An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non‐intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non‐stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA_i to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than‐average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.     

Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy

Background  

Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities.     

Regulation of aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii compared to Saccharomyces cerevisiae

To elucidate the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii, an important yeast for the flavor development in soy sauce, was investigated. It was shown that threonine inhibited the growth of Z. rouxii by blocking the methionine synthesis. It seemed that threonine blocked this synthesis by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii, unlike that of Saccharomyces cerevisiae, was not inhibited by the herbicide sulfometuron methyl (SMM). From enzyme assays, it was concluded that the acetohydroxy acid synthase in Z. rouxii, unlike that in S. cerevisiae, was not sensitive to SMM. Furthermore, the enzyme assays demonstrated that the activity of threonine deaminase in Z. rouxii, like in S. cerevisiae, was strongly inhibited by isoleucine and stimulated by valine. From this work, it is clear that the aspartate-derived amino-acid metabolism in Z. rouxii only partly resembles that in S. cerevisiae.     

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.     

Phylogenetic survey of soluble saxitoxin-binding activity in pursuit of the function and molecular evolution of saxiphilin,a relative of transferrin.

Saxiphilin is a soluble protein of unknown function which binds the neurotoxin, saxitoxin (STX), with high affinity. Molecular characterization of saxiphilin from the North American bullfrog, Rana catesbeiana, has previously shown that it is a member of the transferrin family. In this study we surveyed various animal species to investigate the phylogenetic distribution of saxiphilin, as detected by the presence of soluble [3H]STX binding activity in plasma, haemolymph or tissue extracts. We found that saxiphilin activity is readily detectable in a wide variety of arthropods, fish, amphibians, and reptiles. The pharmacological characteristics of [3H]STX binding activity in phylogenetically diverse species indicates that a protein homologous to bullfrog saxiphilin is likely to be constitutively expressed in many ectothermic animals. The results suggest that the saxiphilin gene is evolutionarily as old as an ancestral gene encoding bilobed transferrin, an Fe(2+)-binding and transport protein which has been identified in several arthropods and all the vertebrates which have been studied.