Identification of arsenic-binding proteins in human breast cancer cells.
As a cancer chemotherapeutic drug, arsenic acts on numerous intracellular signal transduction pathways in cancer cells. However, its mechanism of actions is still not fully understood. Previous studies suggest that arsenic reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. In this study, human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with p-aminophenylarsenoxide. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, beta-tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of beta-tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys(12) alone or vicinal Cys residues (Cys(12) and Cys(213)) of beta-tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. On the contrary, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed beta-tubulin and PKM2 as arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins.
Zhang
Xinyan
X
Cancer Research Program, Julius L Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA.
Yang
Fan
F
Shim
Joong-Youn
JY
Kirk
Kenneth L
KL
Anderson
D Eric
DE
Chen
Xiaoxin
X
D000970:Antineoplastic Agents / Q000494:pharmacology; D001151:Arsenic / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D001943:Breast Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D014404:Tubulin; D001710:Biotin; D011770:Pyruvate Kinase; D003545:Cysteine; D001151:Arsenic