Collins et al., 2003; Kwon et al., 2008), which in turn interact with the R-SNAREs AtVAMP721/AtVAMP722 and HvVAMP721, respectively, by adopting an authentic ternary SNARE complex (Kwon et al., 2008). In addition to a range of other polypeptides, the mentioned Arabidopsis and barley SNARE partners focally accumulate below the site of attempted fungal penetration (Assaad et al., 2004; Bhat et al., 2005; Kwon et al., 2008), which probably precedes their extracellular deposition in the paramural space (Meyer et al., 2009).In this study, we combined BiFC-FRET measurements based on cerulean fluorescent protein (CrFP; Rizzo et al., 2004) as a donor and reconstituted yellow fluorescent protein (YFP; Walter et al., 2004; Schütze et al., 2009) as an acceptor to study the formation of a ternary protein assembly. We concentrated on the well-characterized SNARE complexes described above that play a decisive role in antifungal defense. FRET was detected by measuring the fluorescence lifetime of the donor in each pixel of the sample in a fluorescence lifetime imaging (FLIM) setup. Occurrence of FRET was corroborated by APB experiments. Our data demonstrate the technical feasibility of combined BiFC-FRET-FLIM and BiFC-FRET-APB assays to study ternary protein complexes in living plant cells.     

Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response

In vivo phosphorylation sites of the tobacco calcium-dependent protein kinases NtCDPK2 and NtCDPK3 were determined in response to biotic or abiotic stress. Stress-inducible phosphorylation was exclusively located in the variable N termini, where both kinases were phosphorylated differentially despite 91% overall sequence identity. In NtCDPK2, serine 40 and threonine 65 were phosphorylated within 2 min after stress. Whereas Thr⁶⁵ is subjected to intra-molecular in vivo autophosphorylation, Ser⁴⁰ represents a target for a regulatory upstream protein kinase, and correct NtCDPK2 membrane localization is required for Ser⁴⁰ phosphorylation. NtCDPK3 is phosphorylated at least at two sites in the N terminus by upstream kinase(s) upon stress stimulus, first at Ser⁵⁴, a site not present in NtCDPK2, and also at a second undetermined site not identical to Ser⁴⁰. Domain swap experiments established that differential phosphorylation of both kinases is exclusively determined by the respective N termini. A cell death-inducing response was only observed upon expression of a truncated variant lacking the junction and calcium-binding domain of NtCDPK2 (VK2). This response required protein kinase activity and was reduced when subcellular membrane localization was disturbed by a mutation in the myristoylation and palmitoylation site. Our data indicate that CDPKs are integrated in stress-dependent protein kinase signaling cascades, and regulation of CDPK function in response to in vivo stimulation is dependent on its membrane localization.     

Herpes simplex virus US3 protein kinase regulates virus-induced apoptosis in olfactory and vomeronasal chemosensory neurons in vivo

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone,androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.     

Biosynthesis of triacylglycerol molecular species in an oleaginous fungus,Mortierella ramanniana var. angulispora

The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-(14)C]oleic acid at 30 degrees C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, gamma-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-(14)C]oleic acid at 15 degrees C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-(14)C]Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [(14)C(U)]Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-(14)C]oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.