![]() ![]() Similarly, the normal and patho-physiological conditions of the cardiovascular system are highly dependent on Hsp90 machinery. Hsp90 maintains the functional stability of aggregation-prone neuronal proteins and prevents the accumulation of toxic aggregates ( 10). The Hsp90 machine has also been implicated as a target to treat neurodegenerative and cardiovascular diseases in addition to cancer. For example, Cdc37 is thought to recruit the Hsp90 machine to kinases ( 5– 7), and p23 is essential for the activation of steroid hormone receptors ( 6, 8, 9). These co-chaperones are thought to provide Hsp90 selectivity toward different client proteins. Over 20 co-chaperone proteins, such as p23, Cdc37, HIP, HOP, PP5, UNC45A, and immunophilins (FKBP51, FKBP52, and Cyp40), have been shown to regulate the function of the Hsp90 protein-folding machine ( 2– 4). Hsp90 works in concert with a number of chaperones and co-chaperones to modulate the conformation of Hsp90 and its client proteins. Hsp90 is essential for normal cellular homeostasis, but it is also known to play important roles in several pathological conditions ( 1). These results provide important insight into the molecular mechanism of action of this promising lead compound. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Amygdala (AMY), n = 3-4 rats medial prefrontal cortex (mPFC), n = 3-4 HPC, n = 4-5.Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. No significant interactions between diet and the studied brain regions were revealed ( F (2,16) = 2.44, p = 0.12). Although it did not reach statistical significance, we found a trend for more prefrontal cortex FKBP51 levels when compared with the amygdala and the hippocampus ( F (2,16) = 3.58, p = 0.052). Diet did not alter FKBP51 levels in the amygdala or medial prefrontal cortex. D, Analyses revealed a significant increase in FKBP51 levels in the hippocampus of the WDE rats when compared with CDE rats ( F (1,16) = 9.88 p = 0.0063, p = 0.021). C, Representative blot from micropunched anxiety-related brain regions showed distinctive FKBP51 levels. B, Analyses showed a significant main effect of the diet on brain FKBP51 levels (diet effect: F (1,19) = 15.45, p = 0.0009), PS exposure ( F (1,19) = 0.51, p = 0.49), or interaction between diet and PS exposure ( F (1,19) = 0.42, p = 0.52) did not show significant main effects on FKBP51 levels. A, Representative Western blot shows increased FKBP51 levels in the brains of the rats that consumed the WD. WD increases FKBP51 protein levels in the hippocampus. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).įigure 6. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Approximately 51 and 45 kDa bands corresponding to FKBP51 were observed across cell lines tested. The blots were probed with Anti-FKBP51 Rabbit Polyclonal Antibody (Product # PA1-020, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). ![]() Western blot analysis was performed on nuclear enriched extracts (30 µg lysate) of K562 (Lane 1), Hep G2 (Lane 2), PC3 (Lane 3), and MCF-7 (Lane 4). ![]()
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