Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. II. Plastocyanin.

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.     

Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein.

Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution.     

Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye.

The deregulation of E2F activity is thought to contribute to the uncontrolled proliferation of many tumor cells. While the effects of overexpressing E2F genes have been studied extensively in tissue culture, the consequences of elevating E2F activity in vivo are unknown. To address this issue, transgenic lines of Drosophila were studied in which ectopic expression of dE2F and dDP was targeted to the developing eye. The co-expression of dDP or dE2F disrupted normal eye development, resulting in abnormal patterns of bristles, cone cells and photoreceptors. dE2F/dDP expression caused ectopic S phases in post-mitotic cells of the eye imaginal disc but did not disrupt the onset of neuronal differentiation. Most S phases were seen in uncommitted cells, although some cells that had initiated photo-receptor differentiation were also driven into the cell cycle. Elevated expression of dE2F and dDP caused apoptosis in the eye disc. The co-expression of baculovirus p35 protein, an inhibitor of cell death, strongly enhanced the dE2F/dDP-dependent phenotype. These results show that, in this in vivo system, the elevation of E2F activity caused post-mitotic cells to enter the cell cycle. However, these cells failed to proliferate unless rescued from apoptosis.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Aggregation of zinc‐free p53 is inhibited by Hsp90 but not other chaperones

The structured DNA‐binding domain (DBD) of p53 is a well‐known client protein of the chaperone Hsp90. The p53 DBD contains a single zinc ion, coordinated by the side chains of Cys176, His179, Cys238, and Cys242; zinc coordination plays a structural role to stabilize the DBD and is required for its DNA binding. The ambiguous nature of the p53‐Hsp90 interaction, together with the stabilizing role of the zinc in the structure of the DBD, prompted us to examine the interaction of Hsp90 with zinc‐free p53 DBD. NMR spectroscopy and native gel electrophoresis did not show any apparent preference for the interaction of the destabilized zinc‐free form of p53 DBD with Hsp90. Intriguingly, however, at lower protein concentrations, closer to physiological concentrations, the addition of Hsp90, but not other chaperones such as Hsp70, Hsp40, p23, and HOP, appears to slow or prevent the aggregation of zinc‐free p53 DBD. This result suggests that part of the function of the Hsp90‐p53 interaction in the cell may be to stabilize the apoprotein in the absence of zinc.     

Mutation of Drosophila Lsd1 disrupts H3-K4 methylation, resulting in tissue-specific defects during development

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified. Lsd1 is highly conserved in many species, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. Here we describe the effects of Lsd1 mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development.     

Influence of macrofaunal assemblages and environmental heterogeneity on microphytobenthic production in experimental systems

Despite the complexity of natural systems, heterogeneity caused by the fragmentation of habitats has seldom been considered when investigating ecosystem processes. Empirical approaches that have included the influence of heterogeneity tend to be biased towards terrestrial habitats; yet marine systems offer opportunities by virtue of their relative ease of manipulation, rapid response times and the well-understood effects of macrofauna on sediment processes. Here, the influence of heterogeneity on microphytobenthic production in synthetic estuarine assemblages is examined. Heterogeneity was created by enriching patches of sediment with detrital algae (Enteromorpha intestinalis) to provide a source of allochthonous organic matter. A gradient of species density for four numerically dominant intertidal macrofauna (Hediste diversicolor, Hydrobia ulvae, Corophium volutator, Macoma balthica) was constructed, and microphytobenthic biomass at the sediment surface was measured. Statistical analysis using generalized least squares regression indicated that heterogeneity within our system was a significant driving factor that interacted with macrofaunal density and species identity. Microphytobenthic biomass was highest in enriched patches, suggesting that nutrients were obtained locally from the sediment-water interface and not from the water column. Our findings demonstrate that organic enrichment can cause the development of heterogeneity which influences infaunal bioturbation and consequent nutrient generation, a driver of microphytobenthic production.     

WISE 2005: vascular responses to 60-day bed rest in women

WISE-2005 studied 24 women during a 60-day head down bed rest (HDBR) who look part in an exercise countermeasure (LBNP-treadmill plus flywheel, EX) and no-exercise (No-EX). We conducted a series of experiments to explore changes in cardiovascular function and the ability of EX to prevent these changes. Resting arterial diameter in the arm was not affected but the leg arteries (femoral and popliteal) were significantly reduced in Np-EX, but was increased in EX. In this study we report on drug stimulated responses with sublingual nitroglycerin and infused isoproterenol. Heart rate increased in response to nitroglycerin with larger increases in No-EX after HDBR. Likewise during isoproterenol infusion the HR increase was greater after HDBR in the No-EX group. In all cases, the higher HR was associated with lower stroke volume in No-EX while stroke volume was protected in EX. These data do not support a change in sensitivity of beta-adrenergic receptors after HDBR. The leg vascular resistance decreased in response to isoproterenol and it decreased to a greater extent in No-EX than EX. These data were consistent with observations of lower leg vascular resistance during orthostatic challenge tests after HDBR. We conclude that consistent changes in cardiovascular function in the No-EX were detected by different methods that point to mechanisms contributing to orthostatic intolerance after HDBR.     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999     

Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.