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Updated: Sep 4, 2026
Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
Published on: September 20, 2016
In situ photoaffinity labeling of proteasome with photoactive adriamycin analogue
K I Kiyomiya1, S Matsuo, M Kurebe
1Department of Toxicology, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan. kiyomiya@vet.osakafu-u.ac.jp
Abstract:
An intracellular adriamycin (ADM)-binding protein purified from the cytosol of L1210 mouse lymphocytic leukemia cells had a molecular weight of 700-1500 kDa and hydrolyzed Suc-LLVY-MCA. When L1210 cells were incubated with a photoactive ADM analogue, N-(p-azidobenzoyl)-adriamycin (NAB-ADM), most of the NAB-ADM was found to localize in the nuclei. In situ photoaffinity labeling of L1210 cells with NAB-ADM resulted in low protease activity in the cytosol and nuclear extracts and the cells showed selective photoincorporation of NAB-ADM into the proteasome. These results suggest that the proteasome is a translocator of ADM from the cytoplasm to the nucleus and might therefore become a new candidate for cancer chemotherapy.
Insights
Researchers identified a protein that transports adriamycin (ADM) into the nucleus. This proteasome discovery offers a new avenue for cancer chemotherapy drug development.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Adriamycin (ADM) is a chemotherapy drug with nuclear localization.
- The mechanism of ADM transport into the nucleus is not fully understood.
Purpose of the Study:
- To investigate the role of intracellular proteins in ADM transport.
- To identify potential novel targets for ADM-based cancer chemotherapy.
Main Methods:
- Purification and characterization of an ADM-binding protein from L1210 cells.
- Incubation of L1210 cells with a photoactive ADM analogue (NAB-ADM).
- In situ photoaffinity labeling and analysis of protease activity and NAB-ADM incorporation.
Main Results:
- An intracellular ADM-binding protein (700-1500 kDa) with protease activity was identified.
- NAB-ADM predominantly localized to the nucleus in L1210 cells.
- Selective photoincorporation of NAB-ADM into the proteasome was observed, with reduced protease activity in cytosol and nuclear extracts.
Conclusions:
- The proteasome acts as a translocator for ADM from the cytoplasm to the nucleus.
- The proteasome is a potential novel target for adriamycin-based cancer chemotherapy.
