Arresting amyloid with coulomb's law: acetylation of ALS-linked SOD1 by aspirin impedes aggregation.

Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzyme's Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.

Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations.

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.

Paramagnetic 19F NMR and Electrospray Ionization Mass Spectrometric Studies of Substituted Pyridine Complexes of Chromium(III): Models for Potential Use of 19F NMR to Probe Cr(III)-Nucleotide Interaction.

The synthesis and characterization of chromium basic carboxylate complexes, [Cr3(O2CR)6L3]+, containing trifluoroacetate, 3-fluoropyridine, 3-trifluoromethylpyridine, and 4-trifluoromethylpyridine are described. The substituted pyridine ligands are used as models of DNA bases to determine whether 19F NMR would be a potentially useful probe of the binding of Cr3+ to DNA. The 19F NMR resonances of the coordinated ligands, while broadened by delocalization of unpaired electron density from the S=3/2 chromic centers, are readily discernable, and the contact shifts are of sufficient magnitude that the signals from coordinated and free ligands can easily be differentiated. Thus, 19F NMR appears to be a potentially useful probe of the binding of Cr3+ to DNA containing F-labeled bases. Additionally, electrospray MS is shown to be a convenient method to establish the identity of chromium basic carboxylate assemblies.

Chromium is not an essential trace element for mammals: effects of a "low-chromium" diet.

Chromium was proposed to be an essential trace element over 50 years ago and has been accepted as an essential element for over 30 years. However, the studies on which chromium's status are based are methodologically flawed. Whether chromium is an essential element has been examined for the first time in carefully controlled metal-free conditions using a series of purified diets containing various chromium contents. Male Zucker lean rats were housed in specially designed metal-free cages for 6 months and fed the AIN-93G diet with no added chromium in the mineral mix component of the diet, the standard AIN-93G diet, the standard AIN-93G diet supplemented with 200 µg Cr/kg, or the standard AIN-93G diet supplemented with 1,000 µg Cr/kg. The chromium content of the diet had no effect on body mass or food intake. Similarly, the chromium content of the diet had no effect on glucose levels in glucose tolerance or insulin tolerance tests. However, a distinct trend toward lower insulin levels under the curve after a glucose challenge was observed with increasing chromium content in the diet; rats on the supplemented AIN-93G diets had significantly lower areas (P < 0.05) than rats on the low-chromium diet. The studies reveal that a diet with as little chromium as reasonably possible had no effect on body composition, glucose metabolism, or insulin sensitivity compared with a chromium-"sufficient" diet. Together with the results of other recent studies, these results clearly indicate that chromium can no longer be considered an essential element.

Comparison of tissue metal concentrations in Zucker lean, Zucker obese, and Zucker diabetic fatty rats and the effects of chromium supplementation on tissue metal concentrations.

Diabetes results in several metabolic changes, including alterations in the transport, distribution, excretion, and accumulation of metals. While changes have been examined in several rat models of insulin resistance and diabetes, the metal ion concentrations in the tissues of Zucker lean, Zucker obese (an insulin resistance and early stage diabetes model), and Zucker diabetic fatty (ZDF, a type 2 diabetes model) have not previously been examined in detail. The concentration of Cu, Zn, Fe, Mg, and Ca were examined in the liver, kidney, heart and spleen, and Cr concentration in the liver and kidney of these rats were examined. Zucker obese rats have a reduction in the concentration of Cu, Zn, Fe, Mg in the liver compared to ZDF and/or lean Zucker rats, presumably as a result of the increased fat content of the liver of the obese rats. ZDF rats have increased concentrations of kidney Cu compared to the lean rats, while kidney Ca concentrations are increased in the Zucker obese rats. Spleen Fe concentrations are decreased in Zucker obese rats compared to the lean rats. No effects on metal concentrations in the heart were observed between the lean, obese, and ZDF rats, and no effects on Cr concentrations were identified. Cr(III) complexes have previously been shown to have beneficial effects on the signs of insulin resistance in Zucker obese and ZDF rats. The effects of daily gavage administration of chromium picolinate ([Cr(pic)(3)]) (1 mg Cr/kg body mass), CrCl(3) (1 mg Cr/kg body mass), and Cr3 ([Cr(3)O(propionate)(6)(H(2)O)(3)](+)) (33 µg and 1 mg Cr/kg body mass) on metal concentrations in these tissues were examined. Treatment with CrCl(3) and Cr3, but not [Cr(pic)(3)], at 1 mg Cr/kg resulted in a statistically significant accumulation of Cr in the kidney of lean and obese but not ZDF rats but resulted in lowering the elevated levels of kidney Cu in ZDF rats, suggesting a beneficial effect on this symptom of type 2 diabetes.

Monocarboxylate transporters are not responsible for Cr(3+) transport from endosomes.

Cr(3+), similar to Fe(3+), is transported into cells primarily via endocytosis as the metal-transferrin complex. As Cr(3+) ions are not readily reduced under biological conditions, the ion cannot be transported from endosomes by the same mechanism as iron that utilized divalent metal ion transporters. Cr(3+) has been hypothesized to potentially be transported as small ligand complexes with a free carboxylate functionality by monocarboxylate transporters (MCT), in a similar fashion to that proposed for Al(3+). Consequently, mouse C2C12 muscle cells were utilized to determine if Cr(3+) is potentially transported by MCT by examining the effects of MCT inhibitors on Cr and Fe transport and subcellular distribution when the metals are added as their transferrin complexes. The results suggest that Cr is not primarily transported by MCT from the endosomes to the cytosol, and that another mechanism for this transport needs to be identified.

Potential of chromium(III) picolinate for reproductive or developmental toxicity following exposure of male CD-1 mice prior to mating.

Chromium(III) picolinate, [Cr(pic)(3)], is a commonly used nutritional supplement in humans, which has also been approved for use in animals. Health concerns have arisen over the use of [Cr(pic)(3)]. At high [Cr(pic)(3)] doses, developmental toxicity tests in female mice have shown a higher litter incidence of split cervical arch in exposed fetuses, but this was not consistently reproducible. In the current study, male CD-1 mice were used to further assess the potential for reproductive or developmental toxicity. Four weeks prior to mating, the males were fed a diet providing 200 mg/kg/day [Cr(pic)(3)] for comparison with untreated controls. Females were not treated. Each male was mated with two females, which were sacrificed on gestation day 17, and their litters were examined for adverse effects. Mating and fertility indices were not significantly altered by treatment. Male exposure to [Cr(pic)(3)] also had no effect on prenatal mortality, fetal weight, or gross or skeletal morphology. These results suggest that paternal dietary exposure to chromium(III) picolinate has little potential for adverse reproductive effects, even at exposure levels considerably higher than expected human exposures from nutritional supplements (1 mg of Cr per day or less).

Urinary chromium loss associated with diabetes is offset by increases in absorption.

The results of the current study indicate that diabetic rats have increased urinary Cr loss as a result of their diabetes; however, this increased urinary Cr loss is offset by increased absorption of Cr. Insulin resistant, obese rats have alterations in the rates of Cr transport and distribution compared to lean rats but have similar levels of urinary Cr loss and Cr absorption. Thus, any increases in urinary Cr loss associated with insulin resistance or diabetes are offset by increased absorption. Given that dietary chromium is normally absorbed with only approximately 1% efficiency, suitable Cr exists in the diet so that a standard diet possesses sufficient chromium to allow for the increases in absorption associated with diabetes. Consequently, supplementing the diet with nutritionally relevant quantities of chromium is not anticipated to have any beneficial effects. Similarly, beneficial effects on plasma variables, such as cholesterol, triglycerides, and insulin concentrations, from supra-nutritional doses of Cr(III) complexes should not arise from alleviation of chromium deficiency. These beneficial effects must arise from pharmacological effects of high dose Cr(III) administration.

Mass spectrometric and spectroscopic studies of the nutritional supplement chromium(III) nicotinate.

Despite chromium nicotinate's popular use as a chromium nutritional supplement, the structure and composition of chromium nicotinate have only been poorly described. As solid chromium nicotinate is intractable, being insoluble or unstable in common solvents, studies on the solid have been limited, and studies of the solution from which the "compound" precipitates have additionally provided little additional data. The results of mass spectrometric and spectroscopic investigations designed to further elucidate the structure and composition of chromium nicotinate are described. The results demonstrated that the three common methods for producing "chromium nicotinate" all yield different compounds, all of which are polymers of Cr(III), oxygen-bound nicotinate, hydroxide, and water. Implications for interpreting results of nutritional studies of "chromium nicotinate" are discussed.