Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.     

Mitochondrial polymorphism. IV. Phospholipid composition of mitochondria of a wheat hybrid and its parents

Mitochondria of young seedlings of wheat genotypes 28, 31MS, and 31MS |MX 28 differed in total lipid and phospholipid. Hybrid mitochondria had more lipid and phospholipid than did the parents, and the three genotypes differed in fatty acid composition of the phospholipid fraction. Hybrid mitochondria exhibited heterosis in cytochrome oxidase activity. Although depletion of phospholipid greatly reduced cytochrome oxidase activity in hybrid mitochondria, the heterotic property was preserved. Regulatory aspects of lipid and phospholipid in mitochondria of the hybrid and complementing parental mixture are considered.This research was supported by DeKalb Ag-Research Inc. and in part by grant A-338 from Robert A. Welch Foundation of Houston, Texas.The preceding paper in this series is Sarkissian, I. V., and Srivastava, H. K. (1971). Biochem. Genet. 5:57.     

3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein

[³H]-thymidine is commonly used to analyze the accumulation of [³H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [³H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [³H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [³H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [³H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [³H]-thymidine-labeled DNA. They also demonstrate that [³H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na⁺] _i /[K⁺] _i ratio.     

Translational control of the embryonic cell cycle

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.     

N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses

Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Here, we demonstrate that several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N-methyl-D-aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA-binding protein, on a critical residue that is necessary for polyadenylation-induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal-induced mRNA translation, and elucidates an axis connecting the NMDA receptor to localized protein synthesis at synapses.     

Survival of Trauma-Injured Neurons in Rat Brain by Treatment with Proline-Rich Peptide (PRP-1): An Immunohistochemical Study

The objective of this immunohistochemical research was to reveal the distribution of a proline-rich peptide-1 (PRP-1) in various brain structures of intact and trauma-injured rats and to identify the mechanisms of promotion of neuronal recovery processes following PRP-1 treatment. PRP-1, produced by bovine hypothalamic magnocellular cells and consisting of 15 amino acid residues, is a fragment of neurophysin vasopressin associated glycoprotein isolated from bovine neurohypophysis neurosecretory granules. PRP-1-immunoreactivity (PRP-1-IR) was detected in the brain of intact rats in the neurons of paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, in almost all cell groups in the medulla oblongata, in Purkinje and some cerebellar nuclei cells, and in nerve fibers. At 3 weeks after hemisection of the spinal cord (SC) an asymmetry of PRP-1 localization in the PVN and SON was observed: no PRP-1-IR was exhibited at the affected sides of both nuclei. Daily intramuscular administration of PRP-1 for 3 weeks significantly increased the number of PRP-1-immunoreactive (PRP-1-Ir) varicose nerve fibers, and cells in PVN and SON and in cell groups of the limbic system and brain stem. Tanycytes in the median eminence and covering ependyma also demonstrated strong PRP-1-IR. PRP-1 treatment also activated neuropeptide Y-IR (NPY-IR) in nerve fibers and immunophilin fragment-IR (IphF-IR) in lymphocytes and nerve cells. A strong increase of PRP-1-IR was observed in the PVN and SON of SC-injured rats following the treatment with another PRP (PRP-3). Preliminary physiological data demonstrate that PRP-3 is more "aggressive" in the recovery processes than PRP-1. Based on the findings regarding PRP action on neurons survival, axons regeneration, and the number of IphF-Ir lymphocytes and NPY-Ir nerve fibers, PRP is suggested to act as a neuroprotector, functioning as a putative neurotransmitter and immunomodulator.     

Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development.     

Decrease of various enzyme activities in the amniotic fluid of the fetuses with chromosomal anomalies

The enzymatic activities of gamma-glutamyl-transpeptidase and of aminopeptidase M have been determined in amniotic fluids taken in pregnancies with a trisomic conceptus. The low values obtained in these fluids may be the consequence of fetal growth retardation.