Crystal structure of Tk-subtilisin folded without propeptide: requirement of propeptide for acceleration of folding

Tk-subtilisin (a subtilisin homologue from Thermococcus kodakaraensis) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide. To analyze the folding mechanism of Tk-subtilisin, the crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin^∗), which was refolded in the presence of Ca²⁺ and absence of Tk-propeptide, was determined at 2.16 Å resolution. This structure is essentially the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin. S324A-subtilisin^∗ was refolded with a rate constant of 0.17 and 1.8 min⁻¹ at 30 °C in the absence and presence of Tk-propeptide, respectively, indicating that Tk-subtilisin does not require Tk-propeptide for folding but requires it for acceleration of folding.     

A pelagic larva of <Emphasis Type="Italic">Plagiopsetta glossa</Emphasis> (Pleuronectiformes,Samaridae), characterized by a trailing gut and S-shaped pelvic bone: description and phylogenetic considerations

 The pelagic larval stage of a pleuronectiform samarid, Plagiopsetta glossa, is described and illustrated, for the first time, from a 8.4-mm BL postflexion specimen collected off Tosa Bay, southern Japan. The larva is distinctive in having broad, semitransparent pterygiophore zones on the dorsal and anal fins, a trailing gut, flexible S-shaped pelvic bone, and a skin fold under the throat. These characteristics are shared with poecilopsettid larvae, suggesting a close relationship between the two families.     

Larval development of Pseudorhombus oculocirris and P. arsius (Pleuronectiformes, Paralichthyidae) from off southern Japan

 Larvae of two paralichthyids, Pseudorhombus oculocirris and P. arsius, are described and illustrated from specimens collected off Tosa Bay, southern Japan. Peudorhombus oculocirris larvae (5 specimens, 4.5–7.8 mm BL) are characteristic in having 6 or 7 elongated anterior dorsal fin rays and poorly developed head spines and melanophores on the tail. Pseudorhombus arsius larvae (3 specimens, 5.3–8.4 mm BL) are distinctive in having 11 or 12 elongated anterior dorsal fin rays and well-developed head spines, including a row of spines on the sphenotic. Received: June 28, 2001 / Revised: November 2, 2001 / Accepted: November 22, 2001     

Role of Curdlan Sulfate in the Binding of HIV-1 gp120 to CD4 Molecules and the Production of gp120-Mediated TNF-α

To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-α by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4⁺ cells, but also inhibited TNF-α production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions.     

Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.     

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.