Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23.

Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.

Ataxia telangiectasia and rad3-related kinase contributes to cell cycle arrest and survival after cisplatin but not oxaliplatin.

The DNA cross-linking agents cisplatin and oxaliplatin are widely used in the treatment of human cancer. Lesions produced by these agents are widely known to activate the G1 and G2 cell cycle checkpoints. Less is known about the role of the intra-S-phase checkpoint in the response to these agents. In the present study, two different cell lines expressing a dominant-negative kinase dead (kd) version of the ataxia telangiectasia and rad3-related (ATR) kinase in an inducible fashion were examined for their responses to these two platinating agents and a variety of other DNA cross-linking drugs. The expression of the kdATR allele markedly sensitized the cells to cisplatin, but not to oxaliplatin, as assessed by inhibition of colony formation, induction of apoptosis, and cell cycle analysis. Similar differences in survival were noted for melphalan (ATR dependent) and 4-hydroperoxycyclophosphamide (ATR independent). Further experiments showed that ATR function is not necessary for removal of Pt-DNA adducts. The predominant difference between the responses to the two platinum drugs was the presence of a drug-specific ATR-dependent S-phase arrest after cisplatin but not oxaliplatin. These results indicate that involvement of ATR in the response to DNA cross-linking agents is lesion specific. This observation might need to be taken into account in the development and use of ATR or Chk1 inhibitors.

A novel in situ assay for the identification and characterization of soluble nuclear mobility factors.

The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.

Molecular chaperones function as steroid receptor nuclear mobility factors.

Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and/or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.

The influence of ATP and p23 on the conformation of hsp90.

The chaperoning activity of the heat shock protein hsp90 is directed, in part, by the binding and hydrolysis of ATP and also by association with co-chaperone proteins. One co-chaperone, p23, binds to hsp90 only when hsp90 is in a conformation induced by the binding of ATP. Once formed, the p23-hsp90 complex is very stable upon the removal of ATP and dissipates at 30 degrees with a half-life of about 45 min. This was shown to be due to the high stability of the ATP-induced state of hsp90, not to the rate of p23 dissociation. Further stabilization of this ATP-induced state is achieved by including molybdate or by use of the ATP analogue ATPgammaS. This conformational state of hsp90 is correlated with the tight binding of ADP resulting from hydrolysis of bound ATP. Both p23 and molybdate enhance and stabilize the nucleotide-bound state of hsp90, and this state is maximized by the presence of both agents. These results can be explained in a model where the binding of ATP induces a conformational transition in hsp90 that traps the nucleotide and is committed to ATP hydrolysis. p23 specifically recognizes this state and may also facilitate subsequent steps in the chaperoning cycle.

The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex.

The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K(d) = 90 nm), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K(d) = 1.3 microm) on its own, but this affinity is increased (K(d) = 250 nm) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.

Regulation of heat shock protein 90 ATPase activity by sequences in the carboxyl terminus.

Hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k(cat) of 0.02 min(-1) and a K(m) greater than 300 microm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10-100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k(cat)/K(m)) was greater than 100 times that of the full-length protein (k(cat) of 0.24 min(-1) and K(m) of 60 microm). In contrast, an internal deletion mutant, Delta661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.