Hsp90 a specialized but essential protein-folding tool

Although protein folding, in principle is a spontaneous process which depends only upon the amino-acid sequence, the assistance of molecular chaperones is required for many proteins to achieve their final conformation in vivo.

While Hsp90 is one of the major molecular chaperones, it has long been the most mysterious among them. Recent advances in our knowledge regarding Hsp90 structure and function, owing to both detailed biochemical and genetic characterizations of Hsp90 co-chaperones, as well as eminent structural studies have established Hsp90 as an ATPase-dependent chaperone, and have provided a paradigm of the Hsp90 chaperone cycle, which is sequentially tuned and coordinated by a variety of co-chaperones.

Here we summarize the current knowledge regarding the structure and essential activities of Hsp90, which certainly promises a deeper understanding of the functions of Hsp90 in vivo. Most users should sign in with their email address. If you originally registered with a username please use that to sign in. To purchase short term access, please sign in to your Oxford Academic account above. Don't already have an Oxford Academic account?

Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Disruption of cle in Drosophila and vertebrates. Jensen, L. Cui, Y.

Hou, X. Chang, and J. Per- loss of RafRas association. The function of steroid hormone receptors is and accelerates its degradation by the proteasome. Marsh, J. Kalton, and R. Cns1 is an essential protein asso- — Dynamics of heat shock protein progesterone receptor bind- restore cyclophilin dependent functions in cpr7 cells. Maruya, M. Sameshima, T. Nemoto, and I. Monomer arrange- Smith, D.

Stensgard, W. Welch, and D. Assembly of ment in Hsp90 dimer as determined by decoration with N and C-terminal progesterone receptor with heat shock proteins and receptor activation are region specific antibodies. ATP mediated events. Meacham, G. Patterson, W. Zhang, J. Younger, and D. Stancato, L.

Silverstein, J. Owens-Grillo, Y. Chow, R. Jove, and Downloaded from www. The hspbinding antibiotic geldanamycin decreases Raf degradation. Kawasaki, M. Minami, N. Tanahashi, K. Tanaka, and I. A critical role for the proteasome activator PA28 in the Hspdepen- J. Stebbins, C. Russo, C. Schneider, N. Rosen, F. Hartl, and N. Pavle- Morishima, Y. Murphy, D. Li, E. Sanchez, and W. Step- tich. Crystal structure of an Hspgeldanamycin complex: targeting wise assembly of a glucocorticoid receptor.

Leng, S. Parker, and J. Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabi- Nathan, D.

Vos, and S. In vivo functions of the Saccha- lizes Cdk4. Stensgard, G. Caucutt, B. Bartha, N. McMahon, E. Alnemri, G. Litwack, and D. Nucleotides and two functional states of Nemoto, T.

Ohara-Nemoto, T. Takagi, and K. Mechanism hsp ClpB and HtpG facilitate de novo protein 1—8. Obermann, W. Sondermann, A. Russo, N.

Pavletich, and F. Rommel, B. Dickson, and E. The heat shock Panaretou, B. Prodromou, S. Roe, R. Ladbury, P. Piper, Weaver, A. Sullivan, S. Felts, B. Owen, and D. Crystal and L. ATP binding and hydrolysis are essential to the func- structure and activity of human p23, a heat shock protein 90 co-chaperone. Picard, D. Khursheed, M. Garabedian, M. Fortin, S. Lindquist, and K. Weikl, T.

Abelmann, and J. An unstructured C-terminal re- Yamamoto. Reduced levels of hsp90 compromise steroid receptor ac- gion of the Hsp90 co-chaperone p23 is important for its chaperone func- tion in vivo. Pratt, W. Steroid receptor interactions with heat shock Weikl, T. Muschler, K. Richter, T. Veit, J. Reinstein, and J. C-terminal regions of Hsp90 are important for trapping the nucleotide dur- Prodromou, C.

Piper, and L. Mimnaugh, B. De Costa, C. Myers, and L. Siligardi, R. Woolfson, L. Regan, B. Panaretou, formation by benzoquinone ansamycins: essential role for stress proteins in J. Regulation of hsp90 oncogenic transformation. Wickner, S. Maurizi, and S.

Prodromou, C. Panaretou, S. Chohan, G. Xu, Y. Heat-shock protein hsp90 governs the activity of S. Roe, P. The ATPase cycle of Hsp90 pp60v-src kinase. Polypeptide release by Hsp90 involves ATP hy- mains. Rajapandi, T. Greene, and E. The molecular chaperones Young, J. Schneider, and F. In vitro evidence that hsp90 con- Hsp90 and Hsc70 are both necessary and sufficient to activate hormone tains two independent chaperone sites.

FEBS Lett. Young, J. Obermann, and F. Specific binding of tet- Rao, J. Lee, S. Benzeno, C. One interesting question raised by this model is the manner in which substrate proteins are held by Hsp90 in the ATP state.

Consistent with this estimate, a homodimerized kD fragment of topoisomerase II provides a hole between 2. For comparison, the cavity of the bacterial chaperonin GroEL, which encapsulates nonnative proteins, is 6.

Thus, although a space encircled by the Hsp90 dimer is probably too small for an entire protein substrate to fit inside, a sizable domain could be accommodated. It is also possible that Hsp90 does not enclose its substrate but rather that a substrate-binding face is optimally exposed by Hsp90 in the ATP clamp state. Evidence for multiple substrate-binding sites along the length of the Hsp90 protein has been presented, consistent with either model Young et al.

Once the precise substrate-binding mode of Hsp90 has been established, the larger question of how it aids in folding can be addressed. Although the bacterial and endoplasmic reticulum forms of Hsp90 appear to operate independently, Hsp90 in the eukaryotic cytosol interacts with a variety of cochaperone proteins that assemble into a multichaperone complex and regulate the function of Hsp90 and Hsp The largest class of cochaperones bind to Hsp90 via a modular domain containing typically three 34 amino acid, helix-turn-helix tetratricopeptide repeat TPR motifs Fig.

The isolated TPR domain binds to the pentapeptide with specificity and affinity comparable to the interaction between the full-length proteins. Hydrophobic interactions with the divergent NH 2 termini of the peptides account for the specificity of binding Scheufler et al.

The amino acid residues of Hop, which form the carboxylate clamp, are absolutely conserved in the TPR domains of other Hspbinding cochaperones but not in functionally unrelated TPR proteins.

Also, the COOH-terminal sequences of Hsp90 and Hsc70 are conserved within the eukaryotic cytosolic forms of these chaperones Scheufler et al. In addition to linking Hsp90 with the Hsc70 chaperone system, Hop acts as an inhibitor of the Hsp90 ATPase by preventing access to the nucleotide-binding site of Hsp This inhibitory activity of Hop has been proposed to be part of a substrate-loading mechanism for Hsp90, where an HspHop-Hsc70 complex permits transfer of substrate polypeptide from Hsc70 to the nucleotide-free state of Hsp90 Prodromou et al.

Mature complexes of Hsp90 with bound substrate are also characterized by binding of the cochaperone p23 Johnson and Toft, , which is unrelated to the TPR domain proteins.

Although p23 can itself act as a chaperone in binding unfolded polypeptides Bose et al. The binding site for p23 is located within the NH 2 -terminal half of Hsp90 and is supported by induced dimerization of Hsp90 Chadli et al. This is consistent with the structural model of the Hsp90 ATPase cycle discussed above.

Although p23 does not affect the ATPase activity of Hsp90, the cochaperone significantly stimulates the ATP hydrolysis-dependent dissociation of Hsp90—substrate complexes. One possible mechanism is that p23 more stringently couples the nucleotide state of Hsp90 with conformational changes throughout the dimer Young and Hartl, The active core domain of p23 has an immunoglobulin-like fold with several highly conserved residues exposed in one cluster, forming the probable contact site for Hsp90 Weaver et al.

The recently reported Hspindependent effects of p23 on steroid hormone receptor signaling Freeman et al. Cdc37 has some properties of a chaperone and may itself contribute to the folding reaction Kimura et al. Cdc37 also interacts functionally with androgen receptor but not glucocorticoid receptor Rao et al. There may be further cochaperones of Hsp90 yet to be identified. For example, the proteasome activator PA28 has been reported recently to stimulate the Hscmediated refolding of luciferase bound to Hsp90 in vitro Minami et al.

A growing set of signal transduction proteins constitute the majority of known Hsp90 substrates. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance. Disruption of Hsp90 function by mutations or treatment with inhibitors such as the ansamycins led to multiple physiologic defects in live cells, consistent with a contribution of Hsp90 throughout the cellular signaling network.

For example, in the regulation of cell division alone disruption of Hsp90 affects multiple stages of the mitogenic signal cascade, cyclin-dependent progression through both G1 and G2, and centrosome function during mitosis Fisher et al.

An in vivo requirement for Hsp90 has been established for some steroid hormone receptors Picard et al. These proteins are characteristically large and multidomain and require stabilizing interactions with other factors for their function such as steroid ligands for the steroid hormone receptors or cyclins for the cyclin-dependent kinases.

Because signaling proteins with multiple regulatory states often undergo a conformational switch, the structural flexibility needed for these steps may render them inherently less stable and thus more likely to be recognized by Hsp On the other hand, under stress conditions such as heat shock Hsp90 contributes more generally to the refolding of denatured proteins Nathan et al.

Although the exact structural features recognized by Hsp90 are not yet understood, the exposure of these features to Hsp90 is likely a result of intrinsic or stress-induced structural flexibility. Thus, interactions of substrate proteins with Hsp90 arise from structural properties at the molecular level rather than biological function. The best characterized example of an Hspdependent signaling pathway is that of the steroid hormone receptors Pratt and Toft, Fig.

Interaction of the glucocorticoid receptor with Hsp90 is essential for its activity Picard et al. Once the folded monomeric receptor has been released from the chaperones, it either binds the appropriate steroid hormone, resulting in dimerization and activation, or remains unstable and is recognized again by the chaperone machinery Smith, Recently, progesterone receptor and glucocorticoid receptor have been used to reconstitute the typical multichaperone complexes with purified mammalian Hsp90, Hsc70, Hop, and p Although the requirement for cochaperones in vitro is still controversial Morishima et al.

In the case of v-src and other kinases, Hsp90 is thought to stabilize the exposed catalytic domains before assembly of the kinases into the final signaling complex, a mechanism conceptually related to the steroid receptor pathway Xu and Lindquist, ; Aligue et al. A variation of the steroid receptor signaling pathway has been reported for the Drosophila ecdysone receptor, a member of the heterodimeric retinoid X receptor family Fig.

The isolated receptor together with its partner protein USP is already capable of binding ecdysone but not DNA, and the Hsp90 chaperone machinery converts the hormone-bound receptor to the active DNA-binding state Arbeitman and Hogness, Another mechanism allows human Hsp90 to regulate its own expression by sequestering the monomeric inactive form of heat shock transcription factor HSF 1 under nonstress conditions Fig.

Misfolded proteins produced by stress can compete with HSF1 for binding to Hsp90, liberating the transcription factor for trimerization and initiating the heat shock response Zou et al. The hallmark of ansamycin activity in live cells is induced degradation of Hsp90 substrate proteins by the ubiquitin-dependent proteasome pathway Whitesell et al. Although ansamycins were identified historically as tyrosine kinase inhibitors, they are now known to specifically target Hsp90 family members Whitesell et al.

Ansamycins can inhibit the chaperone-mediated folding of Hsp90 substrates by blocking their ATP-dependent dissociation from Hsp90 Schneider et al.

On the other hand, certain substrate proteins are released from Hsp90 by ansamycins. For these proteins, either drug-induced degradation is not observed as in the case of HSF1 Zou et al. How are Hspbound polypeptides targeted for degradation? The TPR cochaperone CHIP, which recognizes both Hsp90 and Hsc70 see above , contains a U-box domain homologous to those of the E4 ubiquitination factors and may present chaperone-bound misfolded proteins for ubiquitination.

Consistent with this possibility, overexpression of CHIP in cultured cells caused increased ubiquitination and degradation of glucocorticoid receptor and cystic fibrosis transmembrane regulator, known substrates of Hsp90 and Hsc70, respectively Connell et al.

In terms of the kinetic model of protein folding and degradation Wickner et al. The existence of CHIP also suggests that like protein folding, protein targeting to the degradation machinery is regulated by additional factors whose functions are not anticipated by a simple kinetic-partitioning model. Defects in cell physiology caused by Hsp90 disruption can lead to defects at the level of tissue and organism. Interestingly, recent work connects Hsp90 function with morphological evolution, a process that often requires the effects of independent genetic changes Rutherford and Lindquist, Hspnull mutants are lethal in eukaryotes, but surprisingly partial disruption of Hsp90 in Drosophila by a temperature-sensitive mutation or low amounts of GA shows a wide assortment of heritable phenotypic variations.

By extension, polymorphisms in all of the proteins participating in an Hspdependent signaling pathway should be buffered by Hsp90 function. Hsp90 with its connection to the cellular signaling network may be particularly suited to such a function. Thus, the mechanisms of chaperone-mediated protein folding at a molecular level can be integrated with cellular processes and with the development of organisms and species.

Structural basis for the interaction of Hsp90 with nucleotides, ansamycin drugs, and TPR domain cochaperones. A Domain organization of Hsp See text for references. The proposed lid sequence over the nucleotide-binding pocket is indicated. The peptides are oriented with the COOH-terminal carboxyl groups at the bottom of the figure top and towards the viewer bottom Scheufler et al.

Different modes of Hsp90 action in signaling pathways. A Inactive steroid hormone receptors are folded by the Hsp90 machinery. Folded receptors either are stabilized and activated by hormone binding or remain unstable and are rebound by chaperones Smith, ; Pratt and Toft, Under stress conditions, misfolded proteins compete for Hsp90, and HSF1 is displaced from Hsp90 and can form the active trimer Zou et al. Sign In or Create an Account. Advanced Search. User Tools. Sign In. Skip Nav Destination Article Navigation.

Mini-Reviews July 23 Hsp90 : a specialized but essential protein-folding tool Jason C. Young , Jason C.

This Site. Google Scholar. Ismail Moarefi , Ismail Moarefi. Ulrich Hartl F. Ulrich Hartl. Author and Article Information. Ismail Moarefi. Address correspondence to Ulrich Hartl, Dept. Fax: E-mail: uhartl biochem. Received: April 19 Revision Received: June 14 Accepted: June 21 Online Issn: The Rockefeller University Press. J Cell Biol 2 : — Article history Received:. Revision Received:.

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