| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
,2
* School of Biological Sciences, University of Wollongong, Wollongong, Australia;
Department of Chemistry, University of Cambridge, Cambridge, UK; and
School of Chemistry and Physics, University of Adelaide, Adelaide, Australia
4Correspondence: Northfields Ave., Wollongong NSW 2522, Australia. E-mail: mrw{at}uow.edu.au
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: protein aggregation • chaperone:substrate ratio • amyloid fibrils • cytoprotection
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. In many cases the deposits formed in these and other protein conformation disorders are located extracellularly, where they exert pathogenic effects by organ disruption or by cytotoxicity. Although the processes that control the folding of proteins inside cells are relatively well understood, little is known about the corresponding extracellular processes. Clusterin is the best-characterized abundant extracellular chaperone and has recently been proposed to form part of an extracellular protein quality control system (2)
.
Amyloid fibrils found in vivo exhibit common structural features independent of the identity of the parent protein. Intracellular amyloid aggregates are found colocalized with components of the intracellular protein quality control system, including chaperones and ubiquitin (3)
. In a remarkable parallel observation, all disease-associated insoluble extracellular protein deposits tested, including those characterized as amyloid, colocalize with clusterin (Table 1
). The roles of clusterin in amyloid formation and protein deposition are, however, poorly understood. Clusterin is a well-conserved secreted glycoprotein found in most extracellular fluids; it has a potent ATP-independent chaperone action similar to that of the small heat shock proteins. It inhibits stress-induced amorphous protein aggregation by binding to exposed regions of hydrophobicity on non-native protein conformations to form high-molecular-weight (HMW) but still soluble complexes (4)
.
|
Limited data from previous studies suggest that clusterin can affect the amyloid-forming process both in vitro and in vivo. Clusterin has been reported to inhibit in vitro amyloid formation by apolipoprotein C-II (apoC-II) (5)
, the Aß peptide (6
7
8)
, and a fragment of the prion protein, PrP (106–126) (9)
. Depending on the conditions, however, it has been reported to either promote or suppress the cytotoxicity of Aß (6
7
8
, 10)
. Similarly, work with PDAPP mice (a transgenic mouse model for Alzheimer’s disease) in which clusterin expression is ablated has provided results that sometimes appear contradictory. For example, when compared with matched littermates, ablation of clusterin expression decreased the levels of thioflavin S staining of material in brain sections and the number of visibly damaged neurons in PDAPP mice. These findings were interpreted to indicate that clusterin expression promoted Aß amyloid formation and toxicity (11)
. However, in a background of apolipoprotein E-negative (apoE–/–) PDAPP mice, the ablation of clusterin expression had the opposite effect, promoting the early onset of Aß deposition and material staining with thioflavin S (12)
. Such evidence shows that the in vivo effects of clusterin on amyloid formation are likely to involve multiple interactions and processes, making it critical to better understand the nature and mechanism(s) of interactions between clusterin and amyloid-forming proteins through in vitro studies. This objective was the global aim of the current study in which the effects of clusterin on amyloid formation by a broad range of unrelated proteins were examined using a variety of complementary approaches. In addition, we selected two protein substrates—one associated with disease (Aß1–42, Alzheimer’s disease) and the other not (a PI3-SH3 domain)—to extend these investigations to examine the effects of clusterin on amyloid-related toxicity and to better characterize the mechanism(s) by which clusterin affects amyloid formation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Hexafluoroisopropanol (HFIP), lysozyme (from hen egg white), bovine serum albumin (BSA), and 
-casein (from bovine milk) were purchased from Sigma (St. Louis, MO, USA). A plasmid encoding 
-synuclein was a gift from Dr. Robert Cappai (Department of Pathology, University of Melbourne, Melbourne, Australia). -Synuclein was expressed in Escherichia coli and purified by acid precipitation as described (14)
. Glutathione-S-transferase (GST) from Schistosoma japonicum was prepared by thrombin cleavage of recombinant Jun leucine zipper-GST fusion protein and purified by GSH-agarose affinity chromatography (15)
. The short coiled-coil ß (ccß) peptide, originally designed de novo as a model that transforms from a helical conformation at 20°C into amyloid fibrils at 37°C (16)
, was modified by adding a tryptophan residue at its N terminus to produce ccßW (a kind gift from Dr. Cait MacPhee, Department of Physics, University of Edinburgh, Edinburgh, UK). The ccß fibrils described herein were indistinguishable from those described previously. Calcitonin was purchased from both Auspep (Melbourne, Australia) and Southampton Polypeptides Limited (Southhampton, UK). ß2-Microglobulin was a kind gift from Prof. Sheena Radford (University of Leeds, UK). A plasmid encoding the amyloidogenic PI3-SH3 domain of bovine phosphatidyl-inositol-3'-kinase (hereafter referred to simply as SH3) as a GST fusion protein was a kind gift from Dr. Jesus Zurdo (University of Cambridge, UK). SH3 was expressed in E. coli, purified, and GST cleaved using a 5 ml GSTTrapFF cartridge (GE Healthcare, Sydney, Australia) following the manufacturer’s instructions. Aß1–42 was purchased from Biopeptide (San Diego, CA, USA), resuspended in HFIP, and divided into aliquots in which the solvent was left to evaporate (the peptide "film" was frozen at –80°C). Monoclonal anti-Aß antibody WO2 supernatant was a kind gift from Dr. Kevin Barnham (Department of Pathology, University of Melbourne, Australia).
Fibril formation in vitro
SH3 solutions (250 µM, unless otherwise indicated) or mixtures of SH3 and clusterin in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM Na2HPO4, pH 7.5) were heated to 60°C while shaking at 500 rpm for 72 h in a Thermo Finemixer SH2000-DX (Finemould Precision Ind. Co., Seoul, Korea). Directly before use, Aß1–42 was resuspended in buffer (two parts 20 mM NaOH diluted in seven parts Milli Q water and 1 part 10 x PBS) and centrifuged to remove any aggregated material. Aß1–42 (10 µM, unless otherwise indicated) in the presence or absence of clusterin was incubated at 37°C in oxidizing buffer that consisted of PBS containing 0.9 mM CaCl2, 0.5 mM MgCl2, 100 µM CuCl2, 600 µM glycine, pH 7.5, while shaking for 8 h in a FLUOstar OPTIMA fluorescence plate reader (BMG Labtech, Mornington, Victoria, Australia). In similar experiments, the relative ability of clusterin to inhibit fibril formation in reactions that were seeded with preformed aggregates of the same protein (sampled at different times during a previous aggregation reaction) was examined. SH3 (500 µM) and Aß1–42 (10 µM) were incubated as described above; samples were then taken from each aggregation reaction at various time points and stored frozen at –20°C. These samples were used to seed fibril formation reactions at a final molar ratio of preaggregated substrate molecules to monomers of 1:9 in the presence or absence of clusterin (at molar ratios of clusterin:SH3=1:100, clusterin:Aß=1:50). The following proteins were all treated as described below, with and without added clusterin. Samples of -synuclein (-syn, 70 µM), lysozyme (lys, 70 µM), and -casein (-cas, 52 µM) in 50 mM Na2HPO4 buffer, pH 7.4, were shaken at 500 rpm and 57°C for 192 h, 172 h, and 72 h, respectively, in an IKA Vibrax VXR orbital shaker (IKA® Works, Inc., Wilmington, NC, USA). ß2-Microglobulin (ß2m, 85 µM) in 25 mM sodium acetate, pH 2.1, was shaken for 336 h at 500 rpm and 37°C in a Thermo finemixer SH2000-DX. ccßW (60 µM) in 0.1 M Na2HPO4, pH 7.8, was shaken for 40 min at 37°C in a FLUOstar OPTIMA fluorescence plate reader. Calcitonin (calc, 150 µM) was incubated in 50 mM Na2HPO4 buffer, pH 7.4, at 37°C for 20 h. In other experiments, to confirm that clusterin did not form thioflavin T reactive aggregates under the conditions used, clusterin (at 1.0–12.5 µM) was incubated alone in 50 mM Na2HPO4 buffer, pH 7.4, for 24 h at 37°C and for up to 192 h at 60°C or in 25 mM sodium acetate, pH 2.1, for 336 h at 37°C.
Thioflavin T fluorescence assays
Thioflavin T (50 µM) was added to aliquots of samples taken at specific time points after the initiation of fibril formation (lys, -cas, -syn, ß2m, SH3) or to the reaction mixture at the beginning of the time course (calc, Aß and ccßW). Fluorescence was measured on a FLUOstar OPTIMA fluorescence plate reader using excitation and emission windows of 450 ± 10 and 490 ± 10 nm, respectively.
Transmission electron microscopy (TEM)
Formvar and carbon-coated nickel electron microscopy grids were prepared by the addition of 2 µl of protein sample at a concentration of 1 mg/ml. After several minutes, the grids were washed with 3 x 10 µl H2O and negatively stained with 10 µl of uranyl acetate [2% (w/v), Agar Scientific Ltd., Stansted, Essex, UK]. The grids were dried with filter paper between each step. Samples were viewed under 20–125 K magnifications at 120 kV excitation voltages using a Philips CM100 transmission electron microscope and images were analyzed using the SIS Megaview II Image Capture system (Olympus, Munich, Germany).
Cytotoxicity
SH-SY5Y cells (a kind gift from Dr. Kevin Barnham, Department of Pathology, University of Melbourne, Melbourne, Australia) were cultured at 37°C and 5% (v/v) CO2 in full medium, which consisted of DMEM:F12 medium containing 2.5% (v/v) fetal bovine serum (FBS) (both from Trace Biosciences, Melbourne, Australia). Cells suspended in full medium were added to a 96-well plate (100 µl/well containing 5000 cells) and left to attach overnight before washing with DMEM:F12. The cells were then cultured as above for 48 h in FBS-free AIM-V medium (Invitrogen, Melbourne, Australia), with or without additives. Fibril formation by SH3 (250 µM) or Aß (10 µM) was initiated as described above; in some reactions, clusterin or a control protein (BSA) was included to give molar ratios of clusterin/BSA:substrate of 1:500 (SH3) or 1:10 (Aß). To analyze cytotoxicity, aliquots of SH3 and Aß reactions (taken at 12 h and 2 h, respectively) were added to cells alone to give final concentrations of 10 µM and 1 µM, respectively, or in some cases (from reactions lacking clusterin/BSA) were supplemented with clusterin or BSA to give clusterin/BSA:substrate = 1:10. In other experiments, clusterin or BSA alone was added to cells to give a final concentration of 1.0 µM. Calcein-AM was used to measure cell viability (17)
. Calcein-AM (1 µM) was added to cells and left to incubate for 30 min before analyzing fluorescence using a FLUOstar OPTIMA plate reader and excitation and emission windows of 485 ± 10 nm and 520 ± 10 nm, respectively. The significance of differences in fluorescence was assessed using the Student’s t test. Calcein-AM is membrane permeable and nonfluorescent; it is cleaved by esterases in the cytoplasm of viable cells to release fluorescent, membrane-impermeable calcein that remains trapped inside viable cells. Thus, the resulting level of cell-associated calcein fluorescence is proportional to the number of viable cells.
Effects of clusterin on the sedimentation properties of substrate protein aggregates
At the conclusion of in vitro fibril formation time courses, samples of SH3 and Aß, with or without clusterin, were centrifuged for 30 min at 10,000 g. The supernatant was removed and the pellet was resuspended, then washed repeatedly in PBS. SH3 samples were analyzed by 15% SDS-PAGE and stained with Coomassie blue. Aß samples were analyzed by 15% SDS-PAGE and subsequent immunoblotting using anti-Aß monoclonal antibody. The presence of clusterin in supernatant and pelleted fractions was tested by applying these fractions to nitrocellulose membranes that were subsequently blocked with HDC [1% (w/v) heat-denatured casein, 0.04% (w/v) thimerosal, in PBS]. The presence of clusterin was detected using a mixture of G7, 41D, and 78E monoclonal anticlusterin antibodies (18)
. Bound anti-Aß and anticlusterin antibodies were detected with HRP-conjugated sheep anti-mouse Ig antibody (Silenus, Melbourne, Australia), followed by enhanced chemiluminescence (ECL) with Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).
Detection of stable clusterin-substrate complexes
Samples of SH3 and Aß, with or without clusterin, taken from the beginning and end of in vitro fibril formation time courses were centrifuged for 30 min at 10,000 g. The supernatants were shaken end-over-end for 1 h at room temperature with anticlusterin antibody coupled to Sepharose beads (G7-Sepharose; 100 µl packed volume) (4)
. The G7-Sepharose was washed by centrifugation three times with PBS, then incubated in 2M guanidine HCl in PBS at pH 7.5 for 15 min to elute bound protein. The mixture was then centrifuged using a 0.45 µm Ultrafree-MC centrifugal filter device (Millipore, Billerica, MA, USA) to separate the beads from the eluted proteins, which were subsequently analyzed by SDS-PAGE under nonreducing conditions. To detect Aß, immunoblotting was performed as described above.
Immuno-dot blots
Samples of amyloidogenic proteins were taken at various time points during fibril formation and frozen at –20°C until required. Samples (1 µg) were spotted onto nitrocellulose membranes (Pall, Pensacola, FL, USA) and allowed to dry; the membranes were then blocked with HDC. The membranes were incubated for 2 h at 37°C in PBS containing 10 µg/ml clusterin or control proteins GST and ovalbumin before being washed with PBS. Bound clusterin was detected using a mixture of G7, 78E, and 41D antibodies. Rabbit anti-GST (Silenus, Melbourne, Australia) and anti-ovalbumin (a gift from S. Easterbrook-Smith, University of Sydney) were used to detect any bound control protein. Bound primary antibodies were detected with sheep anti-mouse Ig-HRP or sheep anti-rabbit Ig-HRP (Silenus, Melbourne, Australia) using ECL as described above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A), 2) green birefringence when stained with Congo Red (data not shown), and 3) fibrillar structures detected by TEM (Fig. 1B
) characteristic of amyloid species. The addition of clusterin to solutions of all proteins tested showed a dose-dependent decrease in thioflavin T fluorescence (Fig. 1A
). However, for calcitonin, -synuclein, and Aß, molar clusterin:substrate ratios of 1:50, 1:100, and 1:500, respectively, produced an increase in thioflavin T fluorescence. The minimum molar ratio of clusterin:substrate required to affect the levels of thioflavin T fluorescence differed greatly among the systems tested, which could reflect differences between the substrates and/or the conditions used. A ratio of clusterin:CCßw of only 1:100 was sufficient to reduce thioflavin T fluorescence to almost background levels. Molar ratios of clusterin:substrate needed to inhibit 70–80% of the thioflavin T fluorescence measured in the absence of clusterin were 1:100 for -casein and 1:50 for ß2-microglobulin, SH3, and Aß. Although under the conditions tested much higher levels of clusterin were needed to similarly inhibit fibril formation of calcitonin, lysozyme, and -synuclein, even in these cases the effect was markedly substoichiometric (clusterin:substrate 
1:10). The presence of a control protein, BSA, had no measurable effect on thioflavin T fluorescence for any of the proteins tested (data not shown). When incubated alone, clusterin did not develop significant thioflavin T reactivity under any of the conditions tested (data not shown).
|
TEM was used to examine the morphology of protein aggregates (at the highest molar ratio of clusterin:substrate shown for each protein in Fig. 1A
). In the absence of clusterin, fibrillar aggregates of dimensions expected for amyloid fibrils in these well-characterized systems were observed for all proteins (Fig. 1B
). Under the conditions tested, clusterin inhibited the formation of fibrillar structures in all cases (Fig. 1B
). A variety of structures was observed in reactions containing clusterin, including spherical particles of differing diameter (Fig. 1B
; -cas, Aß, and CCßw) and amorphous aggregates (Fig. 1B
; Lys, calc, ß2M, and SH3). TEM analysis of samples identified as having increased thioflavin T fluorescence resulting from a low molar ratio of clusterin:substrate showed them to contain fibrillar aggregates (data not shown).
Effects of clusterin on aggregate toxicity
Compared to SH3 alone, at a clusterin:SH3 ratio of 1:10, the toxicity of aggregates generated after 72 h was significantly reduced (Fig. 2
A; P<0.05), but at a clusterin:SH3 ratio of 1:500 the toxicity of aggregates generated after 48 and 72 h was significantly increased (Fig. 2A
; P<0.05). At a clusterin:Aß 1:10 ratio, clusterin significantly suppressed the toxicity of aggregates generated after 2 h and 8 h (P<0.05), but at a clusterin:Aß 1:500 ratio the toxicity of aggregates at 4 h was significantly enhanced (P<0.05). Moreover, when clusterin was added to aggregates of SH3 and Aß preformed in the absence of clusterin, it significantly decreased their cytotoxicity (Fig. 2B
; P<0.001 and P<0.05, respectively). Under the conditions tested, the addition of either clusterin or BSA alone to cultures had no significant effect on cell viability (data not shown).
|
Clusterin binds amyloid-forming proteins and affects their sedimentation properties
Clusterin, at various ratios relative to the substrate, was added to fibril formation reactions of SH3 and Aß; at the end of the time course the samples were centrifuged to separate supernatant and pellet fractions. SH3 fractions were analyzed by SDS-PAGE; in the absence of clusterin most of the SH3 protein was found in the pellet (P) fraction (Fig. 3
A). However, at clusterin:SH3 ratios of 1:100–1:10, almost all the SH3 protein was found in the supernatant (S) fraction. Even at a ratio of clusterin:SH3 of 1:500, most of the SH3 remained in the S fraction (Fig. 3A
). Similarly, immunoblots of Aß fibril formation reactions showed that the proportion of Aß in the S fraction increased as the ratio of clusterin:Aß increased until (at clusterin:Aß=1:10) effectively all of the Aß was present in the S fraction (Fig. 3A
). In the absence of clusterin, Aß was detected as its monomeric form (at 4.5 kDa) or as SDS-resistant high molecular mass aggregates of > 180 kDa (19)
. However, at clusterin:Aß = 1:10, much of the nonsedimenting Aß was detected in a broad HMW band, which may represent an SDS-resistant clusterin-Aß complex (6)
. Indeed, immunoblotting demonstrated the presence of both Aß and clusterin in this HMW band (data not shown). For both SH3 and Aß, at a clusterin:substrate ratio of 1:10, clusterin was detected only in the S fractions, but at a lower ratio (1:500) was found predominantly in the P fractions (Fig. 3B
). To determine whether clusterin was forming stable complexes with the substrate proteins, we immunoadsorbed clusterin (and hence anything to which it was bound) from the initial and final samples of fibril formation reactions of SH3 and Aß, and analyzed this material by SDS-PAGE and immunoblotting. In fractions prepared from samples of clusterin and substrate protein, before incubation no SH3 or Aß was detected; similarly, when incubated in the absence of clusterin for the duration of the respective time courses, neither SH3 nor Aß was bound by immobilized anticlusterin Ig (Fig. 3C
). However, immunoadsorbed fractions prepared from final samples containing clusterin and substrate protein showed bands corresponding to SH3 and Aß (Fig. 3C
). This result demonstrates that clusterin formed stable complexes with the substrate proteins at some point during the course of the aggregation reactions.
|
Clusterin binds to intermediate structures on the fibril-forming pathway
Samples taken at different times from fibril formation reactions performed (in the absence of clusterin) for the same panel of eight proteins previously examined were spotted on nitrocellulose membranes; the thioflavin T reactivity of species present in these reactions was also measured as a function of time (Fig. 4
). When assayed by immuno-dot blot, clusterin did not bind to the native proteins present in samples before incubation, and bound only weakly or not at all to final samples. Clusterin showed no detectable binding to fibrils after they had been centrifuged and washed (data not shown). In most cases, the strongest binding of clusterin detected was to transient "intermediate" species; in the case of Aß, -synuclein, and ccßw, maximum binding was detected in species present during the transition between the lag and growth phases (Fig. 4)
. For lysozyme and -casein, which under these conditions have no detectable lag phase, the strongest binding was to species present early in the growth phase (Fig. 4)
. For SH3, the strongest binding was detected at 4–6 h, the initial stage at which thioflavin T fluorescence reached a maximum value. There was no binding of clusterin detected in species present at any time in ß2-microglobulin and calcitonin fibril formation reactions (data not shown). In addition, there was no detectable binding of the control proteins GST and ovalbumin to any of the samples of all eight proteins tested (data not shown).
|
Clusterin affects nucleation and is less effective at suppressing fibril elongation
Increasing the concentration of amyloidogenic proteins will generally favor the self-association of monomers into oligomers able to nucleate the aggregation process. Therefore, to test whether clusterin affects nucleation, one clusterin:substrate ratio was selected in each case and the concentration of the substrate protein was varied. As expected, as the concentrations of SH3 and Aß were increased, the level of thioflavin T reactive species increased and the preaggregation lag time shortened (Fig. 5
A). Clusterin inhibited the development of thioflavin T reactive species in all cases; as the concentrations of SH3 and Aß were increased, however, this effect became progressively smaller. For samples containing 0.25 mM SH3, a 1:50 M ratio of clusterin:SH3 suppressed the thioflavin T fluorescence measured on the final sample by >90%. However, when SH3 was at a concentration of 1 mM, the same ratio of clusterin:SH3 decreased thioflavin T fluorescence by only 35% (Fig. 5B
). Similarly, a 1:50 M ratio of clusterin:Aß suppressed the final thioflavin T fluorescence by 90% for samples containing 15 µM Aß, but only by 45% when Aß was at 60 µM (Fig. 5B
). As another means of examining the point at which clusterin exerts its effects on amyloid formation, samples were taken at different times during fibril formation and used to seed amyloid formation in subsequent experiments in the presence or absence of clusterin. Under the conditions used in this study, in the absence of preformed amyloid species, SH3 and Aß exhibited lag phases of 6 h and 2 h, respectively (Fig. 6
A). Note that the kinetics of SH3 aggregation in this reaction were faster than in most of the subsequent SH3 reactions reported here (Fig. 6B
), as the latter were performed using a lower substrate concentration (see Fig. 6
legend). In both reactions used to generate preformed amyloid species, the elongation phase was rapid, after which the level of thioflavin T reactive material remained relatively constant. When samples taken early in these time courses or from the transition region between lag and growth phases were used to seed subsequent aggregation reactions, clusterin was able to suppress the generation of thioflavin T reactive species by up to 75–90% (Fig. 6B, C
). However, when the samples used to seed the reaction were taken later in the initial aggregation time courses, the lag phases of the subsequent aggregation reactions were shortened (Fig. 6B
) and the inhibitory effects of clusterin were significantly less (25–50% inhibition, Fig. 6C
).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 7
, 9)
. We have shown here that clusterin can potently inhibit in vitro amyloid formation by a broad range of unrelated proteins, suggesting that this activity is not strongly dependent on the identity of the specific polypeptide substrate or is associated with disease (for a summary of results, see Table 2
and Table 3
). In all cases tested, significantly substoichiometric ratios of clusterin:substrate (from 1:10 to 1:3.5) completely inhibited the formation of fibrils detectable by fluorescence and TEM (Fig. 1B
). For three of the eight substrate proteins tested (calcitonin, -synuclein, and Aß), when clusterin was present at very low levels relative to the substrate it significantly increased the level of thioflavin T reactive material formed (Fig. 1A
). One possible explanation is that at least for calcitonin, -synuclein, and Aß, when present at very low levels clusterin may facilitate amyloid formation by stabilizing the otherwise unstable protein aggregates required to initiate fibril formation.
|
|
Given the demonstration that, under differing conditions, clusterin exerts variable effects on amyloid fibril formation, we explored whether this is also true for its effects on amyloid toxicity. Both SH3 and Aß are known to generate cytotoxic intermediates during amyloidogenesis (20
, 21)
. For both these proteins, at least for some of the time points tested, clusterin enhanced the cytotoxicity of aggregates formed when present during fibril formation at a clusterin:substrate ratio of 1:500, but had the opposite effect when present at the much higher but still substoichiometric ratio of 1:10 (Fig. 2A
). These results clearly indicate that the effects of clusterin on the cytotoxicity of aggregates are complex and depend on both the stage of amyloid formation at which the aggregates are formed and on the clusterin:substrate ratio. Regions of exposed hydrophobicity have been strongly implicated in the cytotoxicity of protein aggregates (1)
. The enhancement of toxicity seen at very low clusterin:substrate ratios may result from there being sufficient clusterin present under these conditions to physically stabilize in solution aggregates bearing multiple hydrophobic surfaces, but insufficient clusterin to bind to and inhibit the cytotoxicity of all hydrophobic surfaces. If this is correct, then it would be expected that at higher clusterin:substrate ratios clusterin would be more effective at masking exposed hydrophobicity on protein aggregates and reducing their cytotoxicity. This was found to be the case when clusterin was present during amyloid formation and when it was added to aggregates of SH3 and Aß preformed in its absence (see clusterin:substrate=1:10 data in Fig. 2A, B
). Earlier studies have reported that clusterin enhances (10)
or suppresses (7)
the cytotoxicity of Aß; but the conditions used in each of these studies differed greatly, including the type of peptide (Aß1–40 vs. Aß1–42), whether or not clusterin was preincubated with Aß, and the effective ratios of clusterin:Aß tested. The results of the present study show that very different effects can be observed under different experimental conditions, suggesting that the findings of both earlier studies may be broadly correct.
When increasing amounts of clusterin were added to fibril formation reactions containing either SH3 or Aß, a progressive increase in the proportion of substrate protein present in the nonsedimenting supernatant (S) fraction at the end of the time course was observed (Fig. 3A
). At a "high" clusterin:substrate ratio (1:10), the substrate remained in the S fraction, but at a much lower ratio (1:500) it was incorporated into the sedimenting pellet (P) fraction (Fig. 3B
). We found that clusterin did not detectably bind to washed, preformed fibrils formed from any of the eight substrate proteins tested (data not shown). These results suggest that under amyloidogenic conditions, as is the case under conditions when amorphous aggregates are formed (4)
, clusterin can enhance substrate protein solubility even when present at significantly substoichiometric ratios. In the case of proteins forming amorphous aggregates, clusterin appears to exert this effect by promoting the formation of soluble HMW complexes with substrate molecules present in non-native conformations (4
, 22)
. The presence of clusterin and substrate molecules in supernatant fractions prepared from amyloid-forming reactions in which clusterin had inhibited fibril formation (Fig. 3B
) suggests that similar clusterin-substrate complexes might be forming under these conditions. This was confirmed for both SH3 and Aß substrates by immunoprecipitation analyses (Fig. 3C
).
Clusterin inhibits the amorphous aggregation of a variety of proteins in vitro when present at molar clusterin:substrate ratios of 1:5.5–1:0.65 (4)
; this action appears to be a consequence of clusterin binding to regions of exposed hydrophobicity on substrate molecules (23)
. In contrast, data presented here and elsewhere (5
, 8)
show that clusterin can influence amyloid formation at much lower clusterin:substrate ratios (as low as 1:500). The apparent difference in stoichiometry between the effects of clusterin on protein aggregation-producing amorphous vs. amyloid-type aggregates likely relates to the low concentration of protein oligomers that nucleate fibril formation thought to be present in amyloid-forming reactions (24)
. The very low clusterin:substrate ratios that affect the amyloid-forming pathway are consistent with clusterin interacting with species present in low abundance such as those that nucleate growth or their precursors. Immuno-dot blot analyses showed that for most proteins tested, clusterin bound most strongly to transient protein species, which are presumably more abundant during fibril nucleation and/or growth (Fig. 4)
. It is not clear why binding to corresponding species present in calcitonin and ß2m fibril formation reactions was not detected, although one possibility is that the interacting species in these cases are at very low levels. In the case of SH3, maximum binding was traced to species present at 4–6 h, corresponding to the initial stage at which maximum thioflavin T fluorescence was obtained. Electron microscopic examination of these samples indicated that they consisted of a mixture of spherical and short fibrillar aggregates (data not shown). Thus, in this case also it is feasible that clusterin was binding to transient intermediate species. In SH3 and Aß, clusterin suppressed amyloid formation on a molar basis more efficiently at lower substrate concentrations (when self-association of substrate molecules with non-native conformations into oligomers is less favored; Fig. 5
). Similarly, clusterin appears more efficient at inhibiting fibril formation reactions that are seeded with species taken from early time points of preceding reactions than with those seeded with species taken from later time points (Fig. 6)
. These results suggest that clusterin exerts its effects on amyloid formation primarily by interacting with transient protein species that are most abundant prior to fibril elongation; these interacting species are probably small oligomers that are either structural precursors to, or fully functional, aggregation nuclei.
Collectively, our results indicate that at substoichiometric levels, clusterin exerts substantial effects on in vitro amyloid formation by a broad variety of substrate proteins, and suggest that these effects result from interactions with prefibrillar species of very low abundance. At least in vitro, clusterin does not bind detectably to either native substrate proteins or mature amyloid fibrils. The relatively low level of substrate specificity characterizing this action suggests that clusterin interacts with species sharing common structural features present on the amyloid-forming pathways of many different proteins. We have shown that clusterin forms stable complexes with fibril-forming proteins and reduces their propensity to sediment from solution. Our results and those from a recent study using human lysozyme as a substrate protein (36)
suggest that clusterin does not bind to native or non-native monomers, but instead support the hypothesis that clusterin interacts with oligomeric species that may function as nucleation points for fibril formation. Our results clearly show that an important determinant of the nature and extent of the effects of clusterin on both amyloid formation and toxicity is the clusterin:substrate ratio. This conclusion has important potential implications for the likely effects of clusterin on amyloid formation in vivo, as clusterin may exert differing effects on the extracellular folding landscape depending on its relative abundance compared to amyloid-forming substrate proteins in certain biological contexts. For example, although clusterin is present in human plasma at 100 µg/ml (25)
, in cerebrospinal fluid it is present at only 2 µg/ml (26)
. It is therefore feasible that in the central nervous system, clusterin might under some conditions enhance amyloid formation and toxicity (11)
and yet elsewhere in the body, where it is more abundant (e.g., in plasma), exert the opposite effect. The evidence that clusterin does not bind to mature amyloid fibrils, but at very low clusterin:substrate ratios is incorporated into insoluble amyloid material, also provides a tenable explanation for the otherwise curious observation that clusterin is associated with a variety of disease-related amyloid deposits in vivo (Table 1)
. It appears likely that the association of clusterin with these deposits represents its failed attempts to maintain the solubility of amyloid-forming species under disease-specific conditions of high molar substrate excess.
In conclusion, the abundant extracellular chaperone clusterin exerts potent effects on the formation of amyloid in vitro by a wide range of proteins, including examples known to be associated with disease and others that are not. The nature and extent of these effects show a limited dependence on the identity of the substrate protein, but (in contrast) vary with the clusterin:substrate ratio. Clusterin appears to interact with prefibrillar species that share common structural features and, depending on the prevailing conditions, can either promote or suppress amyloid formation and toxicity. However, in all cases tested, at molar ratios of clusterin:substrate of 1:10 or greater, clusterin potently inhibited amyloid formation and provided substantial cytoprotection. These findings suggest that clusterin may be an important part of an armory of mechanisms that defend against the consequences of extracellular protein misfolding. They also raise the possibility that, at least in some circumstances, increasing the levels of clusterin in vivo could be a therapeutic tool in the fight against extracellular protein deposition disorders.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Current address: Department of Chemistry, University of Cambridge, Cambridge CB21EW, UK.
3 Current address: Intelligent Polymer Research Institute, University of Wollongong, Wollongong NSW 2522, Australia.
Received for publication December 19, 2006. Accepted for publication March 1, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. R. Wyatt and M. R. Wilson Identification of Human Plasma Proteins as Major Clients for the Extracellular Chaperone Clusterin J. Biol. Chem., February 5, 2010; 285(6): 3532 - 3539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Naiki and Y. Nagai Molecular Pathogenesis of Protein Misfolding Diseases: Pathological Molecular Environments Versus Quality Control Systems Against Misfolded Proteins J. Biochem., December 1, 2009; 146(6): 751 - 756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Wyatt, J. J. Yerbury, and M. R. Wilson Structural Characterization of Clusterin-Chaperone Client Protein Complexes J. Biol. Chem., August 14, 2009; 284(33): 21920 - 21927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Yerbury, J. R. Kumita, S. Meehan, C. M. Dobson, and M. R. Wilson {alpha}2-Macroglobulin and Haptoglobin Suppress Amyloid Formation by Interacting with Prefibrillar Protein Species J. Biol. Chem., February 13, 2009; 284(7): 4246 - 4254. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Herczenik and M. F. B. G. Gebbink Molecular and cellular aspects of protein misfolding and disease FASEB J, July 1, 2008; 22(7): 2115 - 2133. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
