Identification of arsenic-binding proteins in human breast cancer cells

Abstract

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.

Trivalent Arsenic Inhibits the Functions of Chaperonin Complex

“…In particular, As(III) was shown to directly bind to b-tubulin isolated from a human breast cancer cell line and structural modeling suggested the amino acid residue Cys12 is key to such a physical interaction (Zhang et al 2007). This model of arsenic action would be consistent with most of Table S3.…”

Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic

“…Arsenic bound to five of the key enzymes involved in glycolysis, i.e., hexokinase, aldolase, triose phosphate isomerase, GAPDH, and phosphoglycerate kinase. A previous study (10) identified another set of four enzymes involved in glycolysis as targets of arsenic, i.e., PKM2, enolase, pyruvate carboxylase, and L-lactate dehydrogenase B chain. Thus it is clear that the majority of the enzymes in the glycolysis pathway can be targeted directly or indirectly by arsenic.…”

“…1A). Briefly, the arsenic-biotin conjugate, biotinylated p-aminophenylarsenoxide (Biotin-As) (10), was incubated with the human proteome microarray, and proteins with arsenic-binding capacity were identified by adding Cy3-conjugated streptavidin (Cy3-SA). To avoid false-positive detections, we compared this sample with another in which Biotin-As was omitted from the reaction and free biotin was included instead and identified as arsenic-binding candidate proteins those exhibiting markedly greater signals in the former reaction.…”

“…Knowledge of ATO's binding partners is ultimately crucial for any mechanistic understanding of its effects at the cellular level. To date, about 20 validated ATO-binding human proteins have been reported (10), but inspection of their known functions suggests that they cannot account for the wide and profound effects of ATO on cancer cells. We thus hypothesized that more physiologically relevant ATO-binding proteins remain to be discovered.…”

“…Biotin-As was synthesized as described (10). Proteome microarrays were blocked with blocking buffer (1% BSA in 0.1% Tween 20; TBST) for 1 h at room temperature with gentle agitation.…”