Tracking and characterization of arsenic(III)-binding proteins : insight into the molecular mechanism of action of arsenic trixide for leukemia

Abstract

Arsenic compounds have been extensively utilized in traditional Chinese medical recipes for over 2000 years. In particular, arsenic trioxide (ATO) has been proved to be the active component and has been actively used for the treatment of Acute Promyelocytic Leukemia (APL) in clinical. Really Interesting New Genes(RING) motifs of oncoprotein have been proposed as the potential target of arsenic compounds in treating chronic myelogenous leukemia, yet the mechanism of action of ATO has not been fully revealed. A systematic study on tracking the potential protein targets of arsenic(III) compounds and elucidating the mode of arsenic(III) binding on proteins was therefore conducted in live leukemia cells to improve the understanding on the mechanism of action of this therapeutic agent. To reveal the mode of action and explore the application of this arsenic-based drug, the interaction of arsenic(III) compounds and RING domain of X-linked inhibitor of apoptosis protein (XIAP) was investigated in solution. The results revealed that RING protein was natively bound with two zinc ions while it existed as a dimer; upon the introduction of arsenic(III), the protein dimer dissociated into monomers, which affected its normal ubiquitin ligase function. The effect of arsenic(III) species on disrupting the RING structure was further studied and the modeling of As-RING complex structures proved the arsenic(III) species exerts its effect via binding on the native dimeric interface. To track the arsenic binding proteins in living cells, a novel organoarsenic fluorescent probe As-AC(C20H17AsN4O3S2) was designed by conjugating an arsenic-chelating moiety to a coumarine fluorophore reporter with an arylazide functionality. The probe bound specifically to cysteine residues while a covalent linkage was formed by photoactivation of the arylazide, leading to an 8-fold fluorescence enhancement. The As-AC probe showed high selectivity to free cysteine-containing proteins such as human BIR3 and SlyD proteins, in contrast to the cysteine-free proteins such as SlyD CmutA, SlyDΔC and ubiquitin proteins. Two leukemia cell lines, NB4 and HL60, were incubated with As-AC and 69 arsenic binding proteins were identified. Apart from 22 mutual proteins, proteins identified were mainly involved in enzyme-binding for NB4 and RNA processing for HL60. The proteins identified in NB4 mainly localized at specific locations such as exosome and myelin sheath in accordance to GO analysis, which could possibly be attributed to its high sensitivity towards arsenic. Nucleophosmin (NPM) protein was found in both cell lines. The binding of arsenic(III) with NPM proteins was further confirmed by cellular thermal shift assay. The As-AC probe approach was studied and the proteins identified were compared with those tracked by an arsenic-immobilized column approach. Under comparison, the As-AC probe preferentially tracked more acidic proteins and this approach was comparatively suitable for protein mining with a relatively broad range of molecular weight. Proteins identified in overall could be categorized into five functional groups including cellular homeostasis, metabolic process, cell cycle process, skeleton proteins and RNA processing. This study offers new method in mining the putative arsenic binding proteins in leukemia cells and could extend the application of arsenic-based chemotherapeutic compounds.