Georg N. Zimmerli 90 Jahre

European Journal of Wildlife Research -     

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.     

Karl von Frisch zum 90. Geburtstag

Ohne Zusammenfassung     

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90alpha and Hsp90beta, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90alpha was recovered after heat shock. The data demonstrate that Hsp90alpha and Hsp90beta exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.     

Haematological studies on90Sr-90Y-toxicity

Summary Erythropoietic changes were observed, measured by⁵⁹Fe-uptake into red blood cells, and on radioiron turnover from blood plasma, at different time intervals (2–64 days) after treating adult female mice with varying activities of⁹⁰Sr-⁹⁰Y. Activities of 2.5 or 5.0 µCi radiostrontium per animal lead to a depression at time intervals of two and four days, at longer periods there was an overshoot. With activities of 0.5 or 1.0 µCi radiostrontium disturbances in the radioiron uptake are still observed, although these effects are not as pronounced as in experiments with higher burdens. In comparison with results obtained in experiments in which the plasma⁵⁹Fe-turnover was applied, even with an activity of 5 µCi radiostrontium per mouse, no deviation as against the untreated controls was detected.Dedicated to Prof. Dr. Hermann Muth, Homburg/Saar, on the occasion of his 65th birthday     

Hsp90 & Co. - a holding for folding.

Hsp90 is an abundant molecular chaperone that is involved in the folding of a defined set of signalling molecules including steroid-hormone receptors and kinases. Recent in vitro experiments suggest that Hsp90 contains two different binding sites for non-native proteins, which allow it to combine the properties of a promiscuous chaperone with those of a dedicated folding-helper protein. Significant progress has been made in analysing co-chaperones, which form defined, substrate-dependent complexes with Hsp90 in vivo. Structural studies have identified the ATP-binding site in the N-terminal domain of Hsp90, which can be blocked by high-affinity inhibitors. Although a detailed understanding of the mechanism of Hsp90 action is still lacking, recent advances suggest that the protein is the centre of a dynamic, multifunctional and multicomponent chaperone machinery that extends the limits of protein folding in the cell.     

Coordinated ATP hydrolysis by the Hsp90 dimer.

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.     

Thrombin receptor signaling to cytoskeleton requires Hsp90.

Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.