Hsp90 ATPase Research Articles

P53 modulates Hsp90 ATPase activity and regulates aryl hydrocarbon receptor signaling.

The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (Hsp90), is a ligand-activated transcription factor that plays a role in polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Tobacco smoke activates AhR signaling leading to increased transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to mutagens. Recently, p53 was found to regulate Hsp90 ATPase activity via effects on activator of Hsp90 ATPase (Aha1). It is possible, therefore, that AhR-dependent expression of CYP1A1 and CYP1B1 might be affected by p53 status. The main objective of this study was to determine whether p53 modulated AhR-dependent gene expression and PAH metabolism. Here, we show that silencing p53 led to elevated Aha1 levels, increased Hsp90 ATPase activity, and enhanced CYP1A1 and CYP1B1 expression. Overexpression of wild-type p53 suppressed levels of CYP1A1 and CYP1B1. The significance of Aha1 in mediating these p53-dependent effects was determined. Silencing of Aha1 led to reduced Hsp90 ATPase activity and downregulation of CYP1A1 and CYP1B1. In contrast, overexpressing Aha1 was associated with increased Hsp90 ATPase activity and elevated levels of CYP1A1 and CYP1B1. Using p53 heterozygous mutant epithelial cells from patients with Li-Fraumeni syndrome, we show that monoallelic mutation of p53 was associated with elevated levels of CYP1A1 and CYP1B1 under both basal conditions and following treatment with benzo[a]pyrene. Treatment with CP-31398, a p53 rescue compound, suppressed benzo[a]pyrene-mediated induction of CYP1A1 and CYP1B1 and the formation of DNA adducts. Collectively, our results suggest that p53 affects AhR-dependent gene expression, PAH metabolism, and possibly carcinogenesis.

Recessive mutations in CAKUT and VACTERL association

Understanding the complex genetic makeup underlying congenital anomalies of the kidney and urinary tract (CAKUT) is of primary importance to improve diagnosis, stratify risk for later-onset complications, and develop therapeutic strategies. Saisawat et al. used homozygosity mapping coupled with next-generation sequencing to identify recessive mutations in TRAP1 in families with isolated CAKUT and with VACTERL association. This study points to a novel player in kidney development, possibly affecting apoptosis and endoplasmic reticulum stress signaling.

Anticancer Effects of the Hsp90 Inhibitor 17-Demethoxy-Reblastatin in Human Breast Cancer MDA-MB-231 Cells

Triple-negative breast cancer (TNBC) possesses a higher rate of distant recurrence and a poorer prognosis than other breast cancer subtypes. Interestingly, most of the heat shock protein 90 (Hsp90) client proteins are oncoproteins, and some are closely related to unfavorable factors of TNBC patients. 17-Demethoxy-reblastatin (17-DR), a novel nonbenzoquinone- type geldanamycin analog, exhibited potent Hsp90 ATPase inhibition activity. In this study, the anticancer effects of 17-DR on TNBC MDA-MB-231 cells were investigated. These results showed that 17-DR inhibited cell proliferation, induced apoptosis, and suppressed cell invasion and migration in the MDA-MB-231 cells. Down-regulation of the key Hsp90-dependent tumor-driving molecules, such as RIP1 and MMP-9, by 17-DR may be related to these effects. Taken together, our results suggest that 17-DR has potential as a therapeutic agent for the treatment of TNBC.

Investigation of Hsp90 regulation by a conserved N‐terminal motif in the Aha1/Hch1 co‐chaperone family (567.6)

The molecular chaperone Hsp90 functions in a complex cycle for the maturation and activation of a diverse, but specific, group of client proteins. These clients range from transcription factors to kinases and many are key players in cancer. Consequently, understanding the cycle through which Hsp90 acts is imperative. Hsp90 has a large cohort of co‐chaperone proteins which regulate cycle progression and facilitate client maturation. The co‐chaperone Aha1p is of particular importance as it is the only known co‐chaperone which is a potent stimulator of Hsp90 ATPase activity. The co‐chaperone Hch1p, found in some fungi, is a homologue of Aha1p but regulates Hsp90 function differently. The N‐terminal motif, NxxNWHW, is conserved across the Aha1p/Hch1p family. We show that this motif is necessary for Hch1p function in vivo but is dispensable for the in vitro activity of Aha1p. Functional assays developed in both the model organism Saccharomyces cerevisiae and in mammalian cells reveal the importance of this N‐terminal motif. Additionally, isolation of Hsp90/co‐chaperone complexes provides clues regarding the Hsp90 functional cycle. We are able to provide further support for the dual role of the Aha1p/Hch1p co‐chaperone family in the regulation of Hsp90 activity, both early and late in the Hsp90 cycle.Grant Funding Source: Supported by CIHR and NSERC

Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes.

P53 Protein Regulates Hsp90 ATPase Activity and Thereby Wnt Signaling by Modulating Aha1 Expression

The p53 tumor suppressor gene encodes a homotetrameric transcription factor which is activated in response to a variety of cellular stressors, including DNA damage and oncogene activation. p53 mutations occur in >50% of human cancers. Although p53 has been shown to regulate Wnt signaling, the underlying mechanisms are not well understood. Here we show that silencing p53 in colon cancer cells led to increased expression of Aha1, a co-chaperone of Hsp90. Heat shock factor-1 was important for mediating the changes in Aha1 levels. Increased Aha1 levels were associated with enhanced interactions with Hsp90, resulting in increased Hsp90 ATPase activity. Moreover, increased Hsp90 ATPase activity resulted in increased phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), leading to enhanced expression of Wnt target genes. Significantly, levels of Aha1, Hsp90 ATPase activity, Akt, and GSK3β phosphorylation and expression of Wnt target genes were increased in the colons of p53-null as compared with p53 wild type mice. Using p53 heterozygous mutant epithelial cells from Li-Fraumeni syndrome patients, we show that a monoallelic mutation of p53 was sufficient to activate the Aha1/Hsp90 ATPase axis leading to stimulation of Wnt signaling and increased expression of Wnt target genes. Pharmacologic intervention with CP-31398, a p53 rescue agent, inhibited recruitment of Aha1 to Hsp90 and suppressed Wnt-mediated gene expression in colon cancer cells. Taken together, this study provides new insights into the mechanism by which p53 regulates Wnt signaling and raises the intriguing possibility that p53 status may affect the efficacy of anticancer therapies targeting Hsp90 ATPase.

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10−4min−1μM−1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

Molecular docking study, synthesis and biological evaluation of Schiff bases as Hsp90 inhibitors

Heat shock protein 90 (Hsp90) is an emerging attractive target for the discovery of novel cancer therapeutic agents. Docking methods are powerful in silico tools for lead generation and optimization. In our mission to rationally develop novel effective small molecules against Hsp90, we predicted the potency of our designed compounds by Sybyl surflex Geom X docking method. The results of the above studies revealed that Schiff bases derived from 2,4-dihydroxy benzaldehyde/5-chloro-2,4-dihydroxy benzaldehyde demonstrated effective binding with the protein. Subsequently, a few of them were synthesized (1–10) and characterized by IR, 1HNMR and mass spectral analysis. The synthesized molecules were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The anticancer studies were performed by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The software generated results was in satisfactory agreement with the evaluated biological activity.

Targeting the heat shock protein 90

Macrophage migration inhibitory factor (MIF), originally identified as a proinflammatory cytokine, is highly elevated in many human cancer types, independent of their histological origin. MIF's tumour promoting activities correlate with tumour aggressiveness and poor clinical prognosis. Genetic depletion of MIF in mouse cancer models results in significant inhibition of cell proliferation and induction of apoptosis, making it an attractive target for anticancer therapies. Here, we summarize the current possibilities to inhibit MIF function in cancer. All known small molecule MIF inhibitors antagonize MIF's enzymatic function. However, a recent knockin mouse model suggested that protein interactions play a bigger biological role in tumour cell growth regulation than MIF's enzymatic activity. Thus, alternative strategies are important for targeting MIF. Recently, we identified that MIF in cancer cells is highly stabilized through the heat shock protein 90 machinery (HSP90). Thus, MIF is an HSP90 client. Pharmacological inhibition of the Hsp90 ATPase activity results in MIF degradation in several types of cancer cells. This provides a new way to inhibit MIF function independent of its enzymatic activity. Targeting the HSP90 machinery is a promising way to inhibit MIF function in cancer. Along with MIF and dependent on the molecular make-up of the tumour, a large number of other critical tumourigenic proteins are also destabilized by HSP90 inhibition, overall resulting in a profound block of tumour growth.

Mutation in Integrin-Linked Kinase (ILKR211A) and Heat-Shock Protein 70 Comprise a Broadly Cardioprotective Complex

RationaleIntegrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype. However, ILK has been implicated as both a cardioprotective and oncogenic target, which imposes therapeutic constraints that are generally relevant to the translational potential of many kinases.ObjectiveTo study the cardioprotective properties of the activation-resistant, non-oncogenic, mutation of ILK (ILKR211A) against experimental MI in vivo and Doxorubicin induced apoptosis in vitro and it’s relationships to stress induced heat shock proteins.Methods/ResultsThe transgenic mouse heart over-expressing a point mutation in the ILK pleckstrin homology (PH) domain (TgR211A) exhibits a highly cardioprotective phenotype based on LAD-ligation-induced MI reduction in vivo, and on protection against doxorubicin (DOX)-induced cardiomyocyte apoptosis when overexpressed in human induced pluripotent stem cell (iPS)-derived cardiomyocytes in vitro. Intriguingly, the degree of cardioprotection seen with the ILKR211A mutation exceeded that with the ILKS343D mutation. Microarray and immunoprecipitation analyses revealed upregulation of expression levels and specific binding of ILKWT, ILKS343D and ILKR211A to both constitutively active heat-shock protein 70 (Hsc70) and inducible Hsp70 in response to MI, and to acute ILK overexpression in iPSC-cardiomyocytes. ILK-mediated cardioprotection was shown to depend upon Hsp70 ATPase activity.ConclusionsThese findings indicate that wild type ILK and the non-oncogenic ILKR211A mutation comprise a cardioprotective module with Hsp/c70. These results advance a novel target discovery theme in which kinase mutations can be safely engineered to enhance cardioprotective effects.

Identification of the Plant Compound Geraniin as a Novel Hsp90 Inhibitor

Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90) binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino)-17-demethoxygeldanamycin (17AGG). Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose−dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down−regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors.

A Primate Specific Extra Domain in the Molecular Chaperone Hsp90

Hsp90 (heat shock protein 90) is an essential molecular chaperone that mediates folding and quality control of client proteins. Many of them such as protein kinases, steroid receptors and transcription factors are involved in cellular signaling processes. Hsp90 undergoes an ATP hydrolysis dependent conformational cycle to assist folding of the client protein. The canonical Hsp90 shows a typical composition of three distinct domains and interacts with individual cochaperone partners such as Hop, Cdc37 and Aha1 (activator of Hsp90 ATPase) that regulate the reaction cycle of the molecular chaperone. A bioinformatic survey identified an additional domain of 122 amino acids in front of the canonical Hsp90 sequence. This extra domain (E domain) is specific to the Catarrhini or drooping nose monkeys, a subdivision of the higher primates that includes man, the great apes and the old world monkeys but is absent from all other species. Our biochemical analysis reveals that Hsp103 associates with cochaperone proteins such as Hop, Cdc37 and Aha1 similar to Hsp90. However, the extra domain reduces the ATP hydrolysis rate to about half when compared to Hsp90 thereby acting as a negative regulator of the molecular chaperonés intrinsic ATPase activity.

CLC604 preferentially inhibits the growth of HER2-overexpressing cancer cells and sensitizes these cells to the inhibitory effect of Taxol in vitro and in vivo

HER2 has become a solicitous therapeutic target in metastatic and clinical drug-resistant cancer. Here, we evaluated whether or not 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) and its furopyrazole and thienopyrazole analogues repress the expression of the HER2 protein. Among the test compounds, (1-benzyl-3-(p-hydroxymethylphenyl)-5-methylfuro[3,2-c]pyrazol) (CLC604), an isosteric analogue of YC-1, significantly suppressed the expression of HER2, and preferentially inhibited cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Our results revealed that CLC604 reduced HER2 expression through a post-transcriptional mechanism and involvement of proteasomal activity. CLC604 disrupted the association of 90-kDa heat shock protein (Hsp90) with HER2 resulting from the inhibition of Hsp90 ATPase activity. Moreover, we found that CLC604 significantly enhanced the antitumor efficacy of clinical drugs against HER2-overexpressing tumors and efficiently reduced HER2-induced drug resistance in vitro and in vivo. These findings suggest that CLC604 should be developed further as a novel antitumor drug candidate for the treatment of drug-resistant cancer.

Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90.

The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins’ interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90.

Contributions of Co-Chaperones and Post-Translational Modifications Towards Hsp90 Drug Sensitivity

Hsp90 is a molecular chaperone and important driver of stabilization and activation of several oncogenic proteins that are involved in the malignant transformation of tumor cells. Therefore, it is not surprising that Hsp90 has been reported to be a promising target for the treatment of several neoplasias, such as non-small-cell lung cancer and HER2-positive breast cancer. Hsp90 chaperone function depends on its ability to bind and hydrolyze ATP and Hsp90 inhibitors have been shown to compete with nucleotides for binding to Hsp90. Multiple factors, such as co-chaperones and post-translational modification, are involved in regulating Hsp90 ATPase activity. Here, the impact of post-translational modifications and co-chaperones on the efficacy of Hsp90 inhibitors are reviewed.

Functional characterization of heat shock protein 90 targeted compounds

Heat shock protein 90 (Hsp90) is an attractive cancer target that possesses two potential binding domains at the C and N termini. Nevertheless, assays that can reliably distinguish between the C- and N-terminal Hsp90 inhibitors and quantitatively characterize candidate drugs are limited. Here we report an Hsp90 binding assay and ATPase inhibition assay that can not only separate the C- and N-terminal inhibitors but also quantitatively determine their respective IC50 values.

Integration of the accelerator Aha1 in the Hsp90 co-chaperone cycle

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that associates dynamically with various co-chaperones during its chaperone cycle. Here we analyzed the role of the activating co-chaperone Aha1 in the progression of the yeast Hsp90 chaperone cycle and identified a critical ternary Hsp90 complex containing the co-chaperones Aha1 and Cpr6. Aha1 accelerates the intrinsically slow conformational transitions of Hsp90 to an N-terminally associated state but does not fully close the nucleotide-binding pocket yet. Cpr6 increases the affinity between Aha1 and Hsp90 and further stimulates the Hsp90 ATPase activity. Synergistically, Aha1 and Cpr6 displace the inhibitory co-chaperone Sti1 from Hsp90. To complete the cycle, Aha1 is released by the co-chaperone p23. Thus, at distinct steps during the Hsp90 chaperone cycle, co-chaperones selectively trap statistically distributed Hsp90 conformers and thus turn Hsp90 into a deterministic machine.

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed.

Proteomic Analysis of the Mice Hippocampus After Preconditioning Induced by N-Methyl-d-Aspartate (NMDA)

Preconditioning induced by N-methyl-D-aspartate (NMDA) has been used as a therapeutic tool against later neuronal insults. NMDA preconditioning affords neuroprotection against convulsions and cellular damage induced by the NMDA receptor agonist, quinolinic acid (QA) with time-window dependence. This study aimed to evaluate the molecular alterations promoted by NMDA and to compare these alterations in different periods of time that are related to the presence or lack of neuroprotection. Putative mechanisms related to NMDA preconditioning were evaluated via a proteomic analysis by using a time-window study. After a subconvulsant and protective dose of NMDA administration mice, hippocampi were removed (1, 24 or 72 h) and total protein analyzed by 2DE gels and identified by MALDI-TOF. Differential protein expression among the time induction of NMDA preconditioning was observed. In the hippocampus of protected mice (24 h), four proteins: HSP70(B), aspartyl-tRNA synthetase, phosphatidylethanolamine binding protein and creatine kinase were found to be up-regulated. Two other proteins, HSP70(A) and V-type proton ATPase were found down-regulated. Proteomic analysis showed that the neuroprotection induced by NMDA preconditioning altered signaling pathways, cell energy maintenance and protein synthesis and processing. These events may occur in a sense to attenuate the excitotoxicity process during the activation of neuroprotection promoted by NMDA preconditioning.