Compensatory Evolution of a WW Domain Variant Lacking the Strictly Conserved Trp Residue

Replacement of conserved amino acid residues during evolution of proteins can lead to divergence and the formation of new families with novel functions, but is often deleterious to both protein structure and function. Using the WW domain, we experimentally examined whether and to what degree second-site mutations can compensate for the reduction of function and loss of structure that accompany substitution of a strictly conserved amino acid residue. The W17F mutant of the WW domain, with substitution of the most strictly conserved Trp residue, is known to lack a specific three-dimensional structure and shows reduced binding affinity in comparison to the wild type. To obtain second-site revertants, we performed a selection experiment based on the proline-rich peptide (PY ligand) binding affinity using the W17F mutant as the initial sequence. After selection by ribosome display, we were able to select revertants that exhibited a maximum ninefold higher affinity to the PY ligand than the W17F mutant and showed an even better affinity than the wild type. In addition, we found that the functional restoration resulted in increased binding specificity in selected revertants, and the structures were more compact, with increased amounts of secondary structure, in comparison to the W17F mutant. Our results suggest that the defective structure and function of the proteins caused by mutations in highly conserved residues occurring through divergent evolution not only can be restored but can be further improved by compensatory mutations.     

Intracerebroventricular administration of urotensin II regulates food intake and sympathetic nerve activity in brown adipose tissue

To clarify the functional roles of urotensin II in regulating energy balance, we investigated the effects of a central infusion of urotensin II on food intake, uncoupling protein (UCP) 1 mRNA expression, temperature, and sympathetic nervous system activity in brown adipose tissue (BAT), a site that regulates energy expenditure in rodents. A bolus central infusion of urotensin II at a dose of 1 nmol/rat into the third cerebral ventricle decreased food intake (p<0.05). Additionally, urotensin II induced c-Fos-like-immunoreactivity (c-FLI) in the paraventricular nucleus (PVN) as compared with that in the control (phosphate buffered saline [PBS]-treated) group. Furthermore, urotensin II increased BAT UCP 1 mRNA expression (p<0.05). Finally, central infusion of urotensin II significantly increased BAT sympathetic nerve activity, which was accompanied by a significant elevation in BAT temperature (p<0.05) in rats. Taken together, central infusion of urotensin II regulates food intake and BAT sympathetic nerve activity in rats.     

Comparison of sex-steroid synthesis between neonatal and adult rat hippocampus

Sex-steroid synthesis in the hippocampus had been thought to be much more active at the neonatal stage than at the adult stage. However, the detailed comparison between these two stages had not been demonstrated yet. Here we performed the comparison about the mRNA level of steroidogenic enzymes and the rate of steroid metabolism between these two stages of the hippocampus. The relative expression level of P450(17α), 17β- or 3β-hydroxysteroid dehydrogenase, or P450arom was approximately 1.3-1.5-fold higher at the neonatal than at the adult stage. The rate of sex-steroid metabolism (from dehydroepiandrosterone to estradiol) was 2-7-fold (depending on different steps) more rapid at the neonatal than at the adult stage. Taken together, neonatal steroidogenesis is moderately more active than adult steroidogenesis.     

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.     

Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins and wild-type and lectin-gene-transfected tumor cells as targets

Multivalent glycoclusters have the potential to become pharmaceuticals by virtue of their target specificity toward clinically relevant sugar receptors. Their application can also provide fundamental insights into the impact of two spatial factors on binding, i.e., topologies of ligand (branching mode, cluster presentation) and carbohydrate recognition domains in lectins. Persubstituted macrocycles derived from nucleophilic substitution of iodide from heptakis 6-deoxy-6-iodo-beta-cyclodextrin by the unprotected sodium thiolate of 3-(3-thioacetyl propionamido)propyl glycosides (galactose, lactose and N-acetyllactosamine) were prepared. The produced glycoclusters were first tested as competitive inhibitors in solid-phase assays. A plant toxin from mistletoe and an immunoglobulin G fraction from human serum were markedly susceptible. A nearly 400-fold increase in inhibitory potency of each galactose moiety in the heptavalent form relative to free lactose (217-fold relative to free galactose) was detected. Thus, these glycoclusters can efficiently interfere, for example, with xenoantigen-dependent hyperacute rejection. Among the tested galectins selected from this family of adhesion- and growth-regulatory endogenous lectins, the substituted beta-cyclodextrins acted as sensors to delineate topological differences between the two dimeric prototype proteins. The relatively strong reactivity with chimera-type galectin-3, a mediator of tumor metastasis, disclosed selectivity for glycocluster binding among galectins. Equally important, the geometry of ligand display (maxiclusters, bi- or triantennary N-glycans) made its mark on the inhibitory potency. To further determine the sensitivity of a distinct galectin presented on the cell surface and not in solution, we established a stably transfected tumor cell clone. We detected a significant response to presence of the multivalent inhibitor. This type of chemical scaffold with favorable pharmacologic properties might thus be exploited for the design of galectin- and ligand-type-selective glycoclusters.     

Evolution of an Arbitrary Sequence in Solubility

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His₆ tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.