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

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.


RKO cell line with inducible expression system
HepG2 (human hepatocellular carcinoma) and RKO (human colon carcinoma) cells were maintained in RPMI 1640 supplied with 10% FBS. Hela (human cervical carcinoma) cells were maintained in DMEM medium supplied with 10% FBS.
The level of knock down of protein and the homogeneity of clones were determined by Western blotting and confocal microscopy, respectively. Clones with homologous expression of shRNAs were isolated and expanded.

RNA and miRNA isolation, real-time PCR
Total RNA was isolated by Trizal ® (Invitrogen). cDNA was synthesized from total RNA using random primer according to the protocol of iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA), and expression level of specific genes was quantitated by real-time PCR using the SsoFastTM EvaGreen supermix (Bio-Rad Laboratories).
The level of β-actin was used as internal control. Expression levels of microRNAs were quantified by the TaqMan ® microRNA Assays (Applied Biosystems, Foster City, CA). The level of RNU-6B was used as internal control.

OptiPrep iodixanol continuous density gradient centrifugation
OptiPrep iodixanol density media was obtained from Sigma-Aldrich. Continuous density gradient centrifugation was performed as described previously 5  Post-nuclear supernatant was centrifuged at 36,000 rpm at 4°C using a SW 41Ti Beckman Coulter rotor for 80 minutes. Each sample was divided into 12 fractions and collected by ISCO density gradient fractionation system (ISCO).

Liquid chromatography-mass spectrometry (LC-MS)
Proteins in each fraction obtained from the OptiPrep iodixanol gradient centrifugation were removed by acetonitrile:methanol
The cells were then incubated with optimized dilution of primary antibodies overnight, and followed by fluorescent conjugated secondary antibody in 1%BSA for 1 hour at room temperature. After washing with PBS for three times, the cells were saved in ProLong® Gold antifade-reagent (Life Technologies).

Immunoprecipitation of HSC70 associated protein and mRNA complexes (RNA-IP)
RNA-IP was performed as described previously 6

Characterization of ATP hydrolysis activity of HSC70
Characterization of the ATP hydrolysis activity of HSC70 was described previously 7 .

Expression and purification of recombinant HSC70 and cBAG
Primers for the construction of expression vectors of His-tagged full-length Hsc70, cBAG, and firefly luciferase are shown in Table S1. Recombinant proteins were overproduced in E.coli BL21 (DE3) pLysS cells (Promega, Madison, WI) at 22°C for 4 hours, and purified by Ni 2+ -NTA agarose 8 .

Refolding of denatured luciferase
Recombinant firefly luciferase was dissolved in 6 M guanidine•HCl, 100 mM Tris•HCl, 1 mM DTT, pH 7.7 9 . The refolding reaction was started by addition of 2 μl denatured luciferase (0.1 μM) to refolding buffer with a final dilution factor of 1:100. DCB-3503 and/or cBAG were added at the beginning of the assay. Luciferase activity was measured for the time indicated on Figure 6A&6B for a total of 60 minutes. Equal concentration of BSA was used as a negative control.