Tylophorine Analog DCB-3503 Inhibited Cyclin D1 Translation through Allosteric Regulation of Heat Shock Cognate Protein 70

Abstract

Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3′-untranslated region (3′ UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3′ UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1.

Structure-activity relationships of cryptopleurine analogs with E-ring modifications as anti-hepatitis C virus agents

“…24 Mutation of the ATPase pocket abolished the chaperone activity of Hsc70 and its binding to RNA. 25,26 By promoting the ATPase activity of Hsc70, rac-cryptopleurine and DCB-3503 inhibited HCV. 6,26 Thus, we next determined the impact of cryptopleurine analogs on the ATP/ADP turnover of Hsc70.…”

“…25,26 By promoting the ATPase activity of Hsc70, rac-cryptopleurine and DCB-3503 inhibited HCV. 6,26 Thus, we next determined the impact of cryptopleurine analogs on the ATP/ADP turnover of Hsc70. Addition of rac-cryptopleurine enhanced ADP production by promoting the ATPase activity of Hsc70 at 10 mM (Fig.…”

“…1 We previously that rac-cryptopleurine bound physically but non-covalently to the NBD of Hsc70 6,26 ; therefore, functional analog(s) could interfere with the binding between rac-cryptopleurine and Hsc70. We next performed an affinity binding assay using biotinylated-rac-cryptopleurine, 6,26 and eluted Hsc70 bound to the affinity resin with rac-cryptopleurine or its analogs. YXM-110, but not YXM-109, YXM-139, nor YXM-140, eluted Hsc70 bound to the affinity resin in the similar fashion as that of rac-cryptopleurine ( Fig.…”

“…27 Co-chaperone protein nucleotide exchange factor c-terminal of Bag1 (cBag, or Bag-1 M) increased the ATPase activity of Hsc70 in combination with rac-cryptopleurine or DCB-3503. 26 We then determined whether rac-cryptopleurine and YXM-110 affected luciferase refolding activity of Hsc70. Purified native luciferase was first denatured with 6 M guanidine-HCl and incubated with Hsc70 in the presence or absence of rac-cryptopleurine or YXM-110.…”

“…The affinity purification was performed as described previously. 26 Huh-luc/neo-ET cells were lysed in protein-binding buffer (PBB, 20 mM Hepes, pH 7.9, 100 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40, 6 mM MgCl 2 , 5 mM 2-mercaptoethanol, complete protease inhibitor (Roche)). Biotinylated-rac-cryptopleurine was bound overnight at room temperature to a 50% slurry of streptavidin resin (Invitrogen) in buffer containing 50% DMSO and 50% PBB.…”