The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP ...

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effectively protecting hA against bulk misfolding and irreversible aggregation. ... 1 To whom correspondence should be addressed (email [email protected]). ... from α-synuclein [37,38], APP (amyloid precursor proteins). [39,40], polyQ ...
Biochem. J. (2010) 432, 113–121 (Printed in Great Britain)

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doi:10.1042/BJ20100434

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of β-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes Vita CHIEN*, Jacqueline F. AITKEN*, Shaoping ZHANG*†, Christina M. BUCHANAN*, Anthony HICKEY*, Thomas BRITTAIN*, Garth J. S. COOPER*†‡ and Kerry M. LOOMES*†1 *School of Biological Sciences, Faculty of Science, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, †Maurice Wilkins Centre for Molecular BioDiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, and ‡Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.

Misfolding of the islet β-cell peptide hA (human amylin) into βsheet-containing oligomers is linked to β-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavychain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent β-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA,

but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective β-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

INTRODUCTION

or transgenic rodent models susceptible to hA-evoked diabetes [19,20,22]. Cytotoxic hA aggregates have been reported in both the intracellular and extracellular spaces in islets [20,23,24], but the detailed molecular mechanisms by which such misfolding might trigger apoptosis remain to be fully elucidated. Previous studies have indicated that exposure of cultured islets or β-cells to extracellular fibrillogenic hA can result in cell death and suggested that direct contact of hA aggregates with cell membranes may be required to elicit apoptosis [20,25–28]. Amylin-evoked membrane instability and leakage-induction via cell membrane interactions are considered to provide one potential cytotoxic mechanism [29,30]. Other mechanisms include increased cellular pro-oxidant responses [18], LDL (low-density lipoprotein) uptake evoked by hA aggregate–cell interactions [31] and activation of the ER (endoplasmic reticulum) stress response [32,33]. Amyloid deposition is a pathological feature not only observed in T2DM, but also in several other diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease (and related polyglutamine disorders), transmissible spongiform encephalopathy and amyotrophic lateral sclerosis [34–36]. Although unrelated proteins are involved in each case, the resulting amyloid structures share morphological and biochemical commonalities, such as the generation of β-fibril structures and their high resistance to proteolytic degradation [34,35].

T2DM (Type 2 diabetes mellitus) is characterized by pancreatic islet β-cell dysfunction [1] and loss of β-cell mass [2,3], which leads to progressive impairment of insulin secretion and, ultimately, to overt diabetes. Amylin, also known as IAPP (islet amyloid polypeptide), is a 37-amino-acid polypeptide [4–6] secreted from pancreatic islet β-cells [7,8] and is a physiological component of insulin granules [9], wherein it is secreted through the cell membrane via the regulated secretory pathway. Amylin self-associates to form aggregates in solution [10,11] and pancreatic islet amyloid [12,13] in humans with T2DM [4]. Islet amyloid has been reported in 40–90 % of patients with T2DM studied post-mortem [4,12,14,15], and has been linked to decreased β-cell mass, β-cell dysfunction and the aetiopathogenesis of T2DM [14,16]. In aqueous solution, hA (human amylin) spontaneously forms β-sheets and aggregates, whereas rA (rat amylin) does not [10,11,17,18], and hA aggregation has been linked to β-cell degeneration in T2DM [16,19–21]. Mature amyloid fibrils, however, may not contribute directly to its cytotoxicity, since there is no strong correlation between the extent of amyloid deposits and disease severity [19,22], and mature fibrils are not cytotoxic when assessed by in vitro assays [18]. Misfolding of hA into β-sheetcontaining oligomers evokes apoptosis originating on the surface and/or within cells, as shown by studies using cultured β-cells

Key words: aggregation, amylin, chaperone, glucoseregulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein (GRP78/BiP), heat-shock protein 70 (HSP70), misfolding, Type 2 diabetes mellitus.

Abbreviations used: BiP, immunoglobulin heavy-chain-binding protein; DTT, dithiothreitol; ER, endoplasmic reticulum; GRP78, glucose-regulated protein of 78 kDa; hA, human amylin; HFIP, hexafluoroisopropanol; HSC70, heat-shock cognate 70 kDa protein; HSP, heat-shock protein; rA, rat amylin; T2DM, Type 2 diabetes mellitus; ThT, thioflavin-T. 1 To whom correspondence should be addressed (email [email protected]).  c The Authors Journal compilation  c 2010 Biochemical Society

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V. Chien and others

Similarly, an increasing body of evidence suggests that endogenous chaperones play important protective roles in the pathophysiology of amyloid deposition in several diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease (and related polyglutamine disorders), transmissible spongiform encephalopathy and amyorophic lateral sclerosis [34–36]. These cellular components can recognize non-native proteins and assist protein folding or remove the proteins by proteolytic machineries, such as the ubiquitin/proteasome system, and thus prevent cellular damage caused by protein aggregation. Several studies have now shown that chaperones inhibit amyloid fibril formation derived from α-synuclein [37,38], APP (amyloid precursor proteins) [39,40], polyQ (polyglutamine) [41–43] and immunoglobulin light chain [44], which are primary peptides of amyloid associated with Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and systemic/modular amyloidosis respectively. At present, the cellular processes that lead to or cause hA to misfold and form cytotoxic aggregates remain poorly understood. Clearly, there is a pressing need to better understand the molecular interactions between hA itself and components of the cellular machinery that control and regulate protein folding. Prominent among these processes could be those regulated by molecular chaperones, which bind non-native states of other proteins and assist them to achieve physiological conformations, and stabilize them against irreversible multimeric aggregation [45,46]. Previous studies with pancreatic islet sections have provided evidence for an increased expression of chaperone proteins in diabetes [47] and that overexpression of the HSP (heat-shock protein) HSP70 can attenuate ER stress in cultured islets [48]. Both amylin and insulin are co-localized within the human pancreatic β-cell granule [49] and co-purify in purified granule preparations from cultured insulinoma β-cells [50]. In addition, multiple chaperones including HSP70 were identified within highly purified β-cell granules derived from INS-1E islet βcells [51]. These secretory granules also contained insulin [51] and amylin (C. Buchanan and A. Hickey, unpublished work), supporting the co-localization of all of these components to the same organelle. As hA misfolding can induce ER stress and β-cell apoptosis [52,53] and therefore probably encounter chaperones along the regulated secretory pathway, we therefore decided to probe their intrinsic functional relationships. We analysed solution-based hA–chaperone interactions using a time-dependent CD assay, which measures hA β-sheet formation, and a ThT (thioflavin-T) assay that quantifies hA aggregation [18], both key features of hA misfolding. We now report that the ER-resident chaperone GRP78 (glucose-regulated protein of 78 kDa), the cytosolic chaperone HSP70 and the co-chaperone HSP40 separately suppressed misfolding and aggregation of hA. These findings demonstrate the existence of direct functional interactions by which GRP78, HSP70 and HSP40 can suppress hA misfolding. We conclude that these chaperones could well exert the physiologically significant regulation of hA during the processes by which it is synthesized and folded. EXPERIMENTAL Materials

Experiments were performed in water (18.2 M · cm resistivity; Millipore). All reagents were of analytical grade, unless otherwise stated. Synthetic hA and rA were from Bachem. hA and rA were column-purified to give monomeric amylin as described previously [54]. Peptides thus treated are designated ‘purified’. Recombinant HSP70, HSP40 and GRP78 were from Stressgen.  c The Authors Journal compilation  c 2010 Biochemical Society

Recombinant human HSP70 and human HSP40 were in 1 × DPBS [2.7 mM KCl, 1.5 mM KH2 PO4 , 137 mM NaCl, 8.1 mM Na2 HPO4 (pH 7.2–7.4)], and recombinant hamster GRP78/BiP (immunoglobulin heavy-chain-binding protein) in 37 mM Tris/HCl (pH 7.5) and 37 mM NaCl. BSA (low endotoxin; ICPbio) was dissolved in water (1 mg/ml stock). Methods CD

CD spectra were collected (π*−180; Applied Photophysics) as described previously [18]. Chaperone effects on amylin folding in solution (by measuring the rate of β-sheet formation at 217 nm/s for 1 h) were performed in buffer [100 mM KCl and 50 mM KH2 PO4 (pH 7.4)] with purified hA or rA, relevant controls at the stated concentrations, molar ratios and other conditions, and without or with Mg3 ATP (Sigma), as described in the Figure legends. Kinetic experiments were initiated by the addition of purified amylin (6.4 μM) and 2.5 % (v/v) HFIP (hexafluoroisopropanol) (final concentration) to the buffer containing chaperones and Mg3 ATP, where indicated. Chaperones were used at concentrations at which they did not contribute to any observed CD. ThT assay

ThT fluorescence intensity correlates directly with hA aggregates in solution [18]. End point fluorescence signals were collected (Spectra-MAX Gemini-XS; Molecular Devices) as described previously [55]. Chaperone/hA molar ratios were constituted by adjusting chaperone concentrations, and duplicate group measurements were made. Chaperone binding to amylin by co-immunoprecipitation

Purified amylin was diluted in buffer [100 mM KCl and 50 mM KH2 PO4 (pH 7.4) per 1500 μl] and then hA or rA (6.4 μM, final concentration) and 2.5 % (v/v) HFIP (final concentration) was added, followed by HSP70 or GRP78/BiP (0.032 μM, final concentration), incubated for 1 h at ambient temperature before the addition of a rabbit anti-amylin antibody (1:1000 dilution; in-house preparation). Following incubation for 24 h at 4 ◦ C with gentle rotation, 50 μl of Protein G–SepharoseTM fastflow (GE Healthcare) was added and a second 24-h incubation was performed (under the same conditions). Amylin–chaperone complexes were collected by centrifugation (12 000 g for 20 s). Pellets were washed three times in 1 ml of lysis buffer [500 mM NaCl, 1 % NP-40 (Nonidet P40), 50 mM Tris/HCl (pH 8.0) and 1 mM PMSF] then once with 50 mM Tris/HCl (pH 8.0), and retrieved by centrifugation (12 000 g for 20 s). Pellets were suspended in 20 μl of reducing buffer [1 % (w/v) SDS, 100 mM DTT (dithiothreitol) and 50 mM Tris/HCl (pH 7.5)], heated (95 ◦ C for 3 min) and the mixture was then centrifuged (12 000 g for 20 s). Western blotting

Immunoprecipitates (10 μl) were mixed with NuPAGE® LDS sample agent 10 × (2 μl) (Invitrogen) and 1 M DTT (1 μl). Samples and SeeBlue® (5 μl) plus pre-stained standards (Invitrogen) were loaded on to a 4–12 % Bis-Tris gradient gel (NuPAGE® Novex; Invitrogen) in 1 × Mes buffer (Invitrogen) and developed (120 V/70 mA for 35 min). Proteins were transferred {30 V/110 mA in transfer buffer [100 mM Tris,

Chaperones suppress human amylin misfolding

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80 mM glycine and 20 % (v/v) methanol] for 1.25 h} on to a preactivated (100 % methanol) PVDF membrane (GE Healthcare). Membranes were blocked in PBS containing 0.05 % Tween 20 and 5 % (w/v) non-fat milk for 2 h at ambient temperature and washed three times for 10 min each in PBS containing 0.05 % Tween 20. A goat anti-HSP70 antibody (sc-24, 1:200 dilution; Santa Cruz Biotechnology) and a goat anti-GRP78 antibody (sc-1050, 1:200 dilution; Santa Cruz Biotechnology) were diluted in PBS containing 0.05 % Tween 20 and 1 % (w/v) non-fat milk and incubated with the membrane for 2 h at an ambient temperature. The washing steps described above were then repeated. Membranes were then incubated with an HRP (horseradish peroxidase)-conjugated donkey anti-(goat IgG) antibody (sc-2033, 1:2500 dilution; Santa Cruz Biotechnology in PBS containing 0.05 % Tween 20 and 1 % (w/v) non-fat milk for 2 h at an ambient temperature. Membranes were then washed three times for 10 min each as above. ECL (enhanced chemiluminescence) detection reagent (GE Healthcare) was added, membranes stood for 1 min and signals were detected (lumi-film; Roche). Curve fitting and statistical analysis

Kinetic analysis on CD spectra were performed using fourparameter logistic curve-fits (Prism 4; GraphPad). Aggregation rates in the ThT assay were determined from the slopes fitted during the exponential phase. Statistical analysis (Prism 4) was performed according to tests indicated in the Figure legends, and P values