Tomoko S. Misono

Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin.

Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

Background: HtpG, a bacterial heat shock protein 90 (Hsp90), is essential for thermotolerance in some prokaryotes. Results: HtpG functions with DnaK2/DnaJ2/GrpE to assist unfolding/folding of denatured proteins in both ATP-dependent and -independent fashions. Conclusion: The cooperative action of HtpG and DnaK2 might play a key role under stress. Significance: This might be the first sign of a prokaryotic Hsp90 foldosome. In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Prospects of Ligand-Induced Aptamers

Aptamers are rare functional nucleic acid ligands that bind with high affinity and specificity to their target ligands. Selected aptamers have been shown to inhibit the functions of their cognate targets both in vitro and in vivo. As a consequence, the first aptamer-based drug to treat age-related macular degeneration has been developed. On another front, aptamers have also successfully shown potential use in diagnostics and imaging technology. The next wave of aptamer applications involves the development of ligand-induced aptamers. These aptamers rely on the principles commonly observed in many ribonucleic acid (RNA)-ligand interactions. In the present review, we describe the various strategies for designing ligand-induced aptamers and their applications, including monitoring different ligands, regulating gene expression and expanding microarray analyses.