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

Abstract

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.

Figure 1

Arsenicals and their coordination to sulfhydryl species. (A) Some of the monoarsenicals discussed in this work. (B) Coordination of arsonous acid to a trithiol containing motif. (C) Coordination of an alkyl or aryl arsenical to a dithiol. (D) Structure of the bis- and tris-arsenicals used in this work. BA-2 consists of a mixture of regioisomers (see the text).

Figure 2

Oxidative protein folding catalyzed by QSOX and reduced PDI. (A) An assay for oxidative folding used in this work. QSOX inserts disulfides into reduced RfBP. Mispaired disulfides are corrected iteratively by PDI, and the fluorescence of free riboflavin is quenched on binding to active apo-RfBP. (B) Structure of an open conformation of QSOX from Trypanosoma brucei. CxxC motifs in the thioredoxin (blue) and ERV (green) domains are shown by solid yellow spheres. These CxxC motifs are brought together during catalysis by a large-scale rotation involving a flexible interdomain linking region (dashed line). Vertebrate QSOXs appear to be mechanistically identical, although they have an additional redox-inactive thioredoxin domain of unknown function. (C) The two CxxC motifs in the a and a′ domains in one (of multiple) conformation of human PDI (PDB 4EL1) are highlighted.

Figure 3

Inhibition of the oxidative folding of reduced RfBP in the presence of bis- and tris-arsenicals. (A) Oxidative protein folding monitored by the loss of riboflavin fluorescence as apo-RfBP is generated by the combined action of 30 nM QSOX and 30 μM reduced PDI in aerobic buffer, pH 7.5 (see Materials and Methods). BA-1, BA-2, and TA-1 (at an aggregate As(III) concentration of 10 μM) strongly suppress oxidative folding under these conditions. (B) Extent of riboflavin binding as a percentage of that observed in the control recorded at 60 min in the absence of arsenical. Data for PSAO and MMA are taken from Ramadan et al. These experiments were then repeated with the additional presence of 5 mM GSH, and the data are summarized in panel B.

Figure 4

Inhibition of the oxidative refolding of RNase by arsenicals. (A) Schematic representation of the refolding of RNase in the presence of a redox buffer and PDI. Reduced and denatured RNase (10 μM; 80 μM thiols) was incubated with 5 μM reduced PDI and a redox buffer containing either 1 mM GSH and 0.2 mM GSSG (B) or 5 mM GSH and 1 mM GSSG (C). The colors used are as follows: control (no arsenical), black; 5 μM BA-1, blue; 5 μM BA-2, pink; 3.33 μM TA-1, green; and 10 μM PSAO, gray.

Figure 5

Inhibition of QSOX reactivity by bis- and tris-arsenicals. The activity of 30 nM avian QSOX was evaluated in the oxygen electrode assay (see Materials and Methods) 100–400 s after the addition of enzyme to a solution containing either 5 mM GSH (left) or TCEP (right) as substrates and 5 μM BA-1 and BA-2 and 3.33 μM TA-1. Data are expressed as percentages of a control assayed with either GSH or TCEP (left bar).

Figure 6

Effect of multivalent arsenicals on the reductase activity of PDI. (A) Schematic of the assay. Porcine insulin (50 μM) in 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA) was mixed with either 100 μM TCEP (B) or 5 mM GSH (C) in the absence or presence of 10 μM of As(III) in BA-1, BA-2, and TA-1. Time zero corresponds to the addition of 1 μM PDI (see Materials and Methods). The onset of turbidity was observed at 600 nm and is expressed in bar graph form in panels D and E (for B and C, respectively; see Materials and Methods).

Figure 7

Interaction between BA-1 and reduced PDI. The main figure shows a spectrophotometric determination of the net stoichiometry of binding of BA-1 to 10 μM reduced PDI (see Materials and Methods). The inset repeats the titration using 1 μM reduced PDI to provide an estimate of the dissociation constant. The solid curve is fit to a K_d of 54 nM ± 29 nM with a stoichiometry of 0.90 ± 0.08 molecules of the bis-arsenical BA-1 (see Materials and Methods).

Figure 8

Titration of reduced RfBP with multivalent arsenicals. Reduced RfBP was prepared as in Materials and Methods, and 1 μM of the protein (18 μM thiols) was titrated with increasing concentration of the arsenicals. The increase in absorbance at 300 nm was recorded 20 min after each addition for BA-1, BA-2, and TA-1. All data sets gave sharp end points shown by the dotted lines. If all arsenical moieties in these MVAs were captured by the 18 −SH groups in reduced RfBP, then the stoichiometry for the bis-arsenical would be 4.5, and that for the tris-arsenical, 3.0.

Figure 9

Monitoring MVA-induced aggregation of reduced RNase with Thioflavin T and Congo Red. Reduced RNase (30 μM) was mixed with 5 μM thioflavin T dye and MVAs in the absence (A) or presence (B) of 5 mM GSH. The increase in fluorescence was monitored over time (exciting at 450 nm with emission at 485 nm; see Materials and Methods). No significant increase in fluorescence was observed without the inclusion of the multivalent arsenicals in both cases. Control (no arsenical), black squares; 5 μM BA-1, pink circles; 5 μM BA-2, blue triangles; and 3.33 μM TA-1, green diamonds. (C, D) Congo Red spectral shift assay using 20 μM reduced RNase and 10 μM Congo Red dye and MVAs after 30 min in 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA in the absence (C) or presence of 5 mM GSH (D). Control (no arsenical), black; 5 μM BA-1, pink; 5 μM BA-2, blue; and 3.33 μM TA-1, green.

Figure 10

Simulated interaction of MMA with dithiols in the presence of competing GSH. (A) Complexation between 10 μM MMA and 10 μM dithiols (green colored region) and the competing coordination of MMA by 5 mM GSH (blue area; K_d values were calculated from ref (6)). (B) Illustration of how the avidity of dithiol binding to MMA influences the percentage of arsenical complexed with dithiol in competition with binding to GSH at 5 mM.