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Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

ABSTRACT: The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (HsPBGS, a.k.a. 5-aminolevulinate dehydratase, ALAD) functions in heme biosynthesis. HsPBGS catalytic activity is regulated allosterically via an equilibrium of inactive hexamers and active octamers, and we have shown that certain drugs and drug-like small molecules can inhibit HsPBGS in vitro by stabilizing the hexamer. Here we address whether components of the National Toxicology Program library of environmental contaminants can stabilize the HsPBGS hexamer and inhibit activity in vitro. Native polyacrylamide gel electrophoresis was used to screen the library (1,408 compounds) for components that alter the oligomeric distribution of HsPBGS. Freshly purchased samples of 37 preliminary hits were used to confirm the electrophoretic results and to determine the dose-dependence of the perturbation of oligomeric distribution. Seventeen compounds were identified which alter the oligomeric distribution toward the hexamer and also inhibit HsPBGS catalytic activity, including the most potent HsPBGS inhibitor yet characterized (Mutagen X, IC50 = 1.4 ?M). PBGS dysfunction is associated with the inborn error of metabolism know as ALAD porphyria and with lead poisoning. The identified hexamer-stabilizing inhibitors could potentiate these diseases. Allosteric regulation of activity via an equilibrium of alternate oligomers has been proposed for many proteins. Based on the precedent set herein, perturbation of these oligomeric equilibria by small molecules (such as environmental contaminants) can be considered as a mechanism of toxicity.

SUBMITTER: Lawrence SH

PROVIDER: S-EPMC4102012 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

Lawrence Sarah H SH Selwood Trevor T Jaffe Eileen K EK

Current chemical biology 20130101 2

The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (<i>Hs</i>PBGS, a.k.a. 5-aminolevulinate dehydratase, ALAD) functions in heme biosynthesis. <i>Hs</i>PBGS catalytic activity is regulated allosterically via an equilibrium of inactive hexamers and active octamers, and we have shown that certain drugs and drug-like small molec ...[more]

PMID: 25045409

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Project description:Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood-testis barrier (BTB)?Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function.Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo.We examined the effects of two environmental toxicants: cadmium chloride (0.5-20 µM) and bisphenol A (0.4-200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period.Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA).Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics, endocytic vesicle-mediated trafficking and the proper localization of actin regulatory proteins c-Src and annexin II in Sertoli cells were also affected. Results of statistical analysis demonstrate that these findings were not obtained by chance.(i) This study was done in vitro and might not extrapolate to the in vivo state, (ii) conclusions are based on the use of Sertoli cell samples from three men and (iii) it is uncertain if the concentrations of toxicants used in the experiments are reached in vivo.Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction.

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Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

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Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

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