In vivo heteromer formation. Expression of soluble betaA4-crystallin requires coexpression of a heteromeric partner

The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.     

Crystal structure of truncated human betaB1-crystallin

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates

The beta and gamma crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of gamma-crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of gammaN-crystallin, an evolutionary bridge between the beta and gamma families. In all species examined, gammaN-crystallins have a hybrid gene structure, half beta and half gamma, and thus appear to be the 'missing link' between the beta and gamma crystallin lineages. Overall, there are four major classes of gamma-crystallin: the terrestrial group (including mammalian gammaA-F); the aquatic group (the fish gammaM-crystallins); the gammaS group; and the novel gammaN group. Like the evolutionarily ancient beta-crystallins (but unlike the terrestrial gammaA-F and aquatic gammaM groups), both the gammaS and gammaN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, gammaN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the gamma-crystallins, also in the retina. Although well conserved throughout vertebrate evolution, gammaN in primates has apparently undergone major changes and possible loss of functional expression.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

Thesaurus-based disambiguation of gene symbols

Background  

Massive text mining of the biological literature holds great promise of relating disparate information and discovering new knowledge. However, disambiguation of gene symbols is a major bottleneck.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

A temperature-sensitive mutation of Crygs in the murine Opj cataract

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gammaS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of gammaS-crystallin which would be consistent with ideas that members of the betagamma-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.