Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.     

Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution.     

Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.     

Alterations in the GnRH-LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker

Exposure of Atlantic croaker (Micropogonias undulatus) to the polychlorinated biphenyl mixture (Aroclor 1254, PCB; 1 mg/kg body wt/day for 30 days) during the early-recrudescence phase of the gonadal cycle results in the impairment of LH secretion and gonadal growth. In order to determine whether impairment was due to disruption of the stimulatory GnRH neuroendocrine pathway, we compared various parameters of the GnRH-LH system in early recrudescing vs. spermiating (mature) fish. Seabream GnRH (GnRH) content in the preoptic anterior hypothalamic area (POAH) and pituitary, pituitary GnRH receptor concentrations, and basal and GnRH analog (GnRHa)-induced LH secretion were significantly higher in gonadally mature croaker compared to early-recrudescing fish. In a subsequent experiment, the effects of PCB on the same neuroendocrine indices were investigated during the gonadal recrudescence phase of croaker. PCB exposure during the period of testicular maturation prevented the natural increase in GnRH content in the POAH but not in the pituitary. This finding suggests that PCB may impair GnRH synthesis in the POAH. The number of pituitary GnRH receptors also remained significantly lower in the PCB-exposed group, which was likely due to an impairment of GnRH release. The GnRH content in the POAH, number of pituitary GnRH receptors, and LH secretion in the PCB-exposed group were comparable to those in early-recrudescing fish, suggesting an impairment of normal maturation of the GnRH-LH system during the gonadal recrudescence phase. This impairment may be due to a direct action of PCB on GnRH neurons and/or indirectly via interference with other neurotransmitter pathways that modulate GnRH function.     

Peptide immunization of guinea pigs against Chlamydia psittaci (GPIC agent) infection induces good vaginal secretion antibody response, in vitro neutralization and partial protection against live challenge

Immunization of female guinea pigs with a chimeric peptide consisting of variable domain IV (VDIV) and a region known as GP8 from the major outer membrane protein of Chlamydophila caviae, formerly Chlamydia psittaci guinea pig inclusion conjunctivitis strain, was performed to assess whether humoral immune responses could be elicited in the reproductive tracts of immunized animals. The C. caviae strain is able to cause a sexually transmitted infection in the guinea pig that closely parallels C. trachomatis infections in humans. The best anti-VDIV antibody response in vaginal secretions was achieved by intraperitoneal priming with subsequent intravaginal boosting (P < 0.001). Dot-blot analyses of vaginal secretions confirmed that these anti-VDIV antibodies, produced against a linear peptide, were able to recognize and bind to whole conformational C. caviae elementary bodies. Following live intravaginal challenge with C. caviae, a significant reduction in the intensity (P = 0.01) and an apparent reduction in the duration of the infection was evident between the guinea pigs immunized with VDIV-GP8 and non-immunized controls.     

Alzheimer's disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis

Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer's disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPPalpha and sAPPbeta were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human Abeta40 and Abeta42 into the growth media nearly tripled in CD-MPR- and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells. Comparable increases were confirmed for endogenous mouse Abeta40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Abeta generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.     

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Crystallographic study of the recombinant flavin-binding domain of Baker's yeast flavocytochrome b(2): comparison with the intact wild-type enzyme

Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.