Multivalency in the inhibition of oxidative protein folding by arsenic(III) species.

The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein -SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ∼50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function.

Arsenic(III) species inhibit oxidative protein folding in vitro.

The success of arsenic trioxide in the treatment of acute promyelocytic leukemia has renewed interest in the cellular targets of As(III) species. The effects of arsenicals are usually attributed to their ability to bind vicinal thiols or thiol selenols in prefolded proteins thereby compromising cellular function. The present studies suggest an additional, more pleiotropic, contribution to the biological effects of arsenicals. As(III) species, by avid coordination to the cysteine residues of unfolded reduced proteins, can compromise protein folding pathways. Three representative As(III) compounds (arsenite, monomethylarsenous acid (MMA), and an aryl arsenical (PSAO)) have been tested with three reduced secreted proteins (lysozyme, ribonuclease A, and riboflavin binding protein (RfBP)). Using absorbance, fluorescence, and pre-steady-state methods, we show that arsenicals bind tightly to low micromolar concentrations of these unfolded proteins with stoichiometries of 1 As(III) per 2 thiols for MMA and PSAO and 1 As(III) for every 3 thiols with arsenite. Arsenicals, at 10 microM, strongly disrupt the oxidative folding of RfBP even in the presence of 5 mM reduced glutathione, a competing ligand for As(III) species. MMA catalyzes the formation of amyloid-like monodisperse fibrils using reduced RNase. These in vitro data show that As(III) species can slow, or even derail, protein folding pathways. In vivo, the propensity of As(III) species to bind to unfolded cysteine-containing proteins may contribute to oxidative and protein folding stresses that are prominent features of the cellular response to arsenic exposure.

Effects of As(III) binding on beta-hairpin structure.

While arsenic(III) compounds can exert profound toxicological and pharmacological effects, their modes of action and, in particular, the structural consequences of their binding to cysteinyl side chains in proteins, remain poorly understood. To gain an understanding of how arsenic binding influences beta-structure, pairs of cysteines were introduced into a model monomeric beta-hairpin to yield a family of peptides such that coordination occurs either across the strands or within the same strand of the beta-hairpin. Circular dichroism, NMR, UV-vis spectroscopy, and rapid-reaction studies were used to characterize the binding of monomethylarsonous acid or p-succinylamidephenyl arsenoxide (PSAO) to these peptides. Placement of cysteines at non-hydrogen bond (NHB) positions across the beta-hairpin, such that they occupy the same face of the sheet, was found to enhance the structure as assessed by CD. Cross-strand cysteine residues that project on opposite faces close to the termini of the hairpin can still bind arsenic tightly and show modestly increased beta-sheet content. NMR and modeling studies suggest that arsenic can be accommodated at this locus without disrupting the core interactions stabilizing the turn. However, As(III) binding to nonopposed cysteines, or to cysteines at HB and NHB positions along one strand of the hairpin, caused loss of structure. UV-vis titrations show that all these hairpin peptides bind PSAO stoichiometrically with K(d) values from 13 to 106 nM. Further, binding is moderately rapid, with second-order rate constants for association of 10,000-22,000 M(-1) s(-)1 irrespective of the placement of the cysteines within the hairpin and the consequent extent of structural reorganization required as a result of binding. These studies complement recent work with alpha-helices and further demonstrate that capture of a pair of thiols by As(III) may result in significant changes in local secondary structure in the protein targets of these potent bioactive agents.