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Identification of hNopp140 as a binding partner for doxorubicin with a phage display cloning method
Youngnam Jin1, Jaehoon Yu, Yeon Gyu Yu
1Structural Biology Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea.
Abstract:
Doxorubicin is a widely used anti-cancer drug. It is assumed to act by inhibiting DNA replication or transcription, although its precise targets and mechanism of cytotoxicity remain unresolved. A T7 phage library expressing human liver cDNA was screened against immobilized doxorubicin to isolate doxorubicin binding proteins. The selected phage contained the C-terminal region of nucleolar phosphoprotein hNopp140, an important factor in the biogenesis of the nucleolus. When the cloned sequence was expressed in E. coli, the recombinant protein was phosphorylated by casein kinase II and oligomerized in the presence of magnesium and fluoride ions, as occurs in vivo. Doxorubicin bound to the expressed protein with a dissociation constant of 4.5 x 10(-6) M, and this interaction was inhibited by the phosphorylation of hNopp140. These results suggested that doxorubicin might disrupt the cellular function of hNopp140.
Insights
Doxorubicin, a cancer drug, may disrupt the function of nucleolar phosphoprotein hNopp140. This interaction was identified by screening proteins that bind to doxorubicin, revealing a potential new mechanism of action for this chemotherapy agent.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Doxorubicin is a key chemotherapeutic agent with an incompletely understood mechanism of cytotoxicity.
- Its precise molecular targets and pathways leading to cell death remain subjects of ongoing investigation.
Purpose of the Study:
- To identify novel doxorubicin-binding proteins and elucidate potential mechanisms of doxorubicin's action.
- To investigate the interaction between doxorubicin and its binding partners at a molecular level.
Main Methods:
- A T7 phage display library expressing human liver cDNA was screened against immobilized doxorubicin.
- The C-terminal region of human nucleolar phosphoprotein hNopp140 was identified as a doxorubicin-binding protein.
- Recombinant hNopp140 was expressed, phosphorylated by casein kinase II, and its binding to doxorubicin was characterized.
Main Results:
- The C-terminal region of hNopp140 was isolated as a doxorubicin-binding protein.
- Recombinant hNopp140 exhibited phosphorylation and oligomerization, mimicking in vivo conditions.
- Doxorubicin bound to hNopp140 with a dissociation constant of 4.5 x 10(-6) M.
- Protein phosphorylation inhibited doxorubicin binding, suggesting a regulatory role.
Conclusions:
- Doxorubicin may exert its cytotoxic effects by interfering with the cellular functions of hNopp140.
- The interaction between doxorubicin and hNopp140, particularly its phosphorylation state, offers a new perspective on doxorubicin's mechanism of action.
- Further research is warranted to explore the implications of this interaction in cancer therapy.