Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine

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Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine Residues of Tau Saharnaz Rafiee, Kazem Asadollahi, Gholamhossein Riazi, Shahin Ahmadian, and Ali Akbar Saboury ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00230 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017

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ACS Chemical Neuroscience

Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine Residues of Tau Saharnaz Rafiee1, Kazem Asadollahi1, Gholamhossein Riazi1*, Shahin Ahmadian1, Ali Akbar Saboury 1

Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.

*

Corresponding author: Prof. Gholamhossein Riazi

P.O. Box: 13145-1384 Telephone: +98 9121255924 Email address: [email protected]

1 ACS Paragon Plus Environment


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Abstract Two mechanisms underlie the inhibitory/acceleratory action of chemical compounds on tau aggregation including the regulation of cellular kinases and phosphatases activity and direct binding to tau protein. Vitamin B12 is one of the tau polymerization inhibitors that its deficiency is linked to inactivation of protein phosphatase 2A and subsequently hyper phosphorylation and aggregation of tau protein. Regarding to the structure and function of vitamin B12 and tau protein we assumed that vitamin B12 is also able to directly bind to tau protein. Hence we investigated into the interaction of vitamin B12 with tau protein in vitro using fluorometry and circular dichrosim. Interaction studies was followed by investigation into the effect of vitamin B12 on tau aggregation using ThT fluorescence, Circular dichroism, transmission electron microscopy and SDS-PAGE. The results indicated that vitamin B12 interacts with tau protein and prevents fibrillization of tau protein. Blocking the cysteine residues of tau confirmed the cysteine-mediated binding of vitamin B12 to tau and showed that binding to cysteine is essential for inhibitory effect of vitamin B12 on tau aggregation. SDS-PAGE analysis indicated that vitamin B12 inhibits tau aggregation and tau oligomers formed in the presence of vitamin B12 are mostly SDS-soluble. We propose that direct binding of vitamin B12 is another mechanism underlying the inhibitory role of vitamin B12 on tau aggregation and neurodegeneration. Keywords: Vitamin B12; tau protein; Cobalamin; tau aggregation; Alzheimer’s disease


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1. Introduction The presence of protein aggregates is the hallmark of several neurodegenerative diseases including Alzheimer’s and Parkinson’s disease 1. Intrinsically disordered proteins which are prone to aggregation such as tau protein and A-beta form a great portion of these aggregates 2

. However, A-beta aggregates primarily accumulate in extracellular environment and tau

aggregates are present in cytosol 2. Tau protein as the most important axonal microtubule-associated protein is well-known for its role in microtubule assembly and stabilization 3. Tau also plays a critical role in various cell signaling pathways including MAPK signaling and is responsible for protection of DNA from damage upon environmental stresses

4,5

. Interaction of tau with other cell

components such as endoplasmic reticulum and cell membrane presents another aspect of tau functions

6,7

. The broad range of tau functions is regulated by various kinds of post

translational modifications including phosphorylation, glycosylation, ubiquitinylation and truncation 8. Phosphorylation is the most important post-translational modification of tau that regulates binding of tau to microtubules 9. However, hyper-phosphorylation mediates detachment of tau from microtubule polymers and subsequent accumulation of tau as neurofibrillary tangles 10. Phosphorylation of tau is under intense control via a wide range of protein kinases and phosphatases 11. GSK3β and PP2A are two critical enzymes in regulating the phosphorylation state of tau protein 3. Activation of both enzymes is a post-translational modification-dependent process. Several studies have reported the phosphorylation dependency of GSK3β and PP2A activation, while methylation is a more critical process in activation of PP2A

12

. PP2A is a

multimeric protein complex and the major brain serine/threonine phosphatase that dephosphorylates tau at different sites and prevents tau hyper-phosphorylation 13. Decreased methylation at C-terminal leucine residue of C-subunit of PP2A indirectly affects activity and



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assembly of PP2A holoenzyme which finally regulates tau phosphorylation state

14

.

Participation of mentioned enzymes in several signaling pathways explains the mechanism of action of various kinds of active compounds on tau phosphorylation-dephosphorylation such as vitamin B12

15

. Evidences suggest that enzymatic methylation of PP2A is performed by

several enzymes including methionine synthase which converts homocysteine to methionine. Vitamin B12 is converted to methylcobalamin by taking a methyl group from tetrahydrofolate. The resulted methylcobalamin donates its methyl group to homocysteine, which finally is converted to methionine by methionine synthase. Therefore, vitamin B12 deficiency results in PP2A inactivation, tau hyper-phosphorylation, subsequent tau aggregation and finally neural degeneration 15–17. Structural changes of tau upon direct binding to different ligands are responsible for the effect of other groups of biologically active molecules on tau aggregation 18. One of the most important structural changes, which affect tau aggregation, is formation of disulfide bond 19. It has been reported that formation of intermolecular disulfide bonds accelerates tau aggregation, while intramolecular disulfide bonds could prevent aggregation

20

. Hence, it is

anticipated that native and cysteine mutated tau isoforms containing only one cysteine residue accumulate faster than tau isoforms containing 2 cysteine residues

21

. In this context,

investigation of the effect of cysteine blockers, disulfide bond reducers and oxidants on tau aggregation have illustrated the key role of tau cysteine residues in tau aggregation 18–20. Structure and function of vitamin B12 inside the cells suggest the ability of vitamin B12 to directly bind to tau protein and affects its polymerization. In addition, vitamin B12 is able to interact with thiol groups which encourages this assumption since tau protein has two exposed cysteine residues

22

. Hence, we investigated binding of vitamin B12 to tau protein

and assessed the effect of vitamin B12 on tau aggregation in vitro.


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2.

Results and discussion We hypothesized that the role of vitamin B12 in neurodegeneration is not only exerted

via activation of PP2A but also through direct binding of vitamin B12 to tau protein. Hence, at first step vitamin B12 binding to tau was characterized using fluorescence spectroscopy. Intrinsic fluorescence of tau in the presence of vitamin B12 was recorded in two different temperatures, 25 and 37 °C (Fig. 1a and b). Addition of vitamin B12 into the protein solution resulted in quenching of tyrosine fluorescence, the only fluorophore of tau protein, at 304 nm. In addition to the obvious decrease of intensity at 304 nm, two other regions of spectra at 360 and 405 nm were affected as well. Previous investigations on spectroscopic profile of methylcobalamin and thiolatocobalamin have indicated that spectral changes in these regions are due to interaction of cobalamin with thiol groups and formation of thiolatocobalamin from methylcobalamin which suggests participation of cysteine residues of tau in interaction 23

. Higher quantum yield of methylcobalamin in both of these regions and lower and higher

extinction coefficient of methylcobalamin than thiolatocobalamin at 360 and 405 nm, respectively, are responsible for the observed changes 23. In order to confirm participation of thiols in interaction cysteine residues of tau were blocked by acrylamide and titration experiments were performed using cysteine-blocked tau (CB-tau) at the same conditions. If formation of thiolatocobalamin be responsible for fluorescence spectral changes, blockade of cysteine residues of tau would prevent it. Emission spectra of CB-tau protein showed a normal quenching of tau fluorescence at 304 nm with no spectral changes at 360 and 405 nm (Fig. 1 d and e). These data reinforce this suggestion that cobalamin binds to tau protein via cysteine residues. To get a better insight into vitamin B12/tau protein interaction the Stern-Volmer plot was drawn by using the equation 1: 5 ACS Paragon Plus Environment

24


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1:

= 1 + 12

Where F0 and F are the fluorescence intensity in the absence and presence of various concentrations of cobalamin ([B12]) at 304 nm, respectively (Fig. 1c). As figure 1c shows the Stern-Volmer plot has an upward curvature which reduces by temperature increase. The upward curvature of Stern-Volmer plot is due to a combined static and dynamic quenching 24. Thus, it could be concluded that at higher temperatures only one kind of quenching is dominant. If the formation of thiolatocobalamin be responsible for the static quenching, hence blocking of cysteine residues would eliminate the upward curvature of Stern-Volmer plot. Stern-Volmer plot for fluorescence quenching of CB-tau confirmed that the formation of thiolatocobalamin accounts for static mechanism of quenching (Fig. 1f). Omission of upward curvature of Stern-Volmer plot at high temperatures suggested that cysteine residues of tau are inaccessible at higher temperatures which could be due to partial collapse of tau protein at these temperatures

25

. In order to examine this, quenching of tau by vitamin B12 was

performed at 50 °C. The emission spectra of tau protein at 50 °C upon addition of vitamin B12 was very similar to CB-tau emission spectra without spectral change at 360 and 405 nm (Fig. 1g). Moreover, the upward curvature of Stern-Volmer plot was omitted which indicated inaccessibility of cysteine residues at higher temperatures (Fig. 1h). It should be noted that incomplete elimination of upward curvature of Stern-Volmer plot even after blocking of cysteine residues could be due to incomplete blocking of cysteine residues, the Ellman’s essay showed that only 85% of the cysteine residues are blocked.


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Figure 1. Fluorescence quenching of tau and CB-tau protein by B12 at various temperatures. a; emission spectra of tau protein in the presence of various concentrations of B12 at 25 ℃. Tau fluorescence at 304 nm was quenched. Emission spectra at 360 and 405 nm was also changed that is due to conversion of methylcobalamin to thiolatocobalamin. b; fluorescence emission spectra of tau protein at 37 °C which shows quenching at 304 nm. Spectra has changed at 360 and 405 nm as well. However the change at these regions is less intense in comparison to 25 °C. c; SternVolmer plot for interaction of B12 with tau at 25 and 37 °C. The upward curvature of Stern-Volmer plot has decreased by temperature increase. Solid and opened circles represent plots at 25 and 37 °C, respectively. d and e; shows titration of B12 into CB-tau solution at 25 and 37 °C, respectively. Blockade of cysteine residues resulted in omission of spectral change at 360 and 405 nm that shows participation of cysteines in interaction. f; Stern-Volmer plot for titration of B12 into CB-tau solution at 25 and 37 °C, solid and opened circles, respectively. The upward curvature has been removed after blocking of cysteines and both plot are similar in shape. g; titration of B12 into tau solution at 50 °C. 7 ACS Paragon Plus Environment


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Spectral change at 360 and 405 nm has omitted, like CB-tau quenching experiments, due to partial folding of tau at high temperatures that results in inaccessibility of cysteine residues. h; Stern-Volmer plot for quenching of tau fluorescence at 50 °C. The upward curvature has been omitted that indicates absence of thiolatocobalamin formation. In all experiments 1 µM of tau protein have been quenched with various concentrations of B12 up to 20 µM. The arrows indicate the regions of spectra affected by formation of thiolatocobalamin from methyl cobalamin.

During our experiments we also found that thermal collapse of tau protein is concomitant with decrease in fluorescence intensity of tau protein. Since cysteine residues are placed at the vicinity of the collapsed regions of tau, binding of cobalamin to cysteine residues of tau must prevent partial folding of this region and decrease in fluorescence intensity. Tau protein was incubated with various concentrations of vitamin B12 and the fluorescence intensity of tau at 304 nm was recorded by excitation at 275 nm. The slope of graphs in Fig. 2 measures the rate of decrease in fluorescence intensity as a result of thermal collapse of tau. The lower rate of fluorescence intensity decrease in the presence of cobalamin indicated the inhibitory role of vitamin B12 in thermal collapse of tau. However, vitamin B12 could not prevent complete thermal collapse of tau which is due the small overlap between vitamin B12 binding and tau regions involved in thermal collapse 25.


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Figure 2. Vitamin B12 prevents thermal collapse of tau protein. Continuous thermal collapse of tau protein has been prevented in the presence of vitamin B12. 10 µM tau protein, solid line, was incubated with 10 and 20 µM vitamin B12, dashed and long dashed line, respectively. The fluorescence intensities have been normalized against the fluorescence intensity of control sample to compensate the effect of vitamin B12 on fluorescence quenching of tau.

Fluorescence studies on tau and CB-tau emission spectra proposed that cobalamin binds to cysteine residues of tau. To confirm fluorescence data, CD experiments were performed using both tau and CB-tau species. CD spectra of tau protein is determined by a single negative peak around 200 nm due to unfolded nature of tau. Titration of vitamin B12 into tau solution resulted in intense decrease in ellipticity at 200 nm and appearance of a shoulder around 220 nm, which indicated secondary structural changes of tau protein upon vitamin B12 binding (Fig. 3a). However, injection of cobalamin into CB-tau solution illustrated small changes in ellipticity at 200 nm and around 220 nm which means the negligible effect of vitamin B12 on CB-tau secondary structure (Fig. 3b).



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Figure 3. The effect of vitamin B12 on secondary structure of tau. a; secondary structural

change of tau protein in the presence of various concentrations of B12 shows intense decrease in negative ellipticity at 200 nm. b; CD spectra of CB-tau in the presence of B12. The small effects of B12 on the structure of CB-tau shows that blocking of cysteine residues prevents interaction of cobalamin with tau protein. 10 µM of tau and CB-tau, solid line, have been tittered with 10, 30 and 50 µM of B12, dotted, dashed and long dashed lines, respectively.

In order to quantitate the effect of vitamin B12 on the secondary structure content of tau and CB-tau, the CD spectra were deconvoluted using CDNN 2.1 software. The percentage of different secondary structures are presented in table 1. As the values indicate the content of helix, random coil and beta-turns of tau have been increased, while the beta-structures have decreased in the presence of vitamin B12. Although the secondary structure content of tau 10 ACS Paragon Plus Environment

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protein has been affected by cobalamin, CB-tau structure remained uninfluenced which illustrates the key role of free thiols in interaction. Table 1. The secondary structures content of tau and CB-tau in the presence of vitamin B12 α-Helix* β-Antiparallel β-Parallel β-Turn Random Coil B12 Conc. (µM)** Tau CB-tau tau CB-tau tau CB-tau tau CB-tau tau CB-tau 0

5.2

6.3

32.4

32

3.5

3.9

24.4

22.8

34.5

35

10

7

6.7

23.3

32

3.2

4

30.5

22.6

37

34.7

30

7.7

6.4

21.6

31.5

3.2

4

30.9

22.5

36.6

35.6

36

35.4

8.2 6.6 22.4 32 3.5 4 29.9 22 50 *The values represent the percentage of secondary structures. **10 µM of protein has been tittered with various concentrations of vitamin B12

The importance of cysteine residues in tau aggregation process as well as increase in helix and random coil content of tau protein upon binding to vitamin B12 suggest that vitamin B12 is a potential modulator of tau aggregation. To illustrate this, tau was assembled in the presence of various concentrations of vitamin B12 and the aggregation process was followed by recording ThT fluorescence at various time periods. ThT fluorescence was increased rapidly in control samples, while the presence of vitamin B12 effectively inhibited tau fibrillization (Fig. 4a). It can be concluded from ThT results that fibrillization is inhibited intensely in vitamin B12: tau ratio of 1:1 and higher concentrations of vitamin has a small effect on the extent of aggregation which could be due to presence of only one specific binding site for vitamin B12 on tau protein. Carrying out aggregation experiments using CBtau showed that binding of cobalamin to tau protein via cysteine residues is necessary for inhibitory action of vitamin B12 on tau aggregation (Fig. 4b). Although vitamin B12 does not prevent CB-tau aggregation, it affects the nucleation process of tau at high concentrations that could be explained by nonspecific interaction of B12 with tau protein at high concentrations of vitamin.



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Figure 4. Effect of B12 on tau and CB-tau aggregation. a; B12 effectively inhibits formation of tau fibrils. b; aggregation of CB-tau was not affected by methylcobalamin. Circles (black), Squares (Red), Triangles (green), Crosses (purple) and Plus marks (yellow) represent ThT fluorescence of aggregated proteins in the presence of 0, 10, 30, 50 and 80 µM vitamin B12 at various time periods. c; the CD spectra of aggregated tau in the absence and presence of various concentrations of vitamin B12. CD spectra of aggregated tau protein shows decrease in ellipticity and red-shift toward higher wavelengths that indicates tau protein fibrils have been formed. However the absence of redshift in the presence of B12 indicates the inhibition of fibril formation. d; the CD spectra of aggregated CBtau in the presence of various concentrations of vitamin B12 shows that blocking of cysteine residues inhibits protein aggregation. However, decrease in ellipticity at 200 nm could be due to formation of tau oligomers. CD spectra of monomeric protein, solid black line, and aggregated protein in the presence of 0, 10, 30, 50 and 80 µM of vitamin B12 is presented by Circles (black), Squares (Red), Triangles (green), Crosses (purple) and Plus marks (yellow), respectively. 12 ACS Paragon Plus Environment

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Aggregation of tau protein gives rise to higher content of beta-sheet secondary structures which is easily detectable from the red-shifted spectra of tau fibrils

26

. CD spectra of

aggregated tau protein in the presence and absence of vitamin B12 confirmed ThT results indicating the inhibitory effect of B12 in tau fibrillization (Fig. 4c). The red-shift of aggregated tau CD spectrum in concomitant with decreased peak intensity indicated the formation of tau protein fibrils, while the absence of red-shift in the presence of vitamin B12 demonstrated the inhibitory effect of vitamin B12 on tau fibrillization. Although vitamin B12 prevents tau protein fibrillization, the reduced peak intensity of tau CD spectra in the presence of B12 emphasizes on formation of small oligomers. Although ThT results indicated complete fibrillization of CB-tau, CD spectra of aggregated CB-tau showed only a small decrease in ellipticity at 200 nm and no fibrillization (Fig. 4d). The disagreement in CD spectra and ThT fluorescence in the case of CB-tau could be due to the effect of alkylation of cysteine residues, a hydrophobic moiety, on ThT fluorescence properties. It has been shown previously that introduction of hydrophobic residues into protein sequence affects ThT fluorescence

27,28

. It also has been reported that

mutation of cysteine to alanine residues prevents aggregation of tau protein that explain CD results 21. Since both ThT fluorescence and CD spectroscopy are error prone methods for following aggregation progress, electron microscopy was used for observation of fibrils. TEM micrographs show that the presence of vitamin B12 effectively inhibits tau fibrillization, while vitamin B12 has no effect on aggregation extent of CB-tau (Fig. 5). The electron micrographs of aggregated CB-tau illustrate that blocking of cysteine residues of tau prevents formation of tau fibrils and only small oligomers are formed that was confirmed by CD spectra. However, vitamin B12 cannot prevent formation of these small oligomers.



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Figure 5. TEM micrographs of tau filaments in the absence (left panel) and presence (right panel) of vitamin B12. As the figure shows the presence of B12 effectively inhibits tau filament formation while has no effect on CB-tau aggregation.

In order to examine inhibitory effect of vitamin B12 on tau aggregation, aggregated proteins were loaded on a continuous SDS-polyacrylamide gel. SDS-PAGE analysis illustrated that vitamin B12 could efficiently prevents formation of tau aggregation (Fig. 6). The relatively equal intensity of monomeric tau bands on SDS-PAGE in various concentrations of vitamin B12 on one hand and the presence and absence of tau oligomers in TEM micrographs of aggregated tau in the presence of 10 and 80 µM vitamin B12, respectively, on the other hand proposes that tau oligomers are SDS-soluble. Contrary to the effect of vitamin B12 on tau aggregation, vitamin B12 has no significant inhibitory effect on CB-tau aggregation and formed oligomers, observed by TEM, are SDS-insoluble (Fig. 6). It is worth mentioning that the presence of monomeric CB-tau in the presence of 80 µM vitamin B12 could be due to prevention of aggregation at high concentrations of vitamin B12 14 ACS Paragon Plus Environment

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or SDS-solubility of aggregated CB-tau. SDS-PAGE results indicated that binding of tau protein to vitamin B12 causes formation of less condensed aggregates into which SDS could penetrate efficiently and solubilize the aggregates. However, the absence of vitamin B12 in the CB-tau aggregates results in highly condensed SDS-insoluble aggregates.

Figure 6. SDS-PAGE of aggregated proteins in the presence of various concentrations of protein. In each well 5 µg of aggregated protein was loaded. The numbers indicate the concentration of vitamin B12. The arrowhead indicates monomeric tau protein.

3. Conclusion Several studies have reported the deficiency of vitamin B12 in neurodegenerative diseases

15,16

. It has been illustrated that vitamin B12 deficiency causes deactivation of

phosphatase enzymes, especially PP2A, which are responsible for preventing tau protein hyper-phosphorylation 12. However, there is no report on the direct binding of vitamin B12 on tau protein. Tau protein has an extended structure with two accessible cysteine residues that provides tau with this ability to coordinate metal ions 10. Moreover, vitamin B12 is also able



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to interact with thiol groups 23. We hypothesized that vitamin B12 interacts with tau protein via cysteine residues and influences tau structure and function. To examine our hypothesis, interaction of cobalamin with tau and CB-tau and its effect on aggregation of two protein species was investigated. It can be concluded from our results that binding of cobalamin could directly binds to tau protein via cysteine residues of tau. Inhibition of continues thermal collapse of tau by cobalamin also proposes that conformation of tau/cobalamin complex is different from the conformation of temperature-dependent folded tau protein. The conformation of vitamin B12/tau protein complex hinders stacking of tau monomers and fibrillation of tau protein. Moreover, capping of cysteine residues of tau by vitamin B12 also enhances the inhibitory effect of vitamin B12 on tau aggregation. It is also noteworthy that although the effect of vitamin B12 deficiency in the Alzheimer’s brains mostly exerted via inactivation of PP2A and hyperphosphorylation of tau protein, direct binding of vitamin B12 to tau protein and tau aggregation inhibition as a result can be an alternative mechanism.

4. Material and methods Methylcobalamin, dithiothreitol (DTT), Ammonium sulfate and Thioflavin T (ThT) were purchased from Sigma Company. Vitamin B12, cobalamin and methylcobalamin have been used interchangeably throughout the text.

4.1. Tau purification Tau protein was purified by our previously published method

29

. Briefly, 1N/4R tau

protein was expressed in E. coli BL21 (DE3). The bacterial pellet was recovered and heated for 30 min in boiling water. The cooled bacterial suspension was reached to 2.5 % PCA and


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after 15 min at room temperature was centrifuged at 20000 g for 20 min. the supernatant was treated with Ammonium Sulfate (45% saturated) and was centrifuged as above. The pellet was resuspended and treated with 2% trichloroacetic acid. After centrifugation the pellet was treated with 2% TCA and centrifuged. The supernatant was removed as tau protein and buffer exchanged using a Sephadex G-25 desalting column.

4.2. Fluorescence spectroscopy Structural changes of tau protein upon binding of vitamin B12 was assessed by recording emission spectra of tau in the presence of various concentrations of vitamin at 25 and 37 °C. 1 ml of 1 µM tau protein solution in 20 mM Tris buffer containing 20 mM sodium sulfate was poured into a fluorescence cuvette. Protein solution was excited at 275 nm (slit 5 nm) and emission spectra were recorded form 285-380 (slit 10 nm) using a Carry Eclipse Fluorescence Spectrophotometer equipped with a Varian thermal controller. For measurement of melting point of tau protein, tau protein solution (10 µM) was incubated with various concentrations of vitamin B12 (10 and 20 µM). Protein solution was excited at 275 nm and emission was recorded in a gradual temperature increase from 30 to 80 °C.

4.3. Cysteine blocking Tau cysteine residues were alkylated using acrylamide 30. Briefly tau protein in 100 mM Tris buffer at pH 8.8 was reduced using 10 mM DTT for 2 h at 37 °C. Then, 2 M acrylamide was added to a protein solution and incubation was continued for additional 2 h. The buffer of cysteine-blocked tau (CB-tau) solution was exchanged using a Sephadex G-25 desalting column. The yield of blocking reaction was assessed using the Ellman’s assay according to the standard protocol 31, which showed higher than 85% blockade of cysteine residues. 17 ACS Paragon Plus Environment


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4.4. Tau protein assembly Tau protein solution (10 µM) in the presence and absence of 10 µM vitamin B12 and 1 mM DTT was incubated at 37 °C and reaction was initiated by addition of 0.1 mg/ml heparin. (1 mM of DTT was added daily to samples containing DTT till the end of aggregation process). Assembly process was followed by removing desired volume of the reaction mixture and incubated with ThT (20 µM) for 15 min. Increase in ThT fluorescence upon binding to aggregated proteins was recorded using the carry eclipse fluorimeter by excitation and emission at 344 and 370-600 nm, respectively.

4.5. Circular dichroism (CD) spectroscopy CD spectroscopy was used for measurement of conformational changes of tau protein in the presence of vitamin B12 and following protein assembly process in the presence and absence of DTT and B12. To assess the conformational changes of tau protein in the presence of B12, a solution of tau protein containing 10 µM protein was tittered with vitamin B12 and CD spectra was recorded using an AVIV CD Spectrophotometer. In order to recording of CD spectra of aggregated protein, aggregation reaction mixture was removed at the end of aggregation period and CD spectra was recorded.

4.6. Transmission Electron Microscopy (TEM) Aggregated tau protein was mounted on a carbon-coated mesh grid and incubated at ambient temperature for 2 min. the protein filaments were negatively stained using 2% uranyl acetate and were viewed by a Hitachi HU12A transmission electron microscopy.

4.7. SDS-PAGE analysis of aggregated proteins


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Aggregated proteins were loaded and separated on a 6% continuous SDS-PAGE using phosphate buffer system under reducing conditions

32

. In each well 5 µM of aggregated

protein was loaded. After electrophoresis run was finished the gel was stained with Coommassiee brilliant blue G250.

5. Author Contributions G.H. Riazi and K. Asadollahi conceived and managed the project; S. Rafiee performed most of the experiments. K. Asadollahi and S. Rafiee designed experiments, analyzed data and wrote the manuscript. S. Ahmadian contributed to TEM experiments, Saboury AA, contributed to the fluorescence data analysis and manuscript reviewing. G.H. Riazi contributed to data analysis and reviewed the manuscript.

6. Acknowledgement We kindly appreciate Ms. Ghasemi and Ms. Shafieezadeh for their help in performing fluorescence and TEM experiments.

7. References (1) Ballatore, C., Lee, V. M.-Y., and Trojanowski, J. Q. (2007) Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat. Rev. Neurosci. 8, 663–72. (2) LaFerla, F. M., and Oddo, S. (2005) Alzheimer’s disease: Aβ, tau and synaptic dysfunction. Trends Mol. Med. 11, 170–176. (3) Mandelkow, E. M., and Mandelkow, E. (2011) Biochemistry and cell biology of Tau protein in neurofibrillary degeneration. Cold Spring Harb. Perspect. Biol. (4) Leugers, C. J., Koh, J. Y., Hong, W., and Lee, G. (2013) Tau in MAPK activation. Front. 19 ACS Paragon Plus Environment


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Figure 1. Fluorescence quenching of tau and CB-tau protein by B12 at various temperatures. a; emission spectra of tau protein in the presence of various concentrations of B12 at 25 ℃. Tau fluorescence at 304 nm was quenched. Emission spectra at 360 and 405 nm was also changed that is due to conversion of methylcobalamin to thiolatocobalamin. b; fluorescence emission spectra of tau protein at 37 °C which shows quenching at 304 nm. Spectra has changed at 360 and 405 nm as well. However the change at these regions is less intense in comparison to 25 °C. c; Stern-Volmer plot for interaction of B12 with tau at 25 and 37 °C. The upward curvature of Stern-Volmer plot has decreased by temperature increase. Solid and opened circles represent plots at 25 and 37 °C, respectively. d and e; shows titration of B12 into CB-tau solution at 25 and 37 °C, respectively. Blockade of cysteine residues resulted in omission of spectral change at 360 and 405 nm that shows participation of cysteines in interaction. f; Stern-Volmer plot for titration of B12 into CBtau solution at 25 and 37 °C, solid and opened circles, respectively. The upward curvature has been removed after blocking of cysteines and both plot are similar in shape. g; titration of B12 into tau solution at 50 °C. Spectral change at 360 and 405 nm has omitted, like CB-tau quenching experiments, due to partial folding of tau at high temperatures that results in inaccessibility of cysteine residues. h; Stern-Volmer plot for quenching of tau fluorescence at 50 °C. The upward curvature has been omitted that indicates absence of thiolatocobalamin formation. In all experiments 1 µM of tau protein have been quenched with various concentrations of B12 up to 20 µM. The arrows indicate the regions of spectra affected by formation of thiolatocobalamin from methyl cobalamin. 132x97mm (300 x 300 DPI)

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Figure 2. Vitamin B12 prevents thermal collapse of tau protein. Continuous thermal collapse of tau protein has been prevented in the presence of vitamin B12. 10 µM tau protein, solid line, was incubated with 10 and 20 µM vitamin B12, dashed and long dashed line, respectively. The fluorescence intensities have been normalized against the fluorescence intensity of control sample to compensate the effect of vitamin B12 on fluorescence quenching of tau. 60x45mm (300 x 300 DPI)



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Figure 3. The effect of vitamin B12 on secondary structure of tau. a; secondary structural change of tau protein in the presence of various concentrations of B12 shows intense decrease in negative ellipticity at 200 nm. b; CD spectra of CB-tau in the presence of B12. The small effects of B12 on the structure of CB-tau shows that blocking of cysteine residues prevents interaction of cobalamin with tau protein. 10 µM of tau and CB-tau, solid line, have been tittered with 10, 30 and 50 µM of B12, dotted, dashed and long dashed lines, respectively. 131x210mm (300 x 300 DPI)


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Figure 4. Effect of B12 on tau and CB-tau aggregation. a; B12 effectively inhibits formation of tau fibrils. b; aggregation of CB-tau was not affected by methylcobalamin. Circles (black), Squares (Red), Triangles (green), Crosses (purple) and Plus marks (yellow) represent ThT fluorescence of aggregated proteins in the presence of 0, 10, 30, 50 and 80 µM vitamin B12 at various time periods. c; the CD spectra of aggregated tau in the absence and presence of various concentrations of vitamin B12. CD spectra of aggregated tau protein shows decrease in ellipticity and red-shift toward higher wavelengths that indicates tau protein fibrils have been formed. However the absence of redshift in the presence of B12 indicates the inhibition of fibril formation. d; the CD spectra of aggregated CB-tau in the presence of various concentrations of vitamin B12 shows that blocking of cysteine residues inhibits protein aggregation. However, decrease in elipticity at 200 nm could be due to formation of tau oligomers. CD spectra of monomeric protein, solid black line, and aggregated protein in the presence of 0, 10, 30, 50 and 80 µM of vitamin B12 is presented by Circles (black), Squares (Red), Triangles (green), Crosses (purple) and Plus marks (yellow), respectively. 137x108mm (300 x 300 DPI)



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Figure 5. TEM micrographs of tau filaments in the absence (left panel) and presence (right panel) of vitamin B12. As the figure shows the presence of B12 effectively inhibits tau filament formation while has no effect on CB-tau aggregation. 98x114mm (300 x 300 DPI)


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Figure 6. SDS-PAGE of aggregated proteins in the presence of various concentrations of protein. In each well 5 µg of aggregated protein was loaded. The numbers indicates the concentration of vitamin B12. The arrowhead indicates monomeric tau protein. 82x81mm (300 x 300 DPI)



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TOC graphics 39x19mm (600 x 600 DPI)


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Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine

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