Blocking the thiol at cysteine-322 destabilizes tau protein and prevents

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Blocking the thiol at cysteine-322 destabilizes tau protein and prevents its oligomer formation Hui Chen, Simu Liu, Shuiming Li, Jierui Chen, Jiazuan Ni, and Qiong Liu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00003 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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ACS Chemical Neuroscience

1

Blocking the thiol at cysteine-322 destabilizes tau protein and

2

prevents its oligomer formation

3 Hui Chen1,2,*,§, Simu Liu3,4,*, Shuiming Li4, Jierui Chen1, Jiazuan Ni1, Qiong Liu1,§

4 5 6

1

7

College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060,

8

China.

9

2

Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Sciences,

Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and

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Guangdong Province, College of Optoelectronic Engineering, Shenzhen University,

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Shenzhen 518060, China.

12

3

13

and Oceanography, Shenzhen University, Shenzhen 518060, China.

14

4

15

and Oceanography, Shenzhen University, Shenzhen 518060, China.

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*H.C. and S.M.Liu contributed equally to this work.

17

§

Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences

Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences

Corresponding Authors.

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Abnormal accumulation of tau protein into oligomers contributes to

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neuronal dysfunction. Reduction of tau level is potentially able to prevent its accumulation.

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Here we uncover a critical role of the free thiol at Cys-322 in determining tau stability. We

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found that the application of thiol-blocking agents like NEM or MMTS blocks this thiol, by

5

which it destabilizes tau protein and prevents its oligomer formation. Furthermore, we

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identified a tau-interacting protein, selenoprotein W, which attenuates tau accumulation by

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forming disulfide linkage between SelW Cys-37 and tau Cys-322. These findings provide

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a promising strategy to prevent tau accumulation and oligomer formation.

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KEYWORDS: oligomerization, selenoprotein, disulfide linkage, protein destabilization,

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Alzheimer’s disease

11 12

INTRODUCTION

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Aggregation of microtubule-associated protein tau into neurofibrillary tangles is one of the

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defining pathological features of Alzheimer’s disease1,2. Growing evidence suggests that

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soluble tau oligomers are the main toxic species in destroying neuron functions3.

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Interaction between tau molecules by Cysteine-322 (Cys-322)-mediated disulfide linkage

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is thought to initiate tau oligomer formation and its propagation4,5. Consistent with this,

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small molecules that bind to the cysteine residues could prevent the formation of toxic tau

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oligomers by hindering tau intermolecular interaction6.

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Reduction of tau level is beneficial to inhibit neuronal dysfunction in Alzheimer’s disease

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mouse model7-9. Previous studies have identified some endoproteolytic enzymes such as

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asparagine endopeptidase10 and caspase11 which degrade tau protein by proteolytic

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cleavage. However, these cleavages are unlikely to contribute to the specific reduction of

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tau level, on the contrary, the resulting degraded tau proteins generate more oligomers

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that promote development of Alzheimer’s disease. The E3 ubiquitin ligase CHIP (carboxyl

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terminus of Hsp70 interacting protein) targets tau for ubiquitin/26S proteasome-mediated

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protein degradation to defense against tau accumulation12. However, this pathway also

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does not function due to the impairment of 26S proteasome by abnormal aggregation of

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tau proteins under pathological condition13,14. The genome editing technology would be

30

efficient to delete tau gene, but complete deletion of tau protein may introduce unexpected

31

disorders such as brain insulin resistance15. 2


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Thus, we hypothesized that decreasing tau protein appropriately would be helpful to

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attenuate tau accumulation-caused neurotoxicity without bringing new disorders. By

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focusing on the Cys-322 residue, we found that blocking the thiol at Cys-322 can not only

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prevent tau oligomer formation but also initiate the destabilization of tau protein, which

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provides a potential strategy to reduce excessive tau proteins effectively.

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RESULTS AND DISCUSSION

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We first examined tau oligomerization in vitro. The tau proteins tagged with 6×His were

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expressed in E.coli to perform H2O2-induced oligomerization assays in vitro. To avoid

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unnecessary confusion, we numbered tau amino acids based on the longest tau isoform

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(isoform 2, amino acids 1-441)16. though we employed tau isoform 4 (amino acids 1-352)

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containing only one cysteine residue Cys-322 in all our experiments (Figure S1A).

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Full-length tau protein could form oligomer rapidly upon H2O2 treatment (Figure S1B), in

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contrast, substitution of Cys-322 with Ala (C322A) completely abolished the formation of

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tau oligomer (Figure S1C). To further examine whether the free thiol at Cys-322 is

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required for tau oligomer formation, we employed a thiol-blocking agent N-ethylmaleimide

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(NEM) in these assays (Figure S2A) and found it completely prevented H2O2-induced tau

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oligomer formation (Figure S2B). These results suggest the critical role of free thiol at

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Cys-322 in H2O2-induced tau oligomer formation.

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To further study the effects of NEM on tau oligomerization in vivo, HEK293T cells

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expressing Flag-tau proteins were treated with NEM. NEM treatment did not cause

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detectable cell apoptosis within 20 minutes (Figure S2C). Unexpectedly, the protein level

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of Flag-tau was observed to decrease with increasing concentration of NEM (Figure 1A).

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To confirm the observation that thiol-blocking is responsible for tau destabilization, we also

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employed the other two chemicals, S-methyl methanelthiosulfonate (MMTS) and

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isoproterenol (ISO), in this study (Figure S2A). The MMTS mediates a reversible

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thiol-blocking that differs from NEM17, and ISO can bind to the cysteine residues of tau

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protein6. Different from NEM and MMTS, pretreatment with ISO in a short time (10

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minutes) could not block H2O2-induced tau oligomer formation (Figure S2B), suggesting

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that the blockage of thiol by ISO is not a fast chemical reaction. Next, the lysates of

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HEK293T cells expressing Flag-tau were incubated with these chemicals. MMTS, like 3



1

NEM, could induces tau decrease, but ISO could not (Figure 1B), implying that the thiol

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group in the cysteine may contributes to tau stability.

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To test whether NEM could induce the destabilization of tau proteins that extracted from

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brain, lysates from the brain of triple transgenic model mouse of Alzheimer’s disease

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(3xTg mouse)18 were used. Results showed that tau proteins smaller than 180kDa were

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decreased by NEM treatment (Figure S3). As tau oligomers can spread between neurons

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via exosomes19. these findings suggest a potential application of thiol-blocking drugs in

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preventing tau oligomer formation and propagation.

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To provide further evidences that thiol-blocking at Cys-322 is involved in tau

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destabilization in vivo, we tried to identify the proteins, which bind to tau via disulfide

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linkage, would mimic the role of thiol-blocking agents in destabilizing tau protein. We

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identified a tau-interacting protein selenoprotein W (SelW), which is widely and highly

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expressed in brain20,21. by pull-down assay (Figure S4A). SelW contains two cysteine

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residues Cys-10 and Cys-37, and one selenocysteine (Sec, U) residue Sec-13 (Figure

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S5A). Sec is encoded by a traditional termination codon TGA, and is inserted into protein

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by a specific translational mechanism22. To efficiently express SelW proteins in E.coli and

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human cells, the Sec was substituted with Cys (SelWU13C) (Figure S5A). Intriguingly, the

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SelWU13C protein could form oligomers in the presence of H2O2 easily (Figure S5B), and

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the interactions between SelWU13C and tau could be weakened and enhanced by DTT and

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H2O2, respectively (Figure S4B).

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To analyze whether SelWU13C could form disulfide linkage with tau, purified recombinant

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His-tau and His-SelW U13C proteins were incubated with 1 mM H2O2. A protein band with an

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approximate molecular mass of 70 kilodaltons (kDa) was clearly appeared (Figure 2A),

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which we deduced to be a heterodimer formed by the combination of His-tau (~60 kDa)

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and His-SelW U13C (~10 kDa). This was supported by the observation that both His-tau and

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His-SelW U13C monomer were decreased but tau-SelW U13C dimer was increased at a

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time-dependent manner (Figure 2A).

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Since substitution of tau Cys-322 with Ala (C322A) completely destroyed the formation

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of tau-SelWU13C dimer (Figure S6A), we speculated that SelWU13C should bind to tau

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through disulfide linkage. To identify which cysteines are involved in disulfide linkage

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between SelW U13C and tau, SelW U13C proteins with various substitutions at their cysteines 4


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were analyzed. Individual substituting SelWU13C Cys-10 or Cys-13 with Ser (C10S or

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C13S), and co-substituting SelWU13C Cys-10 and Cys-13 with Ser (C10S/C13S) did not

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detectably affect the formation of SelWU13C oligomer (Figure S5C) and tau-SelWU13C dimer

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(Figure S6B). However, substituting SelW U13C Cys-37 with Ser (C37S) distinctly prevented

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the formation of oligomer in all species (Figure S5C and S6B). Thus, the SelWU13C Cys-37

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is uniquely required for formation of disulfide linkage with tau Cys-322.

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To confirm the identified disulfide-linked cysteines, we performed mass spectrometry

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analysis using the tau-SelWU13C dimer samples shown in Figure 2A. An ion with a mass

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matching the theoretic mass of a disulfide linkage between peptides of tau and SelW U13C

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was detected from the sample with tryptic digestion. The expected sequences of tau and

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SelWU13C were identified from peptide fragments by MS/MS (Figure 2B). These data

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support that the disulfide linkage of tau and SelW U13C is formed through tau Cys-322 and

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SelWU13C Cys-37.

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To study whether SelW U13C could attenuate tau accumulation in human cells, plasmids

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expressing tau and SelWU13C tagged with Flag and Myc respectively were co-transfected

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into HEK293T cells. Immunoblot analysis showed that Flag-tau proteins were diminished

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with increasing expression of SelW U13C-Myc proteins (Figure 3A). As expected,

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abolishment of the disulfide linkage broke SelW U13C’s effect on Flag-tau accumulation

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(Figure 3B). Additionally, SelW U13C with Ser substitutions at both Cys-10 and Cys-13 did

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not affect the protein levels of Flag-tau (Figure 3C). Thus, formation of disulfide linkage

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between SelW U13C Cys-37 and tau Cys-322 is a key step to destabilize tau protein.

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As SelW U13C also contains thiol groups, which could be targeted by NEM like tau protein

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(Figure S7A and S7B), we examined whether NEM could also cause SelW U13C

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destabilization. Results showed that the treatment with NEM could only reduce Flag-tau

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level but not SelW U13C-Myc level (Figure S7C and S7D). Thus, protein destabilization

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resulted from thiol-blocking could only be considered as a mechanism specific for

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regulating the abundance of tau protein.

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Finally, we expressed Flag-tau and Flag-tau mutant (C322A) in human cells, and

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detected the levels of corresponding messenger RNAs (mRNA) and proteins by RT-PCR

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and immunoblot analysis, respectively. Although the mRNA levels of Flag-tau and

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Flag-tau (C322A) showed similar results, their protein levels altered significantly (Figure 5



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4A). In addition, we found that treatment with thiol-blocking agents did not lead to

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detectable change of mRNA levels of tau and tau (C322A) (Figure S8). Mutation at

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Cys-322 severely induced tau destabilization, revealing that Cys-322 is a key residue

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responsible for the stability of tau protein. These results support the observations that

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blocking the thiol at Cys-322 destabilizes tau protein.

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Previous studies have reported that ubiquitination of cysteine contributes to protein

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degradation23,24. which reveal a potential role of cysteine in regulating protein stability. Our

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data demonstrate that blocking the thiol at Cys-322 destabilizes tau protein in human cells.

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However, the mechanism involving in this is still unclear. Tau destabilization induced by

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thiol-blocking only happens in cells or cell crude extracts, suggesting that other proteins in

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human cells are also required for the reduction of tau abundance. Blocking the thiol at

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Cys-322 may cause conformational alteration25 which may possibly initiate the

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intracellular signaling for protein degradation or it may facilitate the interaction of tau with

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some proteins in vivo to induce the cleavage of tau. The precise mechanism remains to be

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investigated extensively.

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Considering that the Cys-322 residue is also required for tau oligomer formation, further

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investigations should elucidate the relationship between tau aggregation and stabilization.

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As SelW directly binds to tau through disulfide linkage and facilitates tau destabilization, it

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will be interesting to examine whether SelW could control tau abundance in brain.

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CONCLUSION

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In summary, we present a strategy to prevent tau accumulation and oligomer formation

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by blocking the thiol at tau Cys-322 using the thiol-blocking agents NEM and MMTS.

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Moreover, we identified a tau-interacting protein SelW, which attenuates tau accumulation

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in human cells by forming disulfide linkage between SelW Cys-37 and tau Cys-322. As

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both NEM and MMTS are general thiol-blocking agents, those provide a new way to

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design anti-tauopathy drugs via blocking the thiol at tau Cys-322. In addition, exploring the

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precise biochemical mechanism underlying tau destabilization induced by thiol-blocking

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will be helpful to discover new targets for treating tauopathy-caused disease including

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Alzheimer’s disease.

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METHODS

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Plasmid construction. For recombinant protein expression, full-length tau and SelW

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were amplified by PCR and introduced into the vector pET28a to generate pET8a-tau and

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pET28a-SelW expression plasmids using the EcoRI/XhoI sites. For protein expression in

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human cells, full-length tau and SelW was amplified by PCR and introduced into the

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modified vector pCDNA3.1 (Invitrogen) to generate pCDNA-tau and pCDNA-SelW

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expression plasmids using the EcoRI/XhoI sites. Substitution mutations were generated

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by PCR-mediated site-directed mutagenesis. All constructs were confirmed by DNA

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sequencing. PCR primers used in this study were listed in Table S1.

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Preparation of recombinant proteins. The pET28a-based expression plasmids were

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transformed into E.coli BL21 (DE3). The bacteria were cultured in liquid medium at 37ºC

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till the OD600 reaches to 0.5-0.6, then isopropyl-β-D-thiogalactopyranoside (IPTG) was

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added into the culture at a final concentration of 0.3 mM. The culture was incubated for

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additional 4 hours at 37ºC to induce the expression of recombinant proteins. The bacteria

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were collected then lysed by ultrasonic cell crusher and cleared by filtration. Recombinant

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proteins with His-tags were purified using Ni Sepharose (GE Healthcare) following the

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manufacturer’s instructions.

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Pull down assay. Crude proteins were extracted from E.coli cells expressing

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recombinant His-tau proteins using pull down buffer [PBS containing 0.1% Triton X-100

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and 1 mM phenylmethyl sulfonyl fluoride (PMSF; Sigma)]. The H2O2 or DTT with indicated

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concentration was added into pull down buffer to test their effects. For GST pull down

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assay, GST-SelW U13C proteins bound to Glutathione Sepharose bead were added into the

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crude extracts and incubated at 4ºC for 2 hours with gentle agitation. Sepharose beads

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were collected by centrifugation at 500g for 5 minutes. After washing five times with PBS

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buffer, the beads were resuspended in 2× SDS-PAGE sample buffer without reducing

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agent. The samples were heated at 95ºC for 5 minutes, and then separated by

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electrophoresis on 10% SDS-PAGE gels, followed by immunoblot using anti-His antibody

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(Proteintech). After immunoblotting, the PVDF membrane was stained using ponceau.

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In vitro H2O2-induced protein oligomerization. After His tagged recombinant proteins

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(100 µM) mixing in buffer containing 50 mM NaH2PO4, 300 mM NaCl (pH7.4), H2O2 (1 mM

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or indicated concentration) was added and incubated at 37ºC for 30 minutes. The reaction 7



1

was stopped by adding 5×SDS-PAGE sample buffer without reducing agent. The samples

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were heated at 95ºC for 5 minutes, and then separated by electrophoresis on 12%

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SDS-PAGE gels, followed by coomassie staining.

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Mass spectrometric analysis of disulfide linkage. Proteins in gel slices were

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digested with trypsin. The peptide samples were resuspended in buffer (0.1% formic acid,

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2% acetonitrile), then was subjected to Triple-TOF 5600 mass spectrometry equipped with

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Eksigent nano LC-Ultra 1D plus system. Chromatographic separations were conducted

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on a reverse-phase capillary column (75 µm×15 cm C18-3µm 120 Å, ChromXP Eksigent)

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with a gradient from starting with 5% of buffer B (acetonitrile containing 0.1% formic acid

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and 2% water) in 5 min, 35% of buffer B in 35 min, to final 80% of buffer B in 5 min. Mass

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spectra were obtained in Information Dependent Acquisition mode (IDA), survey scan of

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TOF-MS was obtained in 250 ms, and MS/MS spectra were obtained in high sensitivity

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mode with accumulation time of 80 ms per spectra. A maximum of 30 precursor ions was

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allowed within each cycle. The threshold intensity of the precursor ions was 200 cps with a

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charge state from +2 to +5. Dynamic exclusion was set for 10 s. The ionization

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parameters were set as follows, ion spray voltage of 2300 V, curtain gas of 35 psi,

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nebulizer gas of 5 psi, and interface heat temperature of 150ºC. For protein identification,

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the MS/MS raw data were analyzed using Protein Pilot software v.4.5 (AB Sciex Inc., USA)

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to search against UniProt human proteome database. Disulfide-linked peptides were

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annotated by PEAKS AB software (Bioinformatics Solutions Inc., Waterloo, ON, Canada).

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Cell culture and plasmid transfection. HEK293T cells were routinely cultured at 37ºC

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in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies) supplemented with 10%

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(v/v) fetal bovine serum (Biological Industries), 100 U/ml of penicillin and 100 µg/ml

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streptomycin, in humidified 5% (v/v) CO2 air. HEK293T cells were seeded at a density of

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approximately 2.5×105 cells/well on a 6-well culture plate and transfected with

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Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. Briefly, plasmid

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(1~2 µg) diluted in DMEM medium and mixed well with P3000 reagent (2 µl/µg DNA) was

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added into DMEM-diluted Lipofectamine 3000 reagent (1:1 ratio), and incubated for 15

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min at room temperature before applying to host cells. 2 ml fresh medium containing 200

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µg/ml G418 was added to each well. For pCDNA-tau and pCDNA-SelW co-transfection, 6

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hours after pCDNA-tau transfection, plasmid pCDNA-SelW was transfected into the same 8


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culture plate. After cultured overnight, the medium was replaced with 2 ml fresh medium

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without G418. The cells were usually harvested 48 hr later for RT-PCR or immunoblot

3

analyses.

4

Chemical treatments. For analyzing chemical effects on H2O2-induced protein

5

oligomerization,

100

µM

N-ethylmaleimide

(NEM;

Sangon),

or

S-methyl

6

methanethiosulfonate (MMTS; Sigma), or isoproterenol (ISO; Sigma) was added into the

7

His tagged recombinant proteins mixing in buffer containing 50 mM NaH2PO4, 300 mM

8

NaCl (pH7.4). After pretreated at 37ºC for 10 minutes, H2O2 (1 mM) was added and

9

incubated at 37ºC for 30 minutes. For analyzing chemical effects on protein stability, the

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2.5×106 transfected HEK293T cells were incubated for 10 min with NEM in indicated

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concentration followed by protein extraction, or crude extracts from the cells were

12

incubated for 10 min in 100 µM NEM, MMTS or ISO. Proteins were detected by

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immunoblot analysis. For analyzing chemical effects on gene expression, the 2.5×106

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transfected HEK293T cells were incubated for 10 min with 100 µM NEM or MMTS

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followed by RNA extraction. mRNA levels were detected by RT-PCR analysis.

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Annexin V-EGFP apoptosis detection. Apoptotic HEK293T cells were identified using

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the Anaexin V-EGFP Apoptosis Detection Kit (Beyotime) following the manufacturer’s

18

instructions. Briefly, after cells were incubated with Annexin V-EGFP for 10 minutes, 100

19

µM NEM was supplied to treat cells at indicated time points. The cells were imaged by

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confocal laser-scanning microscopy (CLSM). The apoptosis inducer Kit (C0005; Beyotime)

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was purchased for positive control assays.

22

RNA extraction and RT-PCR. Total RNA was extracted using RNAiso Plus (Takara)

23

following the manufacturer’s instructions. Reverse transcription reaction was carried out

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using PrimeScript RT reagent Kit with gDNA Eraser (Takara) following the manufacturer’s

25

instructions. PCR was performed using the primers listed in Table S2. The relative

26

expression level of tau gene was normalized to Actinβ expression level. Three

27

independent experiments were performed.

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Protein extraction and immunoblot analysis. HEK293T cells or mouse brain tissues

29

were washed once with PBS and lysed in cell lysis buffer [20 mM Tris (pH7.5), 150 mM

30

NaCl, 1% Triton X-100, 1 mM PMSF (Sigma), 1×Protease inhibitor cocktail (Roche),

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1×phosphatase inhibitor cocktail (Roche)]. Cell lysates were placed on ice for 20 min, 9



1

quickly frozen in liquid N2 once, and kept on ice for 30 min. After centrifuging at 13,000g at

2

4ºC for 10 min, the supernatant was collected and heated at 95ºC for 5 minutes with

3

sample loading buffer, and then separated by electrophoresis on 10% SDS-PAGE gels.

4

Mouse monoclonal anti-tau5 (1:5000; Abcam) and anti-Myc (1:2000; Proteintech)

5

antibodies were used to detect Flag-tau and SelW-Myc proteins, respectively. Actin used

6

for loading control was detected by anti-Actinβ (1:1000; Proteintech) antibody. Bands in

7

immunoblots were visualized by Pierce ECL Western Blotting Substrate (Thermo Fisher

8

Scientific) and quantified by densitometry using ImageJ software program.

9 10 11

ASSOCIATED CONTENT

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Supporting Information is available.

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AUTHOR INFORMATION

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Corresponding Authors

16

§

Email: [email protected].

17

§

Email: [email protected].

18

ORCID

19

Hui Chen: 0000-0001-8879-2185

20

Simu Liu: 0000-0002-0374-3454

21

Author Contributions

22

H.C. conceived this study. H.C. and S.M.Liu designed the research and analyzed the data.

23

Q.L. and J.Z.N. supervised the project. H.C. performed most of the experiments, assisted

24

by S.M.Liu and J.R.C. S.M.Li performed mass spectrometry. H.C. interpreted the results.

25

S.M.Liu, Q.L. and J.Z.N. discussed the results. H.C. and Q.L. wrote the manuscript. All

26

authors read and commented on the manuscript.

27

Notes

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The authors declare no competing interest.

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ACKNOWLEDGMENTS

2

We thank K.Q.Ye (Emory University) for providing pET28c-tau plasmids; N.Jin and

3

L.M.Huang (Shenzhen University) for providing 3×Tg mouse materials. This work was

4

supported by National Natural Science Foundation of China (31470804) and Shenzhen

5

Bureau of Science, Technology and Information (JCYJ20150529164656093).

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1

FIGURE LEGENDS

2

Figure 1. Blocking the thiol at Cys-322 destabilizes tau protein. (A) Treatment with NEM

3

reduces Flag-tau abundance within HEK293T cells. (B) Thiol-blocking agents reduce

4

Flag-tau abundance in cell-free system. Protein crude extracts from HEK293T cells

5

expressing Flag-tau were used in these assays. The Flag-tau was detected using anti-tau

6

(tau 5) antibody, and the Flag-tau protein level in the absence of chemical was set as

7

100%. Actin was used as a loading control. Error bars represent SD. Three biological

8

repeats were performed and analyzed.

9 10

Figure 2. SelW binds to tau protein via disulfide linkage. (A) Analysis of the interaction

11

between tau and SelWU13C in the presence of H2O2 (1 mM) over the indicated time course.

12

The SDS-PAGE gel was stained using coomassie. (B) Identification of the cysteines

13

involved in the disulfide linkage between tau and SelW U13C by mass spectrometric

14

analysis. The protein of tau-SelW U13C dimer shown in (A) was digested for MS analysis.

15 16

Figure 3. SelW U13C attenuates tau accumulation by forming disulfide linkage. Protein

17

crude extracts from HEK293T cells co-transfected with Flag-tau and SelWU13C-Myc

18

plasmids with indicated quantity were analyzed in these assays. (A) The levels of Flag-tau

19

protein is decreased with increasing expression of SelW U13C-Myc proteins. (B) The

20

increasing SelW U13C (C37S)-Myc proteins does not significantly affect tau protein levels.

21

(C) Mutants of SelW U13C-Myc with various cysteine substitutions except Cys-37 does not

22

alter Flag-tau protein levels. The Flag-tau was detected using anti-tau (tau 5) antibody,

23

and the level of Flag-tau protein in the absence of SelW U13C-Myc was set as 100%. Actin

24

was used as a loading control. Error bars represent SD. *** P

Blocking the thiol at cysteine-322 destabilizes tau protein and prevents

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