Article pubs.acs.org/crt
Aluminum-Induced Kinesin Inactivation as Potential Molecular Cause of Impairment of Neuronal Transport Processes Konrad J. Böhm,* Mitra Shabanpour, and Nikolina Kalchishkova Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Beutenbergstraße 11, D-07745 Jena, Germany ABSTRACT: It is commonly accepted that aluminum ions may initiate the development of diverse diseases, including neurological disorders. So far, our knowledge of the molecular mechanisms of the interaction of aluminum with defined cellular structures has been still fragmentary. As functional key tasks of neuronal cells essentially depend on the activity of kinesin, we wanted to find out whether this motor protein represents a molecular target for aluminum. We demonstrate that aluminum ions inhibit (IC50 ∼50 μM) the ATPase of the neuron-specific kinesin KIF5A. The ATPase-active center itself, which is located in the kinesin motor domain, does not seem to be directly affected by aluminum. Our results suggest that inhibition is preferentially caused by aluminum binding to some sequence within the kinesin stalk leading to a conformational state of the kinesin molecule, similar to those described in cases of kinesin autoinhibition caused by motor domain-tail binding. Because of the relative high sequence conservation of mammalian kinesin-1 (to which KIF5A belongs), we assume that also in non-neuronal cells the intracellular transport can be affected by aluminum ions.
1. INTRODUCTION Aluminum is the most abundant metal in the earth crust. It is a highly reactive element, which occurs as oxides or salts rather than pure metal. Aluminum has attained great importance not only in industrial applications but also in practically all spheres of daily life. So, it is being widely used in cooking and storage utensils, including household foils. Moreover, aluminum compounds are found in vaccine adjuvants,1 pharmacological drugs, like antacids, and in antiperspirants.2 Also, numerous food products3 and drinking water4 can be contaminated by aluminum. Hence, there are several sources from which aluminum compounds can enter the human organism. Nowadays, it is commonly accepted that aluminum is not as harmless as assumed in the past but that it may have adverse effects on human health, triggering anemia, osteomalacia, hepatic complaints, and neurological disorders.1,5,6 It has been reported that aluminum can accumulate within the brain during life up to levels of about 0.7 μg per gram (wet weight).7 Because of their longevity, neuronal cells are considered to be predisposed to be affected by metal ions, including aluminum. Especially within the past decade, there has been an increasing number of reports considering aluminum to be a severe neurotoxin,8,9 which contributes to the pathogenesis of Alzheimer’s disease and other neurodegenerative neuronal diseases.2,8,10−14 In spite of accumulating evidence in this research area, the molecular mechanisms underlying aluminum-induced neurotoxicity have been poorly elucidated.15 Functional key tasks within neuronal cells essentially depend on motor protein-driven transport events, whereby the kinesinmicrotubule motility-generating system plays a central role. This system is mainly involved in the long-distance anterograde © 2015 American Chemical Society
transport of different intracellular cargos into distal compartments of axons and dendrites.16 It has been concluded that mutations are responsible for the impairment of kinesin function and finally for the development of neuronal disorders.16−18 In addition, also chemical attacks onto kinesin and/or microtubules or any toxic intervention into the interplay between them seem to be potential causes of triggering cellular transport dysfunctions. In the present article, we demonstrate that the ATPase of the neuron-specific kinesin KIF5A is strongly inhibited by aluminum ions, suggesting that direct attacks of aluminum onto the kinesinmicrotubule transport system might initiate the development of neuronal diseases.
2. MATERIALS AND METHODS 2.1. Isolation of Microtubule Protein and Microtubule Formation. Tubulin, accompanied by about 15% MAP, was purified from porcine brain through temperature-dependent disassembly/ reassembly cycles,19,20 using a buffer containing 20 mM PIPES (pH 6.8), 80 mM NaCl, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM DTT. To obtain pure tubulin, the MAPs were removed by phosphocellulose column chromatography.21 For microtubule formation, protein stocks, stored at −80 °C, were diluted in buffer lacking EGTA and DTT, transferred to glass cuvettes, and supplemented with GTP and aluminum chloride (Sigma-Aldrich Chemie GmbH, Germany, anhydrous, 99.999% trace metals basis; final concentrations as indicated in Results and Discussion). The kinetics of microtubule formation was recorded at 37 °C by turbidity measurement at 360 nm.22,23 Received: February 18, 2015 Published: April 14, 2015 1275
DOI: 10.1021/acs.chemrestox.5b00077 Chem. Res. Toxicol. 2015, 28, 1275−1281
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Chemical Research in Toxicology 2.2. Expression and Purification of Motor Protein Constructs. Human neuron-specific kinesin KIF5A24 was expressed in E. coli as a truncated tag-free construct containing amino acids 1−560 and purified as described formerly.25 Like the complete KIF5A (consisting of 1032 amino acids), this construct forms dimers and has been proved to be fully active concerning its ATPase activity and its ability to bind to cargoes and to generate motility under in vitro conditions.25,26 For comparison, the full-length KIF5A and a motor domain construct of KIF5A with amino acids 1−336 have been involved in the study. The latter is a monomer with relatively high ATPase activity.25 The kinesin constructs were adjusted in ATPase assay buffer containing 50 mM PIPES and 2 mM MgCl2 (pH 6.8) and stored at −80 °C. 2.3. ATPase Activity Measurement. The KIF5A, diluted 200-fold with assay buffer, was mixed with paclitaxel-stabilized microtubules reassembled from pure tubulin, ATP (sodium salt, Roche Diagnostics GmbH, Germany), and aluminum chloride. If not indicated otherwise, the reaction mixture, containing 1.5 μM tubulin, 63 nM KIF5A, 1.5 mM ATP, and 30 μM EGTA, was incubated for 30−45 min at 30 °C. The ATPase activity was determined after stopping hydrolysis by the addition of HCl (0.1 N final concentration) by measuring the released free inorganic phosphate, using a Malachite green staining technique.23,27 The results are given either as percentage of activity [%] (100 × the amount of phosphate released in samples in the presence of aluminum divided by the amount of phosphate released in the control sample without aluminum) or as the absolute amount of phosphate released [μM.] It has been proved that aluminum chloride up to 1 mM (final concentration) does not affect phosphate determination under the assay conditions used (results not shown). 2.4. Microtubule Motility Assay. Stock solutions of paclitaxelstabilized microtubules, KIF5A, ATP, and aluminum chloride were transferred into ATPase assay buffer (kept at room temperature), resulting in final concentrations of 0.4 μM tubulin, 0.5 mM ATP, 70 μM EGTA, 100 nM KIF5A, and aluminum chloride as indicated in Results. After 10 min of preincubation at room temperature, 10-μL drops of this mixture were transferred onto microscopic glass slides pretreated with 5 mg/mL bovine serum albumin, covered with a coverslip, and sealed.28 The gliding microtubules were visualized by video-enhanced differential interference contrast microscopy as described.23 The arithmetic mean of gliding velocities and SD were calculated from the data of at least 15 individual microtubules. For each concentration of aluminum chloride, triple independent assays were performed.
Figure 1. Effect of aluminum on the ATPase activity of KIF5A. (a) Concentration-dependent activity inhibition. KIF5A560 (63 nM), 1.5 μM microtubules, and 1.25 mM ATP. The data points represent the mean values and the standard deviations calculated from seven independent experiments. The data were fitted using the sigmoidal DoseResp function of Microcal Origin 9.1 software (Additive GmbH, Germany), yielding an IC50 of 49.8 μM ± 8.6 μM. (b) Reversibility of ATPase inhibition. A: KIF5A560 (170 μM) was incubated for 30 min at 0 °C with 900 μM AlCl3, thereafter 200-fold diluted with ATPase assay buffer, and subjected to ATPase activity measurement (resulting in final concentrations of 63 nM KIF5A and ∼0.4 μM AlCl3, respectively). B: control without AlCl3 incubation.
3. RESULTS AND DISCUSSION Kinesin and microtubules are ubiquitous key elements of intracellular transport processes. With the motor protein and microtubules alone, kinesin-dependent transport processes can be simulated in a cell-free environment. This enables us to investigate direct interactions of effectors, including metal ions, with defined transport-relevant target structures. It is known that aluminum ions bind to the phosphate groups of nucleoside triphosphates and inhibit enzymes which require magnesium as a cofactor.2 As kinesins are magnesium-dependent ATPases and as microtubule formation and stability also demand magnesium, we hypothesized that aluminum ions contribute to the impairment of neuronal functions by direct attacks on molecular elements of the kinesin-microtubule transport system. With this intention, we measured the ATPase activity of kinesin using a simple assay composed of two single protein components only, the neuron-specific KIF5A and microtubules reconstituted from brain tubulin. Aluminum, added as chloride, has been found to decrease the ATPase activity of KIF5A560 in a concentration-dependent manner (Figure 1a). Under the conditions used, a mean IC50 of 49.8 μM ± 8.6 μM was determined. The ATPase inhibition caused by aluminum was found to be reversible (Figure 1b). Kinesin is an enzyme whose ATPase activity is stimulated by microtubules.29 In this context, the question arises as to whether
the ATPase activity drop is caused by microtubule destruction or by blocking kinesin/microtubule binding. To answer this question, microtubule formation has been investigated by the classic assembly assay.22 We found that under the experimental conditions applied in our study (inter alia, 0.5 mM Mg2+) aluminum chloride up to 400 μM does not change the characteristics of paclitaxel-induced microtubule formation from pure tubulin without microtubule-associated proteins (Figure 2a). In the case of magnesium ion deficiency (2 μM), the turbidity steady state level has been observed to increase depending on the aluminum chloride concentration (Figure 2b) suggesting that aluminum promotes microtubule formation, as reported by Macdonald et al.30 However, it is known that especially in the absence of microtubule-associated proteins tubulin often does not form regular microtubules but so-called aberrant or polymorphic assemblies, described by, e.g., Böhm et al.31and Unger et al.32 In such a situation, the turbidity signal at 360 nm does not necessarily reflect the amount of assembled tubulin. Therefore, we additionally determined the amount of assembled tubulin formed under conditions of magnesium ion deficiency by a sedimentation assay and found assembled tubulin on a level independent of the aluminum concentration (Figure 2c). In this context, it raises the question whether the formation of aberrant tubulin assemblies (instead of microtubules) could be responsible for lowering kinesin ATPase activity. In our opinion, 1276
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to aluminum intoxication.35 Therefore, we conclude that the inhibition of kinesin ATPase activity is not causally due to microtubule destruction. Another reason for decreased ATPase activity might be disturbed kinesin−microtubule binding. It is known that the critical microtubule-interacting residues of kinesin are predominantly positively charged, which is consistent with a primarily electrostatic interaction of kinesin with the negatively charged microtubule surface.36,37 So, it might be possible that aluminum ions prevent kinesin binding by masking the negative charges of microtubules. To prove whether damaged kinesin−microtubule binding contributes to the aluminum-induced ATPase inhibition, KIF5A was allowed to react with microtubules under conditions similar to those of the ATPase assay and submitted to high-speed ultracentrifugation. Analyzing sediments and supernatants of the microtubule-kinesin complexes formed with and without aluminum by polyacrylamide gel electrophoresis revealed that aluminum chloride up to 300 μM did not significantly prevent kinesin binding. Practically all KIF5A applied cosedimented with the microtubules, independently of the aluminum concentration (Figure 3a). Moreover, the ATPase activities of KIF5A have been measured at varying microtubule concentration in the presence of
Figure 2. Microtubule formation in the presence of aluminum chloride. (a) 1.3 mg/mL tubulin, 100 mM Pipes (pH 6.8), 0.25 M glycerol, 3.8 μM EGTA, 500 μM MgCl2, and 250 μM GTP. At the time point of 10 min, paclitaxel (16.7 μM, final concentration) was added. (b) 0.5 mg/ mL tubulin, 100 mM Pipes (pH 6.8), 0.25 M glycerol, 3.8 μM EGTA, 2 μM MgCl2, and 125 μM GTP. At time point 1 min, paclitaxel (16.7 μM, final concentration) was added. (c) Determination of the amount of tubulin assembled. Assembly conditions are the same as those in b. After 45 min, the samples were centrifuged in a Beckman-Coulter TLA55rotor (30 min, 40 000 rpm, 32 °C). The sediments were dissolved in an equal volume of 0.1 N NaOH. The total amount of protein within the sediments was measured by the Lowry procedure.44 Figure 3. KIF5A binding to microtubules in the presence of aluminum chloride. (a) 3.0 μM KIF5A, paclitaxel-stabilized microtubules (2.5 μM tubulin), and 2 mM ATP were incubated for 30 min at 30 °C. Centrifugation in the TLA55-rotor for 30 min, at 40 000 rpm and 30 °C. The sediments were resuspended in an equal volume of incubation buffer. Electrophoretic separation was performed in a 7.5% polyacrylamide gel, followed by Coomassie Brillant Blue staining. Lane 0*, KIF5A without microtubules and without AlCl3; lanes 0, 50, and 300, microtubule sediments with KIF5A plus 0, 50, and 300 μM AlCl3, respectively. (b) Dependence of aluminum-induced ATPase inhibition on microtubule concentration; 63 nM KIF5A560 and 1.25 mM ATP.
such inhibition mechanisms can be excluded as it was proved that single tubulin protofilaments (constituting both microtubules and aberrant assemblies) are sufficient to generate kinesin activity.33,34 Taking into account our results and considerations on microtubule formation, we conclude that within the experimental frame applied in our study, aluminum ions do not eliminate the microtubules. Also, other authors reported that possible impaired brain microtubule function cannot be primarily related 1277
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Figure 4. Dependence of aluminum-induced ATPase inhibition on different additives; KIF5A 63 nM, 1.5 μM microtubules, and 1.25 mM ATP. (a) Inhibition characteristics in the presence of thiols and EGTA. (b) Effect of EDTA on aluminum-induced inhibition. (c) Effect of BSA and alpha casein (Sigma C6780) on aluminum-induced inhibition. (d) Concentration-dependent compensation of aluminum-induced inhibition by casein.
μM AlCl3) neutralized the inhibition effect completely (Figure 4 c,d). Recently, it has been demonstrated that the level of aluminum in patients can be significantly lowered by intravenous administration of EDTA.14 In this context, we tried to find out whether EDTA is able to reduce the inhibition effect of aluminum on kinesin ATPase. We observed that EDTA did not compensate aluminum-induced ATPase inhibition (Figure 4b) but itself reduced the ATPase activity in a concentrationdependent fashion (Figure 4b, insert). Though the stability constant of aluminum-EDTA complexes (16.13) is remarkably higher than that of magnesium-EDTA (8.69),41 the effect of EDTA on kinesin activity might be explained by chelating magnesium ions required as the cofactor for kinesin. Aluminum ions have a ligand-exchange rate which is about 105 times lower than that of magnesium ions.2 Therefore, one might expect that changing the aluminum/magnesium ratio in the ATPase assay could modulate inhibition characteristics. However, our measurements at constant ATP, microtubule, and kinesin concentrations but with varying magnesium ion concentration revealed similar ATPase activity curves, regardless of the presence of aluminum ions (Figure 5a). Both, at 0 and 125 μM aluminum chloride, the activity curves pass an optimum at about 2.25 mM magnesium, whereby total activity was proportionally lowered by aluminum ions. This suggests that under the conditions used in our ATPase assay competition between aluminum and magnesium does not essentially contribute to kinesin ATPase inhibition. Aluminum ion binding to ATP42 might be another reaction mechanism to explain ATPase inhibition. To elucidate the possible contribution of ATP−aluminum complexation to the inhibition effect, we measured the dependence of ATPase activity
aluminum chloride and without it. As mentioned above, kinesin activity depends on microtubule binding, whereby at constant ATP and kinesin concentration the ATPase activity of KIF5A increases with increasing microtubule concentration (Figure 3b). The dependence of the ATPase activity on microtubule concentration follows Michaelis−Menten characteristics, yielding Km values of 1.22 μM and 1.18 μM (tubulin) at 50 μM aluminum chloride and without it, respectively. Independently of the microtubule concentration, the ATPase activity was decreased by a factor of 2 (Figure 3b). These results additionally corroborate our conclusion that the aluminum-induced ATPase inhibition is not due to disturbed kinesin−microtubule binding, strongly suggesting that the motor protein itself is the molecular target of aluminum attacks. It is commonly known that sulfhydryl group-containing compounds, among them cysteine, bind metal ions, causing the loss of protein activity, e.g., the inhibition of kinesin ATPase upon the binding of copper ions.38 Aluminum, however, is assumed to form only weak complexes with sulfhydryl groups.39 Correspondingly, our measurements showed that, unlike copper, neither cysteine nor dithiothreitol compensated KIF5A ATPase inhibition (Figure 4a). This means that as expected the effect of aluminum ions on kinesin is not based on reactions with sulfhydryl groups. In contrast to the effects of the bivalent cadmium23 and copper ions,38 the aluminum-induced ATPase inhibition characteristics was also not changed by EGTA (Figure 4a). According to Martin, phosphate groups are strong aluminum ion binders.40 On this basis, we assumed that phosphate group-rich proteins, like casein, could be able to reduce aluminum-induced ATPase inhibition. Indeed, we found that, unlike bovine serum albumin, casein (∼40 μg/mL at 250 1278
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the ATPase of the KIF5A motor domain, comprising amino acids 1 to 336, i.e., the kinesin head and neck,25 has been found to be rather insensitive to aluminum ions. Accordingly, we conclude that aluminum ions bind preferentially to regions between amino acids 337 to 560. It could be speculated that aluminum binding changes the kinesin conformation in a fashion which is similar to that described in cases of kinesin autoinhibition by motor domain/tail domain binding.43 In addition to the ATPase measurements, we have tried to verify the inhibition of kinesin by an in vitro assay simulating its motility activity. For this, the gliding behavior of kinesin-bound microtubules across a glass surface coated with BSA was studied. It has been observed that up to 125 μM aluminum chloride there were no changes concerning both velocity and the amount of microtubules bound to the BSA-coated surface. However, further increasing the aluminum level prevented microtubule binding. At concentrations above 250 μM, microtubule binding to the BSAcoated surface was practically completely inhibited (Table 1). Table 1. Effects of Aluminum Chloride on the Motility Activity of KIF5A, Examined by an in Vitro Gliding Assay, in Which Microtubules Were Driven by the Kinesin across a BSA-Coated Glass Surfacea
Figure 5. Dependence of aluminum-induced ATPase inhibition on magnesium ions (a) and on ATP (b); 63 nM KIF5A and 1.5 μM microtubules.
AlCl3 [μM]
velocity ± SD [nm/s]
binding
0 125 250 325 400
1209 ± 141 1261 ± 128 1195 ± 152
normal normal weak extremely weak no
0.4 μM tubulin, 0.5 mM ATP, 70 μM EGTA, 0.1 μM KIF5A, and aluminum chloride. The 3rd column describes qualitatively the binding efficiency of the kinesin-loaded microtubules to the surface. a
on ATP concentration with (75 μM) and without aluminum chloride. Comparing the corresponding Km values for ATP (656 μM and 327 μM, respectively) revealed that aluminum chloride lowered the substrate affinity significantly (Figure 5b). The results described above have been obtained with the truncated KIF5A construct (amino acids 1 to 560) comprising the motor domain and a part of the stalk. Therefore, it is reasonable to ask whether KIF5A with the complete amino acid sequence could also be inhibited by aluminum ions. Measuring the ATPase activities of the full-length kinesin revealed a quite similar inhibition profile like that of KIF5A560 (Figure 6), except for a small IC50 increase (from 52 μM to 85 μM). Surprisingly,
To study kinesin-mediated motility in the gliding assay, two conditions should be fulfilled: kinesin should be able to bind to microtubules, and its cargo binding site should be able to bind to the substrate (i.e., to the BSA-coated glass surface). The detachment of microtubules observed in the gliding assay at high aluminum concentration can be explained as follows: first, kinesin binding to microtubules is inhibited, and/or second, the kinesin cargo binding site is damaged, preventing kinesin attachment to the substrate. As the kinesin/microtubule binding has been shown to be not affected (see above), also the gliding assay provides evidence that a kinesin region involved in cargo binding is altered by aluminum ions.
4. CONCLUSIONS Our experimental study demonstrates that aluminum ions are able to inhibit ATPase of neuron-specific kinesin. The ATPaseactive center, which is located in the motor domain of the kinesin, seems to be not affected directly. Our results suggest that inhibition is preferentially caused by aluminum binding to some sequence within the kinesin stalk (corresponding to amino acids 337−560), leading to a conformational state similar to that described in cases of kinesin autoinhibition caused by motor domain-tail binding.43 As kinesin is an obligatory element of the anterograde axonal transport in neurons, it is suggested that the direct molecular interaction of aluminum ions with kinesin might substantially contribute to the development of neuronal disorders. Because of the relative high sequence conservation of mammalian kinesin-1 (to which KIF5A belongs), we assume that also transport
Figure 6. Effect of aluminum on KIF5A ATPase activity dependent on construct length; 63 nM KIF5A336 and KIF5A560; 479 nM full-length KIF5A (KIF5A1032); 1.5 μM microtubules; and 1.25 mM ATP. IC50 values: KIF5A1032, 87.5 μM; KIF5A560, 54.7 μM; KIF5A336, ≫500 μM. 1279
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(12) Walton, J. R. (2014) Chronic aluminum intake causes Alzheimer’s disease: applying Sir Austin Bradford Hill’s causality criteria. J. Alzheimer’s Dis. 40, 765−838. (13) Erazi, H., Ahboucha, S., and Gamrani, H. (2011) Chronic exposure to aluminum reduces tyrosine hydroxylase expression in the substantia nigra and locomotor performance in rats. Neurosci. Lett. 487, 8−11. (14) Fulgenzi, A., Vietti, D., and Ferrero, M. E. (2014) Aluminium involvement in neurotoxicity. BioMed. Res. Int. 2014, 758323. (15) Verstraeten, S. V., Aimo, L., and Oteiza, P. I. (2008) Aluminium and lead: molecular mechanisms of brain toxicity. Arch. Toxicol. 82, 789−802. (16) Franker, M. A., and Hoogenraad, C. C. (2013) Microtubule-based transport - basic mechanisms, traffic rules and role in neurological pathogenesis. J. Cell Sci. 126, 2319−2329. (17) Ebbing, B., Mann, K., Starosta, A., Jaud, J., Schols, L., Schule, R., and Woehlke, G. (2008) Effect of spastic paraplegia mutations in KIF5A kinesin on transport activity. Hum. Mol. Genet. 17, 1245−1252. (18) Hirokawa, N., Niwa, S., and Tanaka, Y. (2010) Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron 68, 610−638. (19) Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. U.S.A. 70, 765−768. (20) Vater, W., Böhm, K. J., and Unger, E. (1986) A simple method to obtain brain microtubule protein poor in microtubule-associated proteins. Acta Histochem. Suppl. 33, 123−129. (21) Weingarten, M. D., Lockwood, A. H., Hwo, S. Y., and Kirschner, M. W. (1975) A protein factor essential for microtubule assembly. Proc. Natl. Acad. Sci. U.S.A. 72, 1858−1862. (22) Gaskin, F., Cantor, C. R., and Shelanski, M. L. (1974) Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J. Mol. Biol. 89, 737−755. (23) Böhm, K. J. (2014) Kinesin-dependent motility generation as target mechanism of cadmium intoxication. Toxicol. Lett. 224, 356−361. (24) Niclas, J., Navone, F., Hom-Booher, N., and Vale, R. D. (1994) Cloning and localization of a conventional kinesin motor expressed exclusively in neurons. Neuron 12, 1059−1072. (25) Kalchishkova, N., and Böhm, K. J. (2008) The role of kinesin neck linker and neck in velocity regulation. J. Mol. Biol. 382, 127−135. (26) Dreblow, K., Kalchishkova, N., and Böhm, K. J. (2010) Kinesin passing permanent blockages along its protofilament track. Biochem. Biophys. Res. Commun. 395, 490−495. (27) Martin, B., Pallen, C. J., Wang, J. H., and Graves, D. J. (1985) Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin. J. Biol. Chem. 260, 14932−14937. (28) Böhm, K. J., Stracke, R., Baum, M., Zieren, M., and Unger, E. (2000) Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity. FEBS Lett. 466, 59−62. (29) Hackney, D. D. (1994) The rate-limiting step in microtubulestimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. J. Biol. Chem. 269, 16508−16511. (30) Macdonald, T. L., Humphreys, W. G., and Martin, R. B. (1987) Promotion of tubulin assembly by aluminum ion in vitro. Science 236, 183−186. (31) Böhm, K. J., Vater, W., Fenske, H., and Unger, E. (1984) Effect of microtubule-associated proteins on the protofilament number of microtubules assembled in vitro. Biochim. Biophys. Acta 800, 119−126. (32) Unger, E., Böhm, K. J., and Vater, W. (1990) Structural diversity and dynamics of microtubules and polymorphic tubulin assemblies. Electron Microsc. Rev. 3, 355−395. (33) Ray, S., Meyhöfer, E., Milligan, R. A., and Howard, J. (1993) Kinesin follows the microtubule’s protofilament axis. J. Cell Biol. 121, 1083−1093. (34) Hoenger, A., Thormählen, M., Diaz-Avalos, R., Doerhoefer, M., Goldie, K. N., Müller, J., and Mandelkow, E. (2000) A new look at the microtubule binding patterns of dimeric kinesins. J. Mol. Biol. 297, 1087−1103.
systems of other cells and organs can be affected by aluminum ions.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +49 3641 656161. Fax: +49 3641 656288. E-mail: kboehm@fli-leibniz.de. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We are very grateful to Mrs. Marina Wollmann for her skillful excellent assistance in technically performing the experiments described in this study.
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ABBREVIATIONS ATP, adenosine 5′-triphosphate; BSA, bovine serum albumin; DTT, dithiothreitol; GTP, guanosine 5′-triphosphate; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene bis (oxyethylenenitrilo) tetraacetic acid; KIF5A, neuron-specific kinesin; PIPES, 1,4-piperazine diethanesulfonic acid; MAP, microtubuleassociated protein
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REFERENCES
(1) Shaw, C. A., Li, D., and Tomljenovic, L. (2014) Are there negative CNS impacts of aluminum adjuvants used in vaccines and immunotherapy? Immunotherapy 6, 1055−1071. (2) Kawahara, M., and Kato-Negishi, M. (2011) Link between aluminum and the pathogenesis of Alzheimer’s disease: The integration of the aluminum and amyloid cascade hypotheses. Int. J. Alzheimer’s Dis. 2011, 276393, doi: 10.4061/2011/276393. (3) Han, S., Lemire, J., Appanna, V. P., Auger, C., Castonguay, Z., and Appanna, V. D. (2013) How aluminum, an intracellular ROS generator promotes hepatic and neurological diseases: the metabolic tale. Cell Biol. Toxicol. 29, 75−84. (4) Rondeau, V., Jacqmin-Gadda, H., Commenges, D., Helmer, C., and Dartigues, J. F. (2009) Aluminum and silica in drinking water and the risk of Alzheimer’s disease or cognitive decline: findings from 15-year follow-up of the PAQUID cohort. Am. J. Epidemiol. 169, 489−496. (5) Willhite, C. C., Karyakina, N. A., Yokel, R. A., Yenugadhati, N., Wisniewski, T. M., Arnold, I. M., Momoli, F., and Krewski, D. (2014) Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts. Crit. Rev. Toxicol. 44 (Suppl. 4), 1−80. (6) Krewski, D., Yokel, R. A., Nieboer, E., Borchelt, D., Cohen, J., Harry, J., Kacew, S., Lindsay, J., Mahfouz, A. M., and Rondeau, V. (2007) Human health risk assessment for aluminium, aluminium oxide, and aluminium hydroxide. J. Toxicol. Environ. Health, Part B 10 (Suppl. 1), 1−269. (7) Markesbery, W. R., Ehmann, W. D., Alauddin, M., and Hossain, T. I. (1984) Brain trace element concentrations in aging. Neurobiol. Aging 5, 19−28. (8) Exley, C. (2014) What is the risk of aluminium as a neurotoxin? Expert Rev. Neurother. 14, 589−591. (9) Bondy, S. C. (2014) Prolonged exposure to low levels of aluminum leads to changes associated with brain aging and neurodegeneration. Toxicology 315, 1−7. (10) Kawahara, M. (2005) Effects of aluminum on the nervous system and its possible link with neurodegenerative diseases. J. Alzheimer’s Dis. 8, 171−182. (11) Exley, C., and Vickers, T. (2014) Elevated brain aluminium and early onset Alzheimer’s disease in an individual occupationally exposed to aluminium: a case report. J. Med. Case Rep. 8, 41. 1280
DOI: 10.1021/acs.chemrestox.5b00077 Chem. Res. Toxicol. 2015, 28, 1275−1281
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Chemical Research in Toxicology (35) Oteiza, P. I., Golub, M. S., Gershwin, M. E., Donald, J. M., and Keen, C. L. (1989) The influence of high dietary aluminum on brain microtubule polymerization in mice. Toxicol. Lett. 47, 279−285. (36) Tucker, C., and Goldstein, L. S. B. (1997) Probing the kinesinmicrotubule interaction. J. Biol. Chem. 272, 9481−9488. (37) Woehlke, G., Ruby, A. K., Hart, C. L., Ly, B., HomBooher, N., and Vale, R. D. (1997) Microtubule interaction site of the kinesin motor. Cell 90, 207−216. (38) Böhm, K. J. (2015) Elevated copper ion levels as potential cause of impaired kinesin-dependent transport processes. Arch. Toxicol. 89, 565− 572. (39) Martin, R. B. (1986) The chemistry of aluminum as related to biology and medicine. Clin. Chem. 32, 1797−1806. (40) Martin, R. B. (1992) Aluminum speciation in biology. Ciba Found. Symp. 169, 5−25. (41) Furia, T. E. (1973) Sequestrants in Food, in CRC Handbook of Food Additives (Furia, T. E., Ed.) 2nd ed., pp 271−294, CRC Press, Boca Raton, FL. (42) Kiss, T., Sovago, I., and Martin, R. B. (1991) Al3+ binding by adenosine 5′-phosphates - AMP, ADP, and ATP. Inorg. Chem. 30, 2130− 2132. (43) Coy, D. L., Hancock, W. O., Wagenbach, M., and Howard, J. (1999) Kinesin’s tail domain is an inhibitory regulator of the motor domain. Nat. Cell Biol. 1, 288−292. (44) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265−275.
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DOI: 10.1021/acs.chemrestox.5b00077 Chem. Res. Toxicol. 2015, 28, 1275−1281
