Related Experiment Videos
Radicicol binds and inhibits mammalian ATP citrate lyase
S W Ki1, K Ishigami, T Kitahara
1Departments of Biotechnology and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Abstract:
Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase with K(i) values for citrate and ATP of 13 and 7 microm, respectively.
Insights
Researchers synthesized biotinylated radicicol derivatives to identify cellular proteins. Radicicol binds to ATP citrate lyase and Hsp90 through distinct structural interactions, affecting ATP citrate lyase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Radicicol is a known inhibitor of cellular signaling pathways.
- Identifying specific protein targets of radicicol is crucial for understanding its mechanism of action.
- Biotinylated probes are valuable tools for affinity-based protein identification.
Purpose of the Study:
- To synthesize and characterize biotinylated radicicol derivatives as affinity probes.
- To identify cellular proteins that bind to radicicol.
- To elucidate the binding interactions and functional consequences of radicicol binding to its targets.
Main Methods:
- Synthesis of six different biotinylated radicicol derivatives.
- Affinity purification and detection of radicicol-binding proteins using biotinylated probes (BR-1 and BR-6).
- Protein identification via immunoblotting and amino acid sequencing.
- Enzymatic activity assays and kinetic analysis of ATP citrate lyase inhibition.
Main Results:
- Biotinylated radicicol derivatives BR-1 and BR-6 retained biological activity.
- Two major radicicol-binding proteins of 120 kDa and 90 kDa were identified.
- The 90 kDa protein was identified as Heat Shock Protein 90 (Hsp90).
- The 120 kDa protein was identified as ATP citrate lyase.
- Radicicol binding to ATP citrate lyase and Hsp90 involved different molecular regions.
- Radicicol and BR-1 inhibited ATP citrate lyase activity, while BR-6 did not.
- Radicicol acted as a non-competitive inhibitor of ATP citrate lyase.
Conclusions:
- Radicicol binds to distinct cellular targets, including ATP citrate lyase and Hsp90.
- The binding sites for radicicol on these two proteins differ structurally.
- Radicicol's inhibition of ATP citrate lyase suggests a role in metabolic regulation.
- Biotinylated radicicol derivatives are effective tools for target identification and mechanistic studies.