Computational aided mechanistic understanding of Camellia sinensis bioactive compounds against co-chaperone p23 as potential anticancer agent

Co-chaperon p23 has been well established as molecular chaperon for the heat shock protein 90 (Hsp90) that further leads to immorality in cancer cells by providing defense against Hsp90 inhibitors, and as stimulating agent for generating overexpressed antiapoptotic proteins, that is, Hsp70 and Hsp27. The natural compounds such as catechins from Camellia sinensis (green tea) are also well known for inhibition activity against various cancer. However, molecular interaction profile and potential lead bioactive compounds against co-chaperon p23 from green tea are not yet reported. To this context, we study the various secondary metabolites of green tea against co-chaperon p23 using structure-based virtual screening from Traditional Chinese Medicine (TCM) database. Following 26 compounds were obtained from TCM database and further studied for extra precision molecular docking that showed binding score between −10.221 and −2.276 kcal/mol with co-chaperon p23. However, relative docking score to known inhibitors, that is, ailanthone (−4.54 kcal/mol) and gedunin ( 3.60 kcal/mol) along with ADME profile analysis concluded epicatechin (−7.013 kcal/mol) and cis-theaspirone (−4.495 kcal/mol) as potential lead inhibitors from green tea against co-chaperone p23. Furthermore, molecular dynamics simulation and molecular mechanics generalized born surface area calculations validated that epicatechin and cis-theaspirone have significantly occupied the active region of co-chaperone p23 by hydrogen and hydrophobic interactions with various residues including most substantial amino acids, that is, Thr90, Ala94, and Lys95. Hence, these results supported the fact that green tea contained potential compounds with an ability to inhibit the cancer by disrupting the co-chaperon p23 activity.     

Extracellular heat shock protein (Hsp)70 and Hsp90α assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90.     

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.     

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.     

S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex

Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.     

Distinct but overlapping functions of Hsp70, Hsp90, and an Hsp70 nucleotide exchange factor during protein biogenesis in yeast

Hsp70 and Hsp90 molecular chaperones play essential roles in protein expression and maturation, and while catalyzing protein folding they can "decide" to target mis-folded substrates for degradation. In this report, we show for the first time distinct but partially overlapping requirements for Hsp90, Hsp70, and an Hsp70 nucleotide exchange factor (NEF) at different steps during the biogenesis of a model substrate, firefly luciferase (FFLux), in yeast. By examining the inducible expression of FFLux in wild type cells and in specific yeast mutants, we find that the Fes1p NEF is required for efficient FFLux folding, whereas the Hsp70, Ssa1p, is required for both protein folding and stability, and to maintain maximal FFLux mRNA levels. In contrast, Hsp90 function was primarily necessary to express the FFLux-encoding gene from an inducible promoter. Together, these data indicate previously unknown roles for these proteins and point to the complexity with which chaperones and cochaperones function in the cell.     

Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53.

Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein. Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H. The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes. The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP. The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex. The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein.     

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.     

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1–3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.     

The Hsp organizer protein hop enhances the rate of but is not essential for glucocorticoid receptor folding by the multiprotein Hsp90-based chaperone system

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR).Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90.Hop.Hsp70.Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR.Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358-7366). Hop has been considered essential for the formation of receptor.Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR.Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.