The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg 2؉ -induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.Molecular chaperones are essential for the correct folding of neo-synthesized proteins in vivo, from their emergence from the ribosomal tunnel to their acquisition of a functional state. Among chaperone proteins, the 90-kDa heat shock protein (Hsp90) 2 is one of the most abundant proteins in the cytosol and is essential for cell survival (1). The Hsp90 functions require the cooperation of cochaperones and other chaperones within macro complexes (2). In contrast to most of the chaperone protein families, Hsp90 is not involved in de novo folding but operates at later stages of the folding process, assisting the maturation and activation of numerous client proteins, including steroid receptors and cell cycle kinases (3, 4). The effectiveness of Hsp90 in protecting and activating such proteins has led researchers to focus on the Hsp90 hub as a target for anticancer drugs (5-7). Hsp90 is ubiquitous and shows an extraordinary conservation from bacteria to higher eukaryotes. In the cell, Hsp90 amounts to 1-2% of the total proteins under nonstress conditions depending on the organism and cell type (2-25 mg/ml). Hsp90 is overexpressed under stress conditions such as heat or hypoxia (8 -10). In eukaryotes, the cytosol contains two isoforms, ␣ and ␤, encoded by distinct genes (11). These isoforms coexist in the cytosol; Hsp90␤ is constitutively expressed, whereas Hsp90␣ is stress-inducible. Th...

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.Chaperones are essential for cell life because of their involvement in the folding of newly synthesized proteins. Among the chaperones, the heat-shock protein of 90 kDa (Hsp90) 3 is one of the most abundant and ubiquitously expressed cellular proteins (10 -50 M (1)) with a main function of helping other proteins maintain their conformation in an active state. Several client proteins of Hsp90 involved in cell proliferation and apoptosis (such as protein kinases and transcription factors) have been described (2-5). Although Hsp90 is fundamental for the life of normal cells, it also protects mutated proteins against cellular degradation, thus promoting cancer cell survival (6). Therefore, considerable efforts have been made to find inhibitors of its activity (7,8).Over the past decade several studies have suggested a possible relationship between Hsp90 and the microtubule cytoskeleton (9 -15). Tubulin is a highly concentrated cellular protein (10 -20 M (16)) and is the building block of microtubules, which are involved in several crucial processes such as cell division and morphogenesis, compartmentalization, and organelle movement. Its fundamental role in the architecture and functioning of the mitotic spindle has also made this protein a classical target of antimitotic drugs. A direct interaction between Hsp90 and tubulin has previously been described (9), but Hsp90 is also known to interact with other chaperones and co-chaperones to perform its function (17-19). Therefore, whether Hsp90 can protect tubulin against denaturation in the absence of other factors remains unknown.In this study we investigated the direct interaction between tubulin and Hsp90, both purified from porcine brain. Tubulin was subjected to thermal denaturation in the absence and in the pres...

Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.