Arsenic(III) species inhibit oxidative protein folding in vitro

Abstract

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.

FIGURE 1

Three arsenicals and their reactions with di- and tri-thiol motifs. Panel A shows the fully protonated forms of the As(III) species used in this work. Panels B and C depict the reactions of alkyl/aryl As(III) species and of arsenous acid with di- and trithiols respectively.

FIGURE 2

As(III) species inhibit oxidative protein folding of riboflavin binding protein. Panel A shows the oxidative folding system for reduced RfBP. QSOX (30 nM) introduces disulfide bonds into the reduced unfolded protein, and reduced PDI (30 μM) isomerizes incorrectly paired disulfide bridges. The fluorescence of riboflavin (0.8 μM) is quenched on binding to the native apo-RfBP allowing oxidative folding to be followed continuously (control). Panel B and C depict the inhibition of oxidative folding by 10 μM arsenite (green), MMA (pink) and PSAO (blue) in the absence or presence of 5 mM GSH, respectively. Red curves are controls in the absence of arsenicals. The starting fluorescence is set arbitrarily to 100 percent in these experiments.

FIGURE 3

Binding of PSAO to reduced PDI and the effect of As(III) species on the reductase activity of PDI. Panel A represents the changes in fluorescence emission intensity at 337 nm (exciting at 290 nm) on the addition of PSAO to 1 μM reduced human PDI (2 μM binding sites). The data were fit (solid line) to a K_d of 1.1 μM (see Experimental Procedures). The inset to panel A shows selected emission spectra (corresponding to 0, 2, 4, and 10 μM PSAO). Panel B: 10 μM arsenite (open square), MMA (cross) or PSAO (closed diamond) do not appreciably inhibit the activity of PDI in the insulin reduction assay (closed squares; in 50 mM potassium phosphate, pH 7.5, containing 1 mM EDTA, 5 mM GSH, and 100 μM insulin). For comparison, the traces labeled 125 and 500 nM PDI are controls in the absence of arsenicals (open diamond and circles respectively).

FIGURE 4

Absorbance and fluorescence measurements of the binding of As(III) species to reduced RfBP. Panel A: a solution of reduced RfBP (5 μM in 50 mM phosphate buffer, pH 7.5, 25°C; see Methods) was titrated with the arsenicals and followed by absorbance increase at 300 nm (PSAO: blue) or 252 nm (arsenite and MMA: green and pink respectively). Dashed lines are drawn at 6 and 9 equivalents per RfBP. Panel B: MMA quenches the fluorescence emission of 5 μM reduced RfBP (exciting at 295 nm; emission 344 nm) in the absence of 5 mM GSH. The inset compares fluorescence quenching with or without GSH (open and closed symbols respectively). The absence of an inner-filter effect was confirmed by repeating the titration using 1 μM RfBP (not shown). Panel C: corresponding fluorescence titrations for arsenite (green) and PSAO (blue).

FIGURE 5

Reactivity of the cysteine residues of reduced RfBP towards DTNB. The reduced protein (5 μM, 90 μM thiols with or without 50 μM arsenite or MMA) was mixed with an equal volume of 4 mM DTNB in phosphate buffer, pH 7.5, 25°C. The increase in absorbance at 412 nm was followed in the stopped-flow. The initial absorbance readings of about 0.38 reflect background DTNB absorbance.

FIGURE 6

Reduced avian lysozyme binds As(III) species. Panel A: PSAO titrations of 5 μM protein (in 100 mM Tris buffer, pH 7.5, containing 100 mM NaCl, 1 mM EDTA and 3 M urea) followed by absorbance (closed squares) and fluorescence (excitation 295 nm; emission 348 nm) in the absence and presence of 5 mM GSH (closed and open squares, respectively). Panel B: regain of enzyme activity during oxidative refolding of 2 μM reduced lysozyme (closed squares; in the same buffer adjusted to pH 8.0 plus 1 mM GSH and 0.2 mM GSSH) is impaired by 10 μM arsenite (open squares).

FIGURE 7

Arsenicals and reduced RNase. Panels A and B: arsenite and PSAO binding to 5 μM reduced RNase measured by absorbance (closed squares) and fluorescence emission (excitation 276 nm; emission 303 nm) in the absence and presence of 5 mM GSH (closed and open circles, respectively). Panel C: sub-stoichiometric concentrations of MMA (here 10 μM) cause the aggregation of 50 μM reduced RNase. The precipitate was collected and found to be formed of monodisperse fibrils by transmission electron microscopy (see Experimental Procedures).

FIGURE 8

Schematic depiction of the oxidative folding of a 14-cysteine containing protein and outcomes of concurrent exposure to a monoalkyl/aryl arsenic (III) species. Forms A and B are unfolded reduced and native 7-disulfide folded protein respectively. Capture of A before disulfide bond formation generates species C with a full complement of bound arsenical. D represents a hybrid protein containing both arsenical and disulfide crosslinks. E depicts arsenic-induced formation of ordered aggregates following the precedent set in Figure 7C.