N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.     

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

A Ubiquitin-Binding Domain that Binds a Structural Fold Distinct from that of Ubiquitin

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The external promoter in the guinea pig 5S rRNA gene is different from the rodent promoter

The guinea pig has about 100 copies of the 5S rRNA gene per haploid genome and they are present in 2.1 kb tandem repeats. Three bona fide 5S rRNA genes and four pseudo genes were sequenced. The conserved external promoter (D box) found in rodents and primates is only partially present in the guinea pig. The "D box like" sequence in guinea pig only has eight of the 12 nucleotides in the conserved D box. The results are in accordance with investigations showing that the guinea pig is not a rodent. Conserved sequences in the non-transcribed spacer can therefore be useful in phylogenetic studies.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Involvement of the L6-7 loop in SERCA1a Ca2+-ATPase activation by Ca2+ (or Sr2+) and ATP

Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.