ARTICLE
Negatively Charged Metal Oxide Nanoparticles Interact with the 20S Proteasome and Differentially Modulate Its Biologic Functional Effects )
)
)
Christine A. Falaschetti,† Tatjana Paunesku,† Jasmina Kurepa,‡ Dhaval Nanavati,§ Stanley S. Chou,^ Mrinmoy De,^ MinHa Song, Jung-tak Jang, Aiguo Wu,z Vinayak P. Dravid,^ Jinwoo Cheon, Jan Smalle,‡ and Gayle E. Woloschak†,* Feinberg School of Medicine, Department of Radiation Oncology, Northwestern University, Chicago, Illinois 60611, United States, ‡Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, United States, §Chemistry of Life Processes Institute, Proteomics Core, Northwestern University, Evanston, Illinois 60208, United States, ^Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208, United States, Department of Chemistry, Yonsei University, Seoul 120-749, South Korea, and zDivision of Functional Materials and Nano-Devices, Ningbo Institute of Materials Technology & Engineering, Ningbo 315201, China
)
†
ABSTRACT The multicatalytic ubiquitinproteasome system
(UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, such as carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, such as neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding “hotspots” possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected. KEYWORDS: iron oxide nanoparticles . titanium dioxide nanoparticles . protein adsorption . ubiquitinproteasome system . proteasome activation
T
he ubiquitinproteasome system (UPS) is conserved in all eukaryotic species and is responsible for the timely and orderly degradation of the majority of cellular proteins, regulating in that way most cellular processes.13 To maintain cellular homeostasis, the UPS targets not only misfolded and oxidized proteins but also cyclins, DNA repair proteins, and apoptosis proteins when their presence interferes with the ongoing cellular events.4 Polyubiquitination most often triggers proteolysis, and ubiquitin modification is carried out by ubiquitinactivating (E1), ubiquitin-conjugating (E2), and ubiquitinprotein ligase (E3) enzymes.5,6 FALASCHETTI ET AL.
Protein degradation is executed by the 26S proteasome, which receives and processes polyubiquitinated proteins. Two subcomplexes complete the 26S proteasome: the 28-subunit core particle (20S proteasome) and the 19subunit regulatory particle (19S proteasome).7,8 The 20S proteasome is a stable protein complex, barrel-shaped, and composed of four stacked heptameric rings of R and β subunits arranged as R17β17β17R17.9 The two outer rings are composed of proteolytically inactive R subunits forming a narrow pore that allows entrance of unfolded substrate proteins into the inner space of the complex, the proteolytic chamber. The active sites of VOL. XXX
’
NO. XX
’
* Address correspondence to
[email protected]. Received for review May 13, 2013 and accepted August 9, 2013. Published online 10.1021/nn402416h C XXXX American Chemical Society
000–000
’
XXXX
A www.acsnano.org
average diameter
hydrodynamic diameter
surface area per mol
chemical composition
(nm)
(nm)
(nm2/mol)
Fe3O4 Fe3O4 Fe3O4 Fe3O4 TiO2 TiO2 Fe3O4/Fe2O3e
10.5 ( 1.1 10.5 ( 1.1 4.1 ( 0.6 4.1 ( 0.6 20.2 3.0 ( 4.4 0.2 5.1 2.8 ( 1.4 0.1 ∼35
42.6 ( 1.3 43.6 ( 0.8 31.2 ( 0.7 27.1 ( 1.1 N/A N/A 59.5 ( 1.2
2.1 10 2.1 1026 3.3 1025 3.3 1025 1.2 1026 3.7 1025 2.3 1027 26
shape
sphere sphere sphere sphere rod rod sphere
zeta-potential
fwhm
(mV)
(mV)
54 ( 1 76 ( 3 36 ( 1 31 ( 1 64 ( 1 51 ( 6 24 ( 1
21 ( 1 26 ( 3 18 ( 2 27 ( 3 19 ( 1 14 ( 2 21 ( 1
surface coating b
TEGc PEG600cc TEGc PEG600c OHd OH carboxydextranf
ARTICLE
a
TABLE 1. Summary of the Nanoparticles Used in This Work
a Measurements shown here were done in nanopure H2O; additional zeta-potential measurements in other buffers and in the presence or absence of proteasome 20S complex are given in Supporting Information Table S1. b TEGc: tetraethylene glycol-carboxylate (average Mn 250). c PEG600c: polyethylene glycol-carboxylate (average Mn 600). d Hydroxyl groups are formed as an outcome of tetramethylammonium hydroxide treatment following nanorod synthesis.49 e FeraSpin R, purchased from Miltenyi Biotec. f Average MW 70 000.
the β1, β2, and β5 subunits face this inner space and exhibit caspase-, trypsin-, and chymotrypsin-like activity, respectively.10 The 19S proteasome, an allosteric stimulator of 20S proteolytic activity, recognizes polyubiquitinated proteins, removes ubiquitin moieties, and unfolds substrates to be degraded by the 20S proteasome.1114 In organisms from yeast to humans, activity of the UPS regulates cell cycle progression, signal transduction, and differentiation.15,16 Diseases as varied as cardiac dysfunction, autoimmune disorders, and viral infections often involve deregulated proteasome activity or expression.1719 A decline of proteasome activity often correlates with the appearance of protein aggregates in age-related neurodegenerative diseases.2022 In addition, many cancers are linked with increased polyubiquitination and/or proteasome quantity or activity.2325 The investigation of nanomaterials as diagnostic and therapeutic agents in medicine is expanding exponentially. Therefore, it is important to evaluate interactions of nanoparticles with different cellular components and biomolecules.26 In most biological environments, nanomaterials have the opportunity to interact with local proteins. This leads to the creation of a protein corona, which alters the biological identity of the nanoparticles and modulates biological responses to nanomaterials.2730 On the other hand, the folding and activity of the bound proteins often change, as well.31,32 Most previous studies on protein coronas have focused on plasma proteins that interact with the nanomaterial rather than on intracellular proteins.3336 Among intracellular proteins, the proteasome is present in high concentrations;37,38 therefore, intracellular contact between nanoparticles and the 20S proteasome complex is likely. A recent study indicated the presence of 20S proteasome subunits in nanoparticle coronas,39 but the functional effects of these interactions still await full exploration. Nanoparticles made of gold, iron oxide, and titanium dioxide are among the most abundantly used metallic nanomaterials. Nevertheless, protein interaction studies for the latter two nanoparticle types lag behind FALASCHETTI ET AL.
work done on Au nanomaterials. For example, a literature search for nanoparticleprotein corona articles published this year results in only one paper on iron oxide, while there are four such articles on Au nanoparticles.4044 For work presented here, we assembled a small collection of Fe3O4 and TiO2 nanoparticles (Table 1, Supporting Information Figure S1). Nanoparticle size and shape have been shown to influence interactions with proteins;45,46 therefore, we selected spherical 4.1 and 10.5 nm Fe3O4 nanoparticles as well as FeraSpinR, commercially available nanoparticles with a spherical 35 nm iron oxide core and rod-shaped 20.2 3 and 5.1 2.8 nm TiO2 nanoparticles. While large iron oxide nanoparticles such as FeraSpinR found their place in in vivo studies as magnetic resonance contrast agents, smaller Fe3O4 and Fe2O3 nanoparticles are used in different non-MR applications, as well, such as gene delivery.47 With regard to TiO2 nanoparticles, rod-shaped nanoparticles were selected because their synthesis allows for a more controlled, monodisperse preparation.48 All of these nanoparticles were prepared as monodisperse and surface modified with hydroxyl groups or carboxyl groups carried by ethylene glycol or dextran (Table 1). Zeta-potential measurements of these nanoparticles in H2O and other buffers used in this study, in the presence and absence of the 20S proteasome, are shown in Table 1 and Supporting Information Table S1. Several approaches were used to evaluate the interactions between selected nanoparticles and the 20S proteasome complex (Figure 1). We investigated the depletion of individual 20S proteasome subunits and their component peptides using Western blot and mass spectrometry. We also studied fluctuations of the 20S proteasome proteolytic activities in the presence of nanoparticles. RESULTS AND DISCUSSION Interaction between the 20S proteasome as a whole and nanoparticles was investigated by mixing the 20S proteasome with 10.5 nm Fe3O4 nanoparticles coated with tetraethylene glycol-carboxylate (TEGc). These VOL. XXX
’
NO. XX
’
000–000
’
B
XXXX www.acsnano.org
ARTICLE Figure 1. Schematic representation of experimental design. Metal oxide nanoparticles (1) adsorb to the 20S proteasome, (2) show preferential adsorption to several peptide sequences, and (3) induce fluctuations in 20S proteasome activity. (1) Following co-incubation of nanoparticles and 20S proteasome complexes, nanoparticles were pelleted, and unbound 20S proteasome was quantified by Western blot and mass spectrometry (Figures 2 and 3). (2) 20S proteasome co-incubated with nanoparticles was digested either by trypsin, chymotrypsin, or Asp-N; the nanoparticles were subsequently pelleted, and free peptides were analyzed by mass spectrometry (Figures 4 and 5). (3) Cleavable luminogenic peptides were used to evaluate three major catalytic activities of the 20S proteasome in the presence of nanoparticles (Figures 68).
“super-complexes” made of nanoparticles and the 20S proteasome were visualized with transmission electron microscopy (Supporting Information Figure S2). Co-incubated nanoparticles and the 20S proteasome were applied to grids, stained with uranyl acetate, and dried. Imaged samples show an interspersed distribution of the 20S proteasome and nanoparticles, suggesting that the nanoparticles preferentially interact with the 20S proteasome rather than with each other. Next, the 20S proteasome was co-incubated with increasing concentrations of 10.5 and 4.1 nm Fe3O4 nanoparticles coated with TEGc or polyethylene glycolcarboxylate (PEG600c). In these experiments, the concentration of proteasome was constant at 440 nM (2 μg of purified 20S proteasome). After 17 h incubations, nanoparticles were pelleted together with the adsorbed 20S proteasome complex. Because of the high colloidal stability of these nanoparticles, it was necessary to induce flocculation before centrifugation. This was done with the addition of 4.5 M NaCl; nanoparticles precipitated in this manner formed an insoluble pellet together with the 20S proteosome complexes adsorbed on their surfaces. The same experimental conditions applied to the 20S proteasome did not lead to free protein precipitation (Supporting Information Figure S3). Next, the supernatant was desalted and 20S proteasome subunits were resolved by SDS-PAGE. The R2 subunit was assessed by a Western blot as a representative protein for the 20S proteasome complex. In this assay setup, where irreversible co-precipitation of nanoparticles and proteins is obtained after centrifugation, greater adsorption of protein to the nanoparticles corresponds to a reduction in the presence of protein in the supernatant. Using a Western blot for the R2 subunit, we found that the nanoparticles FALASCHETTI ET AL.
adsorb the 20S proteasome in a concentration-dependent manner (Figure 2). Both 62.3 and 103.8 nM concentrations of 10.5 nm Fe3O4 TEGc and PEG600c nanoparticles measurably depleted the R2 20S proteasome subunit from the samples (Figure 2a,d). Only a 600 nM concentration of 4.1 nm Fe3O4 TEGc nanoparticles achieved similar levels of 20S proteasome adsorption (Figure 2b,c). The nanoparticle surface area per mol is very different for these two nanomaterials, and the cumulative surface of 103.8 nM 10.5 nm Fe3O4 TEGc nanoparticles and 600 nM 4.1 nm Fe3O4 TEGc nanoparticles is almost equal at 1.3 1016 and 1.2 1016 nm2, respectively. Therefore, it is likely that surface area plays a significant role in the concentration-dependent nanoparticle20S proteasome binding. However, nanoparticles of different diameters covered with PEG600c did not show a similar result (Figure 2df). Due to its greater length and higher polarity, the PEG600c coating may make a greater contribution to the overall nanomaterial properties than TEGc. However, it is difficult to speculate why the binding capacity of 10.5 nm PEG600c particles would be increased and that of 4.1 nm PEG600c particles would be decreased, compared to their TEGc-covered counterparts. Because the use of high salt concentration and the pelleting step can potentially disrupt nanoparticleprotein interactions,35 surface plasmon resonance (SPR) was used to confirm the adsorption between nanoparticles and the 20S proteasome (Supporting Information Figure S4). The 20S proteasome was immobilized to carboxyl-coated SPR substrate chips via amine-coupling chemistry and exposed to nanoparticles. The 10.5 nm Fe3O4 PEG600c nanoparticles demonstrated the highest adsorption capability, while the 4.1 nm Fe3O4 PEG600c nanoparticles bound to 20S VOL. XXX
’
NO. XX
’
000–000
’
C
XXXX www.acsnano.org
proteasome complexes most weakly (Supporting Information Figure S4). SPR data predicted a nanoparticle/ protein stoichiometry greater than 1:1, making it impossible to obtain accurate kinetic parameters for these interactions. These findings were in keeping with the observations made by TEM (Supporting Information Figure S2), which suggested that more than one nanoparticle can interact with a single 20S proteasome complex and vice versa. To determine the adsorption for each of the 20S proteasome subunits individually, we examined the supernatants of nanoparticle20S proteasome co-incubation experiments by label-free quantification by mass spectrometry (Figure 3). Once again, 10.5 nm Fe3O4 TEGc nanoparticles at 20.8 and 103.8 nM concentrations were incubated with 2 μg of 20S proteasome protein (proteasome concentration of 440 nM) for 17 h. The “super-complex” formed from nanoparticles and the 20S proteasome was precipitated by the addition of salt and centrifugation, and the unbound protein in the supernatant was collected. The unbound 20S proteasome was denatured, digested with trypsin, and analyzed by mass spectrometry. Total spectral counting of each subunit in the 20S proteasome was used to measure quantitative difference between nanoparticle-treated 20S proteasome complexes and controls (Figure 3). The spectral counting relies on a general correlation between the number of peptides sequenced per protein and the amount of sample protein. In this experiment, incubation with 20.8 nM 10.5 nm Fe3O4 TEGc nanoparticles (nanoparticle/protein ratio of 1:21) does not lead to significant removal of the 20S proteasome; however, incubation with 103.8 nM 10.5 nm Fe3O4 TEGc nanoparticles (nanoparticle/ protein ratio of 1:4) reduces the quantity of each of the 20S proteasome subunits in the supernatant to close to 40% (Figure 3). A similar degree of depletion of each of the subunits under these experimental conditions suggests that intact 20S proteasome complexes are pulled down by the nanoparticles and that adsorption of the 20S proteasome complex to nanoparticles does not result in subunit dissociation. To confirm that co-incubation with nanoparticles does not separate the subunits of the 20S proteasome FALASCHETTI ET AL.
ARTICLE
Figure 2. 20S proteasome adsorption to Fe3O4 nanoparticles determined through depletion of the R2 subunit. “Free” R2 subunit was quantified by Western blot following a 17 h incubation of 440 nM 20S proteasome with (a) 10.5 nm Fe3O4 TEGc, (b, c) 4.1 nm Fe3O4 TEGc, (d) 10.5 nm Fe3O4 PEG600c, and (e,f) 4.1 nm Fe3O4 PEG600c nanoparticles. Nanoparticles and the adsorbed 20S proteasome were irreversibly pelleted in the presence of 4.5 M NaCl and removed from the samples; the R2 subunit remaining in solution was detected by Western blot. Duplicate samples per nanoparticle concentration are presented.
Figure 3. Mass spectrometry analysis of unbound 20S proteasome subunits following co-precipitation of the 20S proteasome and 10.5 nm Fe3O4 TEGc nanoparticles. 20.8 nM (red) and 103.8 nM (blue) 10.5 nm Fe3O4 TEGc nanoparticles were incubated with the 20S proteasome (440 nM) for 17 h and the adsorbed 20S and nanoparticles irreversibly precipitated. Unbound protein from the supernatant was trypsin digested; peptide spectral matches (PSMs) of each of the 20S proteasome subunits were compared with the nanoparticle-free control. The standard error of triplicate experiments is shown. Statistical significance between experimental and control samples was assessed with an unpaired t test, where * reflects a p value
