Glucofuranosylation with penta-O-propanoyl-beta-D-glucofuranose

Readily available, crystalline penta-O-propanoyl-beta-D-glucofuranose is shown to be a suitable glycosylating agent for the acid-catalysed, direct synthesis of O-, S- and N-glucofuranosyl compounds. Beta-linked products are formed with good selectivity. Reaction with cyanotrimethylsilane gave the 1,2-O-(1-cyanopropylidene)acetal rather than the C-glycosyl cyanide. By selective acid-catalysed hydrolysis, the title compound was converted to the 1-hydroxy analogue from which the trichloroacetimidates were made as further potential glycosylating agents.     

GRACILE syndrome,a lethal metabolic disorder with iron overload,is caused by a point mutation in BCS1L

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Accelerated molecular evolution in halophilic crustaceans

In contrast to the stable ionic composition of the oceans, inland waters show striking diversity, possessing salt concentrations varying from I mM to 5 M. Although species diversity is highest in fresh water, some lineages have colonized hypersaline environments where they encounter elevated levels of both ultraviolet (UV) radiation and osmotic stress. This study compares rates of evolution in halophilic and freshwater taxa for two groups of microcrustaceans, anostracans and daphniids, from Australia and North America. The results establish that halophilic species show consistent rate acceleration, involving elevated levels of both insertion/deletion events and of nucleotide substitutions. The elevated pace of molecular evolution does not appear to be linked to selection or to other agents that are known to influence the supply rate of mutations, such as UV exposure, generation length, or shifts in metabolic rate. However, variance in ionic strength, which is known to have potent effects on DNA-protein interactions as well as on the structural properties of DNA and proteins, might account for the lowered fidelity of DNA replication in life from hypersaline settings. Regardless of its cause, the consistent rate acceleration in halophiles suggests that past efforts to employ sequence divergences to date events, such as the age of asexual lineages in Artemia, have resulted in serious overestimates. More generally, the results indicate that coordinated shifts in rates of molecular evolution may occur in lineages exposed to extreme environmental conditions.     

Chemoenzymatic synthesis of peptidyl 3,4-dihydroxyphenylalanine for structure-activity relationships in marine invertebrate polypeptides

An improved method for hydroxylating tyrosine-containing sequences in polypeptides to peptidyl 3,4-dihydroxyphenylalanine (DOPA) using mushroom tyrosinase at relatively high enzyme-to-substrate ratios is described. The new method involves incorporating borate into the reaction mixture to stop formation of the unwanted side product 3,4,5-trihydroxyphenylalanine. Using this method, a model for the palindromic central sequence for the antimicrobial peptide family, the styelins, Y*Y*KHKY*Y* (where Y* is DOPA), was successfully synthesized in high yield from YYKHKYY. This sequence represents a particularly challenging target because of the cluster of four precursor tyrosine residues are in close proximity. The method should be readily applied to larger polypeptides produced by either solid-phase synthesis or recombinant techniques and give greater insight into the roles of this unusual posttranslational modification in marine invertebrates such as mussels and ascidians.     

Inhibitors of different structure induce distinguishing conformations in the omega loop,Cys69-Cys96, of mouse acetylcholinesterase

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Omega loop), Cys(69)-Cys(96), of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Omega loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Omega loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors.     

Regulation of cAMP-dependent protein kinase activity by glutathionylation

The catalytic subunit of cAMP-dependent protein kinase (cAPK) is susceptible to inactivation by a number of thiol-modifying reagents. Inactivation occurs through modification of cysteine 199, which is located near the active site. Because cysteine 199 is reactive at physiological pH, and modification of this site inhibits activity, we hypothesized that cAPK is a likely target for regulation by an oxidative mechanism, specifically glutathionylation. In vitro studies demonstrated the susceptibility of kinase activity to the sulfhydryl oxidant diamide, which inhibited by promoting an intramolecular disulfide bond between cysteines 199 and 343. In the presence of a low concentration of diamide and reduced glutathione, the kinase was rapidly and reversibly inhibited by glutathionylation. Mutant kinase containing an alanine to cysteine mutation at position 199 was resistant to inhibition by both diamide and glutathionylation, thus implicating this as the oxidation-sensitive site. Mouse fibroblast cells treated with diamide showed a reversible decrease in cAPK activity. Inhibition was dramatically enhanced when cells were first treated with cAPK activators. Using biotin-cysteine as means for detecting and purifying thiolated cAPK from cells, we were able to show that, under conditions in which cAPK is inactivated by diamide, it is also readily thiolated.     

Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner.

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).