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ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78
Biochemical and Biophysical Research Communications xxx (2017) 1e4
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78 Masafumi Sakono*, Tomoya Kidani Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-855, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 August 2017 Accepted 31 August 2017 Available online xxx
Neuronal cell death induced by an accumulation of amyloid beta (Ab) peptides, which are pathogenic molecules for Alzheimer's disease, is closely related with endoplasmic reticulum (ER) stress. In the ER stress condition, part of the ER resident chaperones is known to be translocated to another cellular location, such as the cell surface. The ER chaperone 78-kDa glucose-regulated protein (GRP78), which shows ATP-dependent chaperone activity, also shows translocation to the cell surface. In this study, we examined the influence of GRP78 on Ab fibrillation in the presence or absence of ATP. We revealed that a small amount of GRP78 effectively inhibited fibrillation of Ab fragments. Intriguingly, the fibrillation inhibition by GRP78 was confirmed in the absence of ATP, suggesting GRP78 exhibited ATP-independent interaction with Ab fragments. © 2017 Elsevier Inc. All rights reserved.
Keywords: Amyloid beta GRP78 Molecular chaperone Proteineprotein interaction
1. Introduction Amyloid beta (Ab) peptides are widely known as pathogenic molecules for Alzheimer's disease (AD) [1]. They form a fibrillar assembly consisting of intermolecular b-sheet formation [2]. Ab aggregates found in hippocampal senile plaques are believed to induce neural cell death, resulting in short-term memory impairment through hippocampal denaturation [3]. Ab cleaved from the transmembrane amyloid precursor protein (APP) is released into extracellular space. Subsequently, amyloid aggregation is caused by deposition of Ab fragments and structural change of Ab on the cell surface, such as with lipid rafts [4]. Neuronal cell death induced by Ab accumulation in the AD is closely related with endoplasmic reticulum (ER) stress [5]. A translocation of the ER chaperone to another cellular location, such as the cell surface, in the ER stress condition was recently found [6], raising the possibility of interaction between Ab fragments and ER chaperones. Our previous report indicated that ER resident chaperones such as calreticulin, protein disulfide isomerase, and ERp57 effectively inhibited Ab fibrillation [7]. This implied that ER resident chaperones exhibited good interaction with Ab fragments. The 78-kDa glucose-regulated protein (GRP78), referred to as
BiP or HSPA5, is one of the ER resident molecular chaperones, and serves multiple roles in maintaining cell viability [8,9]. The major function of GRP78 is facilitating transportation of new-born proteins and their folding assistance. GRP78 is included in the heat shock protein 70 (Hsp70) protein family, and the amino acid sequence of Hsp70 is highly conserved. Similar to Hsp70, both the ATPase and substrate recognition domains are contained within GRP78 [10]. Binding and hydrolysis of ATP play crucial roles in preventing misfolding and aggregation of nascent polypeptides [11e13]. In other words, GRP78 shows ATP-dependent chaperone activity for inhibiting protein aggregation. A number of studies have reported decrease of Ab40 and Ab42 accompanying interaction between GRP78 and APP [14,15]. Moreover, translocation of GRP78 to the cell surface was found in the ER stress condition [16,17], suggesting the possibility that GRP78 interacts with Ab fragments. In the present study, we examined the influence of GRP78 addition on Ab fibrillation in the presence or absence of ATP. Our results indicated that a small amount of GRP78 effectively inhibited Ab elongation. We also confirmed fibrillation inhibition by GRP78 in the absence of ATP, suggesting GRP78 exhibited ATP-independent interaction with Ab fragments.
* Corresponding author. E-mail address: [email protected] (M. Sakono). http://dx.doi.org/10.1016/j.bbrc.2017.08.162 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
2
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
solutions at 80 C. Concentration of Ab monomers was measured using the Bradford protein assay, with bovine serum albumin as a control. Ab monomer solutions were gently thawed on ice and diluted in PBS buffer containing GRP78 and ATP. The mixtures containing 20 mM Ab were incubated for 24 h at 37 C and without agitation. 2.4. Confirmation of Ab fibrillation by ThT assay Ab fibrillation was assessed by ThT assay in accordance with previous reports [7]. Briefly, Ab solution was mixed with PBS buffer containing 20 mM ThT, and the final Ab concentration was adjusted to 0.5 mM. The mixture was incubated for 5 min at room temperature. Emission fluorescent intensity was monitored at 485 nm through excitation at 445 nm using a spectrofluorometer (LS-55; PerkinElmer Inc., Waltham, MA, USA). 2.5. Immunoblotting analysis
Fig. 1. SDS-PAGE profiles of recombinant human GRP78.
2. Materials and methods 2.1. Materials Ammonium solution and adenosine 50 -triphosphate disodium salt trihydrate (ATP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Thioflavin T (ThT) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Human Ab (142) was obtained from Peptide Institute (Osaka, Japan). PVDF membrane was purchased from EMD Millipore (Billerica, MA, USA). Nitrocellulose membrane was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Ab antibody (6E10) was obtained from BioLegend (San Diego, CA, USA). Rabbit anti-mouse IgG conjugated with horseradish peroxidase was purchased from Abcam (Cambridge, UK). ECL Prime Western Blotting Detection Reagent was purchased from GE Healthcare (Little Chalfont, UK).
SDS-PAGE and native-PAGE of Ab samples were performed using 14% Tris-Glycine gels. After gel electrophoresis, separated proteins were transferred to PVDF membranes, using a current constant of 100 mA for 1 h. Incubated Ab samples were centrifuged at 15,000 rpm for 20 min for dot-blotting analysis. Supernatants and precipitates were collected separately, with precipitates resuspended in fresh PBS solution. Dot-blotting was performed by spotting each Ab sample solution directly onto nitrocellulose membrane. For immunodetection, membranes were blocked overnight at 4 C with 5% skimmed milk in PBS. After blocking, membranes were incubated with a mouse monoclonal Ab antibody (6E10, 1:2000) for 1 h at 37 C, followed by secondary horseradish peroxidaseconjugated anti-mouse IgG (1:10000). Proteins were visualized using the ECL Prime Western Blotting Detection Reagent, as per the manufacturer's instructions. 2.6. Morphology observation using transmission electron microscopy Samples were placed on carbon-coated copper grids. Excess sample was removed using filter paper, and the grids were air-
2.2. Expression and purification of recombinant GRP78 A cDNA encoding human GRP78 was cloned into pCold I expression plasmid (Takara Bio Inc., Otsu, Japan), which is designed to produce N-terminally (His)6-tagged proteins. The obtained plasmid was transformed into BL21 cells, and the recombinant proteins were expressed and purified using Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany), as per the manufacturer's instructions. The purified GRP78 was submitted into an NAP-5 column (GE Healthcare) equilibrated with PBS buffer. GRP78 dissolved in PBS was then obtained. 2.3. Preparation of Ab fibril Purchased lyophilized Ab was fully dissolved in 0.1% ammonium solution, and the solution was then centrifuged at 15,000 rpm for 3 h to remove seeds. Supernatants containing Ab monomers were collected into microtubes and stored as Ab monomer stock
Fig. 2. Influence of GRP78 on Ab fibrillation. Fibrillation was assessed using the ThT fluorescence method. Incubated samples containing chaperones with or without ATP were mixed with 20 mM ThT solution, and the final Ab concentration adjusted to 0.5 mM. Emission fluorescent intensity was monitored at 480 nm by excitation at 445 nm, using a spectrofluorometer.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
3
dried. Samples on grids were negatively stained using uranyl acetate, with excess stain removed by air-drying. Samples were observed using an electron microscope (JEM-1400TC; JEOL Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Effect of ATP on inhibition of Ab fibrillation by GRP78 A human GRP78 was prepared by expression from Escherichia coli. An expression and purification of recombinant GRP78 was confirmed by SDS-PAGE (Fig. 1). An obvious single band appeared around the estimated molecular weight (74 kDa). Therefore, a preparation of GRP78 was considered to have been successfully conducted. The effect of GRP78 addition on Ab fibrillation was investigated. The mixtures were subjected to ThT assay after incubation of Ab with appropriate concentration of the GRP78. Fig. 2 shows the effect on Ab fibrillation of GRP78 in the presence or absence of ATP. The intensity was dramatically decreased by addition of GRP78 with ATP compared with the ThT fluorescence intensity of the incubated Ab only sample. Addition of GRP78 greater than 1 mM evidently inhibited aggregation of 20 mM Ab effectively. Therefore, it was found that a small amount of GRP78 in the presence of ATP effectively inhibited Ab fibrillation. Intriguingly, similar reduction of ThT intensity also occurred in the absence of ATP. Dose-dependent addition of GRP78 without ATP decreased ThT intensity, and addition of 1 mM GRP78 fully inhibited ThT binding. This result implied that the inhibition of Ab fibrillation by GRP78 was carried out in an ATP-independent manner.
3.2. Ab structure maintained as monomer state in the presence of GRP78 Various electrophoresis and immunoassays were performed to investigate the details of the structural state of Ab in the presence of GRP78 without ATP. As shown in Fig. 3A, the bands appeared clearly in the presence of >0.1 mM GRP78 at the same position of Ab monomer. SDS-resistant aggregation appeared to have been produced, such as Ab fibril, because disappearance of the band in the lane of GRP78 < 0.01 mM was confirmed. Therefore, manifestation of bands by addition of GRP78 implied inhibition of amyloid aggregates formation. Next, the Ab structure under a non-denaturing condition was investigated. The Ab samples after incubation were applied to dotblot analysis and Native-PAGE. The result of the dot-blot analysis indicated addition of GRP78 did not form insoluble Ab aggregation, as most of the Ab contained within the sample that included >0.1 mM GRP78 was confirmed at the supernatant (Fig. 3B). Similar to the result for SDS-PAGE, appearance of bands in the lane of GRP78 > 0.1 mM was ascertained at the same position of Ab monomer as in the Native-PAGE (Fig. 3C). Production of Ab oligomer was not observed, as the other bands did not appear anywhere in the polyacrylamide gel. Therefore, Ab was suggested to exist as a monomer state, but not oligomer, in the presence of an appropriate concentration of GRP78.
Fig. 3. Analysis of structural state of Ab in the presence of GRP78 without ATP. (A) SDSPAGE analysis of Ab solutions. Samples incubated with 20 mM Ab and various concentrations of GRP78 were separated by SDS-PAGE, probed using Ab antibody, and
visualized using chemiluminescence. Ab M and Ab F represent Ab monomers and Ab fibrils, respectively. (B) Dot-blot analysis of Ab solutions. Samples were spotted onto nitrocellulose membrane, probed using an Ab antibody, and visualized by chemiluminescence. Sup and Pre represent supernatants and precipitates, respectively. (C) Native-PAGE analysis of Ab solutions. Samples incubated with 20 mM Ab and various concentrations of GRP78 were separated by native-PAGE, probed using an Ab antibody, and visualized by chemiluminescence.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
4
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 4. Morphology of Ab aggregates in the presence of GRP78. The scale bar indicates 200 nm.
3.3. Morphology observation of Ab by transmission electron microscopy Morphology of Ab included in the samples after incubation was observed using an electron microscope (Fig. 4). Although a wellknown fibrillar structure was observed in the sample containing Ab only, such characteristic structures were not verified in the presence of GRP78. Moreover, the effect of GRP78 addition on the morphology of Ab was not dependent on the presence of ATP. Thus, microscopic observation also indicated GRP78 effectively inhibited formation of an Ab fibrillar structure regardless of whether ATP was present. Considering the above results, it was revealed Ab elongation was dramatically suppressed by GRP78 even if the ATP was absent. Our findings seemed to not be consistent with the general consensus because GRP78 has been well known as an ATP-dependent molecular chaperone [9]. A previous study reported amyloid aggregation of human amylin (hA) was effectively inhibited by GRP78 and Hsp70 without ATP [18]. This result was quite similar with ours, indicating that the Hsp70 protein family, such as GRP78, probably functioned to inhibit intermolecular b-sheet formation without ATP. Although ATP is generally believed to be required for changing the tertiary structure of GRP78, it was presumed that such structural change were not necessary for interaction between GRP78 and amyloidogenic peptides. Furthermore, it was clarified that inhibition of fibrillation was carried out with a small amount of GRP78 as compared with Ab. A similar tendency was also observed in the case of interaction between hA and GRP78, implying interactions between amyloidogenic peptides and GRP78 repeatedly associate and dissociate in a short period of time. The association and dissociation is presumed to probably cause inhibition of bsheet transition of Ab. Therefore, the Hsp70 family, such as GRP78, would be beneficial for suppressing Ab-induced cellular stress. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgements We thank the University of Toyama Life Science Research Center for its assistance with transmission electron microscopy analysis. The present study was supported by a research grant from the Toyama First Bank Scholarship Foundation (to M.S.).
References [1] M.P. Murphy, H.L. Iii, Alzheimer's disease and the b-amyloid peptide, J. Alzheimer's Dis. 19 (2010) 311e323. [2] M. Sunde, L.C. Serpell, M. Bartlam, P.E. Fraser, M.B. Pepys, C.C.F. Blake, Common core structure of amyloid fibrils by synchrotron x-ray diffraction, J. Mol. Biol. 273 (1997) 729e739. [3] G. Irvine, O. El-Agnaf, Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases, Mol. Med. 14 (2008) 451e464. [4] K. Yanagisawa, Role of gangliosides in Alzheimer's disease, Biochim. Biophys. Acta - Biomembr. 1768 (2007) 1943e1951. [5] A. Salminen, A. Kauppinen, T. Suuronen, K. Kaarniranta, J. Ojala, ER stress in Alzheimer's disease: a novel neuronal trigger for inflammation and Alzheimer's pathology, J. Neuroinflammation 6 (2009) 41. [6] V.R. Wiersma, M. Michalak, T.M. Abdullah, E. Bremer, P. Eggleton, Mechanisms of translocation of ER chaperones to the cell surface and immunomodulatory roles in cancer and autoimmunity, Front. Oncol. 5 (2015) 7. [7] K. Kitauchi, M. Sakono, Glycoprotein quality control-related proteins effectively inhibit fibrillation of amyloid beta 1e42, Biochem. Biophys. Res. Commun. 481 (2016) 227e231. [8] M. Wang, S. Wey, Y. Zhang, R. Ye, A.S. Lee, Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders, Antioxid. Redox Signal. 11 (2009) 2307e2316. [9] C. Casas, GRP78 at the centre of the stage in cancer and neuroprotection, Front. Neurosci. 11 (2017) 177. €€ [10] M. Daugaard, M. Rohde, M. Ja attel€ a, The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions, FEBS Lett. 581 (2007) 3702e3710. [11] C.K. Kassenbrock, R.B. Kelly, Interaction of heavy chain binding protein (BiP/ GRP78) with adenine nucleotides, EMBO J. 8 (1989) 1461e1467. [12] J. Wei, J.R. Gaut, L.M. Hendershot, In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis, J. Biol. Chem. 270 (1995) 26677e26682. [13] L. Hendershot, J. Wei, J. Gaut, J. Melnick, S. Aviel, Y. Argon, Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 5269e5274. [14] Y. Yang, R.S. Turner, J.R. Gaut, The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases a 40 and A 42 secretion, J. Biol. Chem. 273 (1998) 25552e25555. [15] T. Hoshino, T. Nakaya, W. Araki, K. Suzuki, T. Suzuki, T. Mizushima, Endoplasmic reticulum chaperones inhibit the production of amyloid-b peptides, Biochem. J. 402 (2007) 581e589. [16] Y. Zhang, R. Liu, M. Ni, P. Gill, A.S. Lee, Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP, J. Biol. Chem. 285 (2010) 15065e15075. [17] Y.L. Tsai, Y. Zhang, C.C. Tseng, R. Stanciauskas, F. Pinaud, A.S. Lee, Characterization and mechanism of stress-induced translocation of 78-kilodalton glucose-regulated protein (GRP78) to the cell surface, J. Biol. Chem. 290 (2015) 8049e8064. [18] V. Chien, J.F. Aitken, S. Zhang, C.M. Buchanan, A. Hickey, T. Brittain, G.J.S. Cooper, K.M. Loomes, The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of b-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes, Biochem. J. 432 (2010) 113e121.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78 Masafumi Sakono*, Tomoya Kidani Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-855, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 August 2017 Accepted 31 August 2017 Available online xxx
Neuronal cell death induced by an accumulation of amyloid beta (Ab) peptides, which are pathogenic molecules for Alzheimer's disease, is closely related with endoplasmic reticulum (ER) stress. In the ER stress condition, part of the ER resident chaperones is known to be translocated to another cellular location, such as the cell surface. The ER chaperone 78-kDa glucose-regulated protein (GRP78), which shows ATP-dependent chaperone activity, also shows translocation to the cell surface. In this study, we examined the influence of GRP78 on Ab fibrillation in the presence or absence of ATP. We revealed that a small amount of GRP78 effectively inhibited fibrillation of Ab fragments. Intriguingly, the fibrillation inhibition by GRP78 was confirmed in the absence of ATP, suggesting GRP78 exhibited ATP-independent interaction with Ab fragments. © 2017 Elsevier Inc. All rights reserved.
Keywords: Amyloid beta GRP78 Molecular chaperone Proteineprotein interaction
1. Introduction Amyloid beta (Ab) peptides are widely known as pathogenic molecules for Alzheimer's disease (AD) [1]. They form a fibrillar assembly consisting of intermolecular b-sheet formation [2]. Ab aggregates found in hippocampal senile plaques are believed to induce neural cell death, resulting in short-term memory impairment through hippocampal denaturation [3]. Ab cleaved from the transmembrane amyloid precursor protein (APP) is released into extracellular space. Subsequently, amyloid aggregation is caused by deposition of Ab fragments and structural change of Ab on the cell surface, such as with lipid rafts [4]. Neuronal cell death induced by Ab accumulation in the AD is closely related with endoplasmic reticulum (ER) stress [5]. A translocation of the ER chaperone to another cellular location, such as the cell surface, in the ER stress condition was recently found [6], raising the possibility of interaction between Ab fragments and ER chaperones. Our previous report indicated that ER resident chaperones such as calreticulin, protein disulfide isomerase, and ERp57 effectively inhibited Ab fibrillation [7]. This implied that ER resident chaperones exhibited good interaction with Ab fragments. The 78-kDa glucose-regulated protein (GRP78), referred to as
BiP or HSPA5, is one of the ER resident molecular chaperones, and serves multiple roles in maintaining cell viability [8,9]. The major function of GRP78 is facilitating transportation of new-born proteins and their folding assistance. GRP78 is included in the heat shock protein 70 (Hsp70) protein family, and the amino acid sequence of Hsp70 is highly conserved. Similar to Hsp70, both the ATPase and substrate recognition domains are contained within GRP78 [10]. Binding and hydrolysis of ATP play crucial roles in preventing misfolding and aggregation of nascent polypeptides [11e13]. In other words, GRP78 shows ATP-dependent chaperone activity for inhibiting protein aggregation. A number of studies have reported decrease of Ab40 and Ab42 accompanying interaction between GRP78 and APP [14,15]. Moreover, translocation of GRP78 to the cell surface was found in the ER stress condition [16,17], suggesting the possibility that GRP78 interacts with Ab fragments. In the present study, we examined the influence of GRP78 addition on Ab fibrillation in the presence or absence of ATP. Our results indicated that a small amount of GRP78 effectively inhibited Ab elongation. We also confirmed fibrillation inhibition by GRP78 in the absence of ATP, suggesting GRP78 exhibited ATP-independent interaction with Ab fragments.
* Corresponding author. E-mail address: [email protected] (M. Sakono). http://dx.doi.org/10.1016/j.bbrc.2017.08.162 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
2
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
solutions at 80 C. Concentration of Ab monomers was measured using the Bradford protein assay, with bovine serum albumin as a control. Ab monomer solutions were gently thawed on ice and diluted in PBS buffer containing GRP78 and ATP. The mixtures containing 20 mM Ab were incubated for 24 h at 37 C and without agitation. 2.4. Confirmation of Ab fibrillation by ThT assay Ab fibrillation was assessed by ThT assay in accordance with previous reports [7]. Briefly, Ab solution was mixed with PBS buffer containing 20 mM ThT, and the final Ab concentration was adjusted to 0.5 mM. The mixture was incubated for 5 min at room temperature. Emission fluorescent intensity was monitored at 485 nm through excitation at 445 nm using a spectrofluorometer (LS-55; PerkinElmer Inc., Waltham, MA, USA). 2.5. Immunoblotting analysis
Fig. 1. SDS-PAGE profiles of recombinant human GRP78.
2. Materials and methods 2.1. Materials Ammonium solution and adenosine 50 -triphosphate disodium salt trihydrate (ATP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Thioflavin T (ThT) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Human Ab (142) was obtained from Peptide Institute (Osaka, Japan). PVDF membrane was purchased from EMD Millipore (Billerica, MA, USA). Nitrocellulose membrane was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Ab antibody (6E10) was obtained from BioLegend (San Diego, CA, USA). Rabbit anti-mouse IgG conjugated with horseradish peroxidase was purchased from Abcam (Cambridge, UK). ECL Prime Western Blotting Detection Reagent was purchased from GE Healthcare (Little Chalfont, UK).
SDS-PAGE and native-PAGE of Ab samples were performed using 14% Tris-Glycine gels. After gel electrophoresis, separated proteins were transferred to PVDF membranes, using a current constant of 100 mA for 1 h. Incubated Ab samples were centrifuged at 15,000 rpm for 20 min for dot-blotting analysis. Supernatants and precipitates were collected separately, with precipitates resuspended in fresh PBS solution. Dot-blotting was performed by spotting each Ab sample solution directly onto nitrocellulose membrane. For immunodetection, membranes were blocked overnight at 4 C with 5% skimmed milk in PBS. After blocking, membranes were incubated with a mouse monoclonal Ab antibody (6E10, 1:2000) for 1 h at 37 C, followed by secondary horseradish peroxidaseconjugated anti-mouse IgG (1:10000). Proteins were visualized using the ECL Prime Western Blotting Detection Reagent, as per the manufacturer's instructions. 2.6. Morphology observation using transmission electron microscopy Samples were placed on carbon-coated copper grids. Excess sample was removed using filter paper, and the grids were air-
2.2. Expression and purification of recombinant GRP78 A cDNA encoding human GRP78 was cloned into pCold I expression plasmid (Takara Bio Inc., Otsu, Japan), which is designed to produce N-terminally (His)6-tagged proteins. The obtained plasmid was transformed into BL21 cells, and the recombinant proteins were expressed and purified using Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany), as per the manufacturer's instructions. The purified GRP78 was submitted into an NAP-5 column (GE Healthcare) equilibrated with PBS buffer. GRP78 dissolved in PBS was then obtained. 2.3. Preparation of Ab fibril Purchased lyophilized Ab was fully dissolved in 0.1% ammonium solution, and the solution was then centrifuged at 15,000 rpm for 3 h to remove seeds. Supernatants containing Ab monomers were collected into microtubes and stored as Ab monomer stock
Fig. 2. Influence of GRP78 on Ab fibrillation. Fibrillation was assessed using the ThT fluorescence method. Incubated samples containing chaperones with or without ATP were mixed with 20 mM ThT solution, and the final Ab concentration adjusted to 0.5 mM. Emission fluorescent intensity was monitored at 480 nm by excitation at 445 nm, using a spectrofluorometer.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
3
dried. Samples on grids were negatively stained using uranyl acetate, with excess stain removed by air-drying. Samples were observed using an electron microscope (JEM-1400TC; JEOL Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Effect of ATP on inhibition of Ab fibrillation by GRP78 A human GRP78 was prepared by expression from Escherichia coli. An expression and purification of recombinant GRP78 was confirmed by SDS-PAGE (Fig. 1). An obvious single band appeared around the estimated molecular weight (74 kDa). Therefore, a preparation of GRP78 was considered to have been successfully conducted. The effect of GRP78 addition on Ab fibrillation was investigated. The mixtures were subjected to ThT assay after incubation of Ab with appropriate concentration of the GRP78. Fig. 2 shows the effect on Ab fibrillation of GRP78 in the presence or absence of ATP. The intensity was dramatically decreased by addition of GRP78 with ATP compared with the ThT fluorescence intensity of the incubated Ab only sample. Addition of GRP78 greater than 1 mM evidently inhibited aggregation of 20 mM Ab effectively. Therefore, it was found that a small amount of GRP78 in the presence of ATP effectively inhibited Ab fibrillation. Intriguingly, similar reduction of ThT intensity also occurred in the absence of ATP. Dose-dependent addition of GRP78 without ATP decreased ThT intensity, and addition of 1 mM GRP78 fully inhibited ThT binding. This result implied that the inhibition of Ab fibrillation by GRP78 was carried out in an ATP-independent manner.
3.2. Ab structure maintained as monomer state in the presence of GRP78 Various electrophoresis and immunoassays were performed to investigate the details of the structural state of Ab in the presence of GRP78 without ATP. As shown in Fig. 3A, the bands appeared clearly in the presence of >0.1 mM GRP78 at the same position of Ab monomer. SDS-resistant aggregation appeared to have been produced, such as Ab fibril, because disappearance of the band in the lane of GRP78 < 0.01 mM was confirmed. Therefore, manifestation of bands by addition of GRP78 implied inhibition of amyloid aggregates formation. Next, the Ab structure under a non-denaturing condition was investigated. The Ab samples after incubation were applied to dotblot analysis and Native-PAGE. The result of the dot-blot analysis indicated addition of GRP78 did not form insoluble Ab aggregation, as most of the Ab contained within the sample that included >0.1 mM GRP78 was confirmed at the supernatant (Fig. 3B). Similar to the result for SDS-PAGE, appearance of bands in the lane of GRP78 > 0.1 mM was ascertained at the same position of Ab monomer as in the Native-PAGE (Fig. 3C). Production of Ab oligomer was not observed, as the other bands did not appear anywhere in the polyacrylamide gel. Therefore, Ab was suggested to exist as a monomer state, but not oligomer, in the presence of an appropriate concentration of GRP78.
Fig. 3. Analysis of structural state of Ab in the presence of GRP78 without ATP. (A) SDSPAGE analysis of Ab solutions. Samples incubated with 20 mM Ab and various concentrations of GRP78 were separated by SDS-PAGE, probed using Ab antibody, and
visualized using chemiluminescence. Ab M and Ab F represent Ab monomers and Ab fibrils, respectively. (B) Dot-blot analysis of Ab solutions. Samples were spotted onto nitrocellulose membrane, probed using an Ab antibody, and visualized by chemiluminescence. Sup and Pre represent supernatants and precipitates, respectively. (C) Native-PAGE analysis of Ab solutions. Samples incubated with 20 mM Ab and various concentrations of GRP78 were separated by native-PAGE, probed using an Ab antibody, and visualized by chemiluminescence.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162
4
M. Sakono, T. Kidani / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 4. Morphology of Ab aggregates in the presence of GRP78. The scale bar indicates 200 nm.
3.3. Morphology observation of Ab by transmission electron microscopy Morphology of Ab included in the samples after incubation was observed using an electron microscope (Fig. 4). Although a wellknown fibrillar structure was observed in the sample containing Ab only, such characteristic structures were not verified in the presence of GRP78. Moreover, the effect of GRP78 addition on the morphology of Ab was not dependent on the presence of ATP. Thus, microscopic observation also indicated GRP78 effectively inhibited formation of an Ab fibrillar structure regardless of whether ATP was present. Considering the above results, it was revealed Ab elongation was dramatically suppressed by GRP78 even if the ATP was absent. Our findings seemed to not be consistent with the general consensus because GRP78 has been well known as an ATP-dependent molecular chaperone [9]. A previous study reported amyloid aggregation of human amylin (hA) was effectively inhibited by GRP78 and Hsp70 without ATP [18]. This result was quite similar with ours, indicating that the Hsp70 protein family, such as GRP78, probably functioned to inhibit intermolecular b-sheet formation without ATP. Although ATP is generally believed to be required for changing the tertiary structure of GRP78, it was presumed that such structural change were not necessary for interaction between GRP78 and amyloidogenic peptides. Furthermore, it was clarified that inhibition of fibrillation was carried out with a small amount of GRP78 as compared with Ab. A similar tendency was also observed in the case of interaction between hA and GRP78, implying interactions between amyloidogenic peptides and GRP78 repeatedly associate and dissociate in a short period of time. The association and dissociation is presumed to probably cause inhibition of bsheet transition of Ab. Therefore, the Hsp70 family, such as GRP78, would be beneficial for suppressing Ab-induced cellular stress. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgements We thank the University of Toyama Life Science Research Center for its assistance with transmission electron microscopy analysis. The present study was supported by a research grant from the Toyama First Bank Scholarship Foundation (to M.S.).
References [1] M.P. Murphy, H.L. Iii, Alzheimer's disease and the b-amyloid peptide, J. Alzheimer's Dis. 19 (2010) 311e323. [2] M. Sunde, L.C. Serpell, M. Bartlam, P.E. Fraser, M.B. Pepys, C.C.F. Blake, Common core structure of amyloid fibrils by synchrotron x-ray diffraction, J. Mol. Biol. 273 (1997) 729e739. [3] G. Irvine, O. El-Agnaf, Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases, Mol. Med. 14 (2008) 451e464. [4] K. Yanagisawa, Role of gangliosides in Alzheimer's disease, Biochim. Biophys. Acta - Biomembr. 1768 (2007) 1943e1951. [5] A. Salminen, A. Kauppinen, T. Suuronen, K. Kaarniranta, J. Ojala, ER stress in Alzheimer's disease: a novel neuronal trigger for inflammation and Alzheimer's pathology, J. Neuroinflammation 6 (2009) 41. [6] V.R. Wiersma, M. Michalak, T.M. Abdullah, E. Bremer, P. Eggleton, Mechanisms of translocation of ER chaperones to the cell surface and immunomodulatory roles in cancer and autoimmunity, Front. Oncol. 5 (2015) 7. [7] K. Kitauchi, M. Sakono, Glycoprotein quality control-related proteins effectively inhibit fibrillation of amyloid beta 1e42, Biochem. Biophys. Res. Commun. 481 (2016) 227e231. [8] M. Wang, S. Wey, Y. Zhang, R. Ye, A.S. Lee, Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders, Antioxid. Redox Signal. 11 (2009) 2307e2316. [9] C. Casas, GRP78 at the centre of the stage in cancer and neuroprotection, Front. Neurosci. 11 (2017) 177. €€ [10] M. Daugaard, M. Rohde, M. Ja attel€ a, The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions, FEBS Lett. 581 (2007) 3702e3710. [11] C.K. Kassenbrock, R.B. Kelly, Interaction of heavy chain binding protein (BiP/ GRP78) with adenine nucleotides, EMBO J. 8 (1989) 1461e1467. [12] J. Wei, J.R. Gaut, L.M. Hendershot, In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis, J. Biol. Chem. 270 (1995) 26677e26682. [13] L. Hendershot, J. Wei, J. Gaut, J. Melnick, S. Aviel, Y. Argon, Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 5269e5274. [14] Y. Yang, R.S. Turner, J.R. Gaut, The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases a 40 and A 42 secretion, J. Biol. Chem. 273 (1998) 25552e25555. [15] T. Hoshino, T. Nakaya, W. Araki, K. Suzuki, T. Suzuki, T. Mizushima, Endoplasmic reticulum chaperones inhibit the production of amyloid-b peptides, Biochem. J. 402 (2007) 581e589. [16] Y. Zhang, R. Liu, M. Ni, P. Gill, A.S. Lee, Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP, J. Biol. Chem. 285 (2010) 15065e15075. [17] Y.L. Tsai, Y. Zhang, C.C. Tseng, R. Stanciauskas, F. Pinaud, A.S. Lee, Characterization and mechanism of stress-induced translocation of 78-kilodalton glucose-regulated protein (GRP78) to the cell surface, J. Biol. Chem. 290 (2015) 8049e8064. [18] V. Chien, J.F. Aitken, S. Zhang, C.M. Buchanan, A. Hickey, T. Brittain, G.J.S. Cooper, K.M. Loomes, The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of b-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes, Biochem. J. 432 (2010) 113e121.
Please cite this article in press as: M. Sakono, T. Kidani, ATP-independent inhibition of amyloid beta fibrillation by the endoplasmic reticulum resident molecular chaperone GRP78, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.162