期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 287, 期 42, 页码 35768-35778出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.396911
关键词
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资金
- National Institutes of Health Grant [GM29595]
- Damon Runyon Cancer Research Fellowship [DRG2015-09]
- Howard Hughes Medical Institute international student fellowship
Triapine (R) (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the beta(2) subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe-2(III)-Y center dot)(Fe-2(III))] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of beta(2) have been proposed; one involves direct iron chelation from beta(2), where as a second involves Y center dot destruction by reactive oxygen species formed in situ in the presence of O-2 and reductant by Fe(II)-(3-AP). Inactivation of beta(2) can thus arise from cofactor destruction by loss of iron or Y center dot. In vitro kinetic data on the rates of Fe-55 and Y center dot loss from [(Fe-55(2)III-Y center dot)(Fe-55(2)III)]-beta(2) under aerobic and anaerobic conditions reveal that Y center dot loss alone is sufficient for rapid beta(2) inactivation. Oxyblot (TM) and mass spectrometric analyses of trypsin-digested inhibited beta(2), and lack of Y center dot loss from H2O2 and O-2((center dot) over bar). treatment together preclude reactive oxygen species involvement in Y center dot loss. Three mammalian cell lines treated with 5 mu M 3-AP reveal Y center dot loss and beta(2) inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo-over [(Fe-2(III)-Y center dot)(Fe-2(III))]-beta(2) in lysates, similar iron-content in beta(2) immunoprecipitated from 3-AP-treated and untreated [Fe-55]-prelabeled cells, and prolonged (12 h) stability of the inhibited beta(2) are most consistent with Y center dot loss being the predominant mode of inhibition, with beta(2) remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y center dot of the [(Fe-2(III)-Y center dot)(Fe-2(III))] cofactor of beta(2).
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