Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1

A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

<div><p>Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys₆₄ and Cys₃₇₇) and the alpha-domain, where Cys₁₂₁ and Cys₄₆₈ are located.</p></div

Parameters of ligand-induced spin shift in CYP3A4 and its unlabeled and double-labeled mutants^*.

<p>*The values given in the table represent the averages of 2–7 individual measurements, and the ± values show the confidence interval calculated for <i>p</i> = 0.05.</p

Interactions of double-labeled CYP3A4 proteins with testosterone monitored by LRET.

<p>Interactions of C377/C468-DY/ER (circles) and C58/C64/C121-ER/DY (triangles) with testosterone monitored by LRET in steady-state (<i>a</i>) and lifetime (<i>b</i>) setup. Panel <i>a</i> shows the changes in the intensity of donor fluorescence observed in titrations of the double-labeled proteins with testosterone. The lines show the results of fitting of the data sets to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083898#pone.0083898.e004" target="_blank">Eq. 4</a>. Panel <i>b</i> shows the phosphorescence decay traces recorded in the absence of added ligand (open symbols) and in the presence of 100 µM testosterone (closed symbols)</p

Cross-correlation between inter-residue distances in a set of CYP3A4 X-ray structures.

<p>Cross-correlation between the distances between the side chains in Cys₆₄/Cys₄₆₈, Cys₃₇₇/Cys₄₆₈ and Cys₆₄/Ala₁₂₁ pairs of residues observed in a set of 13 structures of CYP3A4. The distances were calculated between the sulfur atoms of cysteines or beta carbon atom of alanine. The lines represent the results of linear regression of these sets, which are characterized by the square correlation coefficients of 0.50, 0.81 and 0.62 for the plots shown in panels <b><i>a</i></b>, <b><i>b</i></b> and <b><i>c</i></b> respectively.</p

Titration of double-labeled derivatives of C64/C468 with testosterone monitored by steady-state fluorescence spectroscopy.

<p>(<i>a</i>) A series of the spectra of delayed emission of C64/C468-ER/DY recorded at increasing concentrations of testosterone; (<i>b</i>) Changes in the intensity of erythrosine phosphorescence in the titrations of C64/C468-ER/DY (circles) and C64/C468-DY/ER (triangles) with testosterone. The solid line represents the results of fitting of the data set with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083898#pone.0083898.e004" target="_blank">Eq. 4</a>.</p

Spectra of delayed emission and the erythrosine phosphorescence decay traces of labeled derivatives of CYP3A4.

<p>Panel <b><i>a</i></b> shows the decay traces obtained with the double labeled C64/C468-ER/DY (black), C64/C468-DY/ER (red), C64/C121-DY/ER (green), C377/C468-DY/ER (blue),and the single-labeled CYP3A4(C468)-ERIA (magenta). Spectra of delayed emission shown in panel <b><i>b</i></b> represent C64/C468-ER/DY (black), C64/C468-DY/ER (red), C64/C121-DY/ER (green) and C377/C468-DY/ER (blue).</p

Spectra of absorbance and delayed fluorescence of the double-labeled C64/C468-ER/DY protein.

<p>Absorbance and fluorescence spectra of C64/C468-ER/DY are shown in solid black and solid blue lines respectively. Spectrum of delayed fluorescence of CYP3A4(C468)-ERIA is shown in blue dashed line.</p

Titration of double-labeled derivatives of C64/C468-ER/DY with ligands monitored by lifetime measurements.

<p>(<i>a</i>) Phosphorescence decay traces obtained with CYP3A4(C468)-ERIA (circles) and C64/C468-ER/DY at no substrate added (open triangles) and in the presence of 100 µM testosterone (closed triangles). Solid lines represent the bi-exponential approximations (ρ²>0.999). (b) Dependence of the average lifetime of phosphorescence <τ_d> in C64/C468-ER/DY on the concentration cholesterol (circles) and ANF (triangles). The lines show the results of fitting of the data sets to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083898#pone.0083898.e004" target="_blank">Eq. 4.</a></p