Cadmium and arsenite binding byN-dihydrolipoyl-aminoethoxydextran: A model study of enzyme dithiol criteria

Gaber, Bruce P.; Fluharty, Arvan L.

doi:10.1016/s0006-3061(00)80157-x

Cited by 13 publications

(8 citation statements)

References 15 publications

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“…Cadmium and zinc differ not only in ionic radii but also in the geometry of their coordination polyhedra (10). Differential affinity for metal ions may reflect differences in local structure around the two mutations (11); for example, the usual order of ligand affinity (Zn2+ > Cd2+) can be reversed in the case of a dithiol ligand (12). Other factors that can influence the metal binding affinity include the electrostatic potential at the binding site, the pKa of the coordinating groups, steric accessibility, and the degree of strain that is introduced into the (17).…”

Section: Methodsmentioning

confidence: 99%

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

Benitah

Tomaselli

Marbán

1996

Proc. Natl. Acad. Sci. U.S.A.

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The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using sitedirected mutagenesis, we introduced pairs of cysteines into the P loops of ,ul rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.The voltage-dependent sodium channel, a protein that underlies excitability in muscle and nerve, is specialized to transport Na+ ions selectively and at high rates (1). This distinctive behavior is imparted by loops that line the channel pore, with one "P" loop being contributed by each of four domains within the a subunit (2). Mutagenesis has pinpointed a number of individual residues that are important determinants of pore properties (3-8). Nevertheless, little is known about the tertiary structure of the pore. Molecular dimensions remain unclear, and the relative orientations of the four P loops are indeterminate. We have introduced pairs of cysteines into the pore to identify residues capable of close-range interactions, as revealed by their ability to form disulfide bonds and high-affinity metal-binding sites. In the gl rat skeletal muscle channel, the first domain mutation Y401C was paired with cysteine mutations in each of the three other P loops. Two remarkable pairings were identified: Y401C+G1530C (spanning domains I and IV) created a high-affinity binding site for cadmium ions, while Y401C+E758C (domains I and II) spontaneously formed an intramolecular disulfide bridge. These residues must be very close to each other within the channel. This generalizable approach can provide unique insights into the threedimensional structure of functional, intact biological proteins. MATERIALS AND METHODSCysteine mutants were made in the Na+ channel a subunit (9) by oligonucleotide-directed mutagenesis and were expressed in Xenopus laevis oocytes as described (8). Whole-cell currents were recorded 2-5 days after injection with a twomicroelectrode voltage clamp at 20 + 2°C. Currents were elicited by 50-ms test pulses from -70 to 20 mV in 5-mV increments from a holding potential of -100 mV, applied every 2 s. The currents were normalized to the maximum current (Imax,) recorded in control solution (ND-96) containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.6). The normalized currents were fitted to a transform of the, where V is the test potential, and the parameters estimated by fit are Vrev, the reversal potential; Gmax, the relative maximal conductance, VO.5, the...

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Section: Methodsmentioning

confidence: 99%

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

Benitah

Tomaselli

Marbán

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Arsenite has a strong affinity for vicinal cysteine-SH groups and it has been suggest that it is the binding of arsenite to such cys-SH groups that subserves its many and diverse pharmacological and toxicological effects (Gaber and Fluharty, 1972;Gitler et al, 2003). In the reporter gene expression experiment illustrated in Figure 4, we tested the effects of dithiol and monothiol compounds in modulating the effects of arsenite in the N-18 mouse neuroblastoma cells.…”

Section: Effect Of Arsenite Requires a Functional Hsf1mentioning

confidence: 99%

“…Central to the biological activity of arsenic is its reactivity to thiol groups (Gaber and Fluharty, 1972;Gitler et al, 2003). A variety of evidence suggests that the toxicological and pharmacological effects of arsenic are likely derived from the interaction of arsenic with vicinal thiol containing proteins.…”

mentioning

confidence: 99%

“…In previous studies, we showed that redoxdependent thiol-disulfide exchange may provide a mechanism regulating the structure and function of heat shock transcription factor 1 ( 1 HSF1) (Manalo and Liu, 2001;Manalo et al, 2002), the transcription factor that mediates the cellular stress response to promote the induction of heat shock protein (HSP) chaperones (Morimoto, 1993;Morimoto et al, 1994;Wu, 1995). Because of the selectivity and avidity of trivalent arsenic for vicinal cysteine-SH (Gaber and Fluharty, 1972;Gitler et al, 2003), we decided to use the sodium arsenite as a probe to further evaluate the role of cysteine in regulating the heat shock transcriptional response. We note that arsenic has been shown to be an effective inducer of HSP expression (Del Razo et al, 2001;Liu et al, 2001;Barnes et al, 2002;Fauconneau et al, 2002), and that induction of the HSP chaperones appear to provide a protective mechanism against oxidative stress, apoptosis, as well as clastogenic and aneugenic activities of arsenic (Barnes et al, 2002;Fauconneau et al, 2002).…”

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confidence: 99%

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Dynamic regulation and involvement of the heat shock transcriptional response in arsenic carcinogenesis

Khalil

Luciano

Chen

et al. 2006

Journal Cellular Physiology

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The objective of this study is to better define induction of the heat shock response by arsenite, and to evaluation if induction of heat shock proteins (HSPs) contributes to the carcinogenic activity of arsenite. We show here that arsenite is a ubiquitous inducer of the heat shock response in mammalian cells: that it activated heat shock transcription factor 1 (HSF1) DNA-binding activity, enhanced hsp 70 promoter, and induced hsp70mRNA and synthesis of HSP chaperones. Using a high throughput hsp70 promoter-luciferase reporter assay, we observed a hormetic dose response where low concentrations of arsenite stimulated and high concentrations inhibited. Further, the response was time-dependent such that with longer times of incubation, the dose response shifted to the left. The effect of arsenite in inducing the hsp 70-luciferase reporter absolutely required a functional HSF1 as it was not observed in HSF1 minus cells but re-instated by expression of HSF1. Consistent with the suggestion that arsenic targets vicinal cysteine-SH, we showed that dithiothreitol blocked the effect of arsenite. Assays of cell viability and caspase showed that arsenite caused a dose-dependent increase in cell death by activation of caspase 3/7 and pre-induction of HSPs blunted these effects. Using anchorage independent cell growth as a late stage tumor promotion assay, we showed that low concentrations of arsenite had a growth promoting effect, which was enhanced by moderate heat shock. Our study provides evidence that induction of the heat shock response is a sensitive biomarker of arsenic exposure and that induction of HSPs likely contributes to the tumor promotion effect of arsenic.

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“…In these precursors, As was bound to proteins, enzymes, etc., mainly as tridentate complexes with the SH À group of cysteine; a configuration that seems to be conserved in the peat structure [25]. The binding of As(III) with thiol is not surprising considering that As(III) is bound to dithiol and trithiol sites in many proteins and peptides [26][27][28][29][30][31][32][33], either completely or partly inhibiting their specific actions in the body.…”

Section: Introductionmentioning

confidence: 99%

Thiol groups controls on arsenite binding by organic matter: New experimental and modeling evidence

Catrouillet

Davranche

Dia

et al. 2015

Journal of Colloid and Interface Science

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Although it has been suggested that several mechanisms can describe the direct binding of As(III) to organic matter (OM), more recently, the thiol functional group of humic acid (HA) was shown to be an important potential binding site for As(III). Isotherm experiments on As(III) sorption to HAs, that have either been grafted with thiol or not, were thus conducted to investigate the preferential As(III) binding sites. There was a low level of binding of As(III) to HA, which was strongly dependent on the abundance of the thiols. Experimental datasets were used to develop a new model (the modified PHREEQC-Model VI), which defines HA as a group of discrete carboxylic, phenolic and thiol sites. Protonation/deprotonation constants were determined for each group of sites (pKA=4.28±0.03; ΔpKA=2.13±0.10; pKB=7.11±0.26; ΔpKB=3.52±0.49; pKS=5.82±0.052; ΔpKS=6.12±0.12 for the carboxylic, phenolic and thiols sites, respectively) from HAs that were either grafted with thiol or not. The pKS value corresponds to that of single thiol-containing organic ligands. Two binding models were tested: the Mono model, which considered that As(III) is bound to the HA thiol site as monodentate complexes, and the Tri model, which considered that As(III) is bound as tridentate complexes. A simulation of the available literature datasets was used to validate the Mono model, with logKMS=2.91±0.04, i.e. the monodentate hypothesis. This study highlighted the importance of thiol groups in OM reactivity and, notably, determined the As(III) concentration bound to OM (considering that Fe is lacking or at least negligible) and was used to develop a model that is able to determine the As(III) concentrations bound to OM.

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Cadmium and arsenite binding byN-dihydrolipoyl-aminoethoxydextran: A model study of enzyme dithiol criteria

Cited by 13 publications

References 15 publications

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

Dynamic regulation and involvement of the heat shock transcriptional response in arsenic carcinogenesis

Thiol groups controls on arsenite binding by organic matter: New experimental and modeling evidence

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