The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of The 90-kDa heat shock protein (Hsp90) 1 is a highly conserved, abundant, and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is specifically involved in the folding and conformational regulation of a limited subset of proteins. Almost all natural substrates of Hsp90 are medically relevant signal transduction molecules, including the nuclear receptors for steroid hormones, several protooncogenic kinases, and disparate proteins such as nitric-oxide synthase (1-4). In order to fulfill its cellular role, Hsp90 cooperates with a cohort of cofactors such as Sti1/Hop/p60, the immunophilins FKBP51/54 and FKBP52, cyclophilin 40, cdc37/ p50, Sba1/p23, and CHIP (5-8) and acts as a part of a multichaperone machine together with Hsp70 (3, 9).Several Hsp90-associated cofactors contain tetratricopeptide repeat motifs, a degenerate 34-aa sequence, that mediate binding to the C-terminal EEVD motif of the molecular chaperone (10). Other cofactors of Hsp90, like Sba1/p23 and cdc37/p50, lack tetratricopeptide repeats and are believed to use unique sequences to associate with the molecular chaperone.The basic mechanism of Hsp90 function is still not fully understood. Recent crystal structures of the N-terminal domain of Hsp90 (11, 12) have identified a conserved binding site for ATP. Based on this structural evidence for nucleotide binding, it was demonstrated that Hsp90 can hydrolyze ATP and that this activity of the molecular chaperone is essential in vivo (13, 14). Thus, Hsp90 was unequivocally identified as an ATPdependent molecular chaperone.Here we demonstrate binding of the novel cofactors Aha1 and Hch1 to the middle domain of Hsp90, currently a poorly investigated region in the molecular chaperone. The intrinsic ATPase activity of Hsp90 can be stimulated 5-fold by the cofactor Aha1. Deletion of the genes encoding Aha1 and Hch1 in Saccharomyces cerevisiae impaired activation of the heterologous Hsp90-dependent substrate protein v-Src and interfered with cell viability under non-optimal conditions. EXPERIMENTAL PROCEDURES Plasmid Construction and Yeast Two-hybrid Experiments-YeastHsp90 (yHsp90) and fragments thereof were amplified from pTGPD/ P82 (a gift from S. Lindquist) and yeast Aha1 (yAha1); Hch1 an...

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1 H-1 H and 1 H-31 P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/ PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP) 2 -IP 4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP) 2 -IP 4 (Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.Signal transduction pathways frequently rely on a specific target protein recognizing a precise spatial arrangement of one or more phosphate groups on either another protein or a small metabolite. The six-carbon inositol ring offers what is arguably the most dramatic example of how even subtle modifications to phosphate topology can impart signaling specificity. The combinatorial manner in which phosphate groups can be arranged around the inositol skeleton creates a large family of phosphorylated molecules, many of which have individual, physiological roles (1). The inositol pyrophosphates, such as diphosphoinositol tetrakisphosphate (also known as PP-IP 4 ), PP-IP 5 2 (also known as IP 7 ) and (PP) 2 -IP 4 (also known as IP 8 ) (2, 3), are a specialized subgroup of the inositol-based signaling family that are distinguished by the presence of diphosphate groups. These particular molecules regulate a diverse range of cellular activities, including phosphate sensing, actin cytoskeleton dynamics, apoptosis, vesicle trafficking, transcription, and DNA repair (see Refs. 4 and 5 for reviews). The different isomers of inositol pyrophosphates can be distinguished by biological receptors (6, 7). Thus, there is great interest in understanding the structure/ function relationships of protein interactions with the inositol pyrophosphate ligands. .). Instrum...

Four main-chain type polybenzoxazine precursors were synthesized from the Mannich-type polycondensation of biphenol A, paraformaldehyde, and four typical aromatic diamines: 1,4-phenylenediamine, 4,4'-diaminodiphenyl methane, 4,4'-diaminodiphenyl ether, and 2,2-bis[4-(4-aminophenoxy)phenyl] propane. The solvent effect on the polycondensation was examined. We found that polybenzoxazine precursors prepared in toluene/ethanol exhibit higher purity, higher molecular weight, and lead to better thermal properties after curing than those prepared in chloroform, as recommended by Takeichi's group (T. Takeichi et al., Polymer, 2005, 46, 12172). The cured polybenzoxazine films prepared by these polybenzoxazine precursors display unusual flexibility, extremely high T-g, and low surface energy. They show T-g values as high as 272-353 degrees C (DMA data), coefficients of thermal expansion as low as 39-49 ppm/degrees C, contact angles as high as 100-102 degrees, and surface energy as low as 17.7-24.5 mJ m(-2)

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.