Negatively charged metal oxide nanoparticles interact with the 20S proteasome and differentially modulate its biologic functional effects

Abstract

The multicatalytic ubiquitin-proteasome 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.

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, 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, 5). 3) Cleavable luminogenic peptides were used to evaluate three major catalytic activities of the 20S proteasome in the presence of nanoparticles (Figures 6–8).

Figure 2

20S proteasome adsorption to Fe₃O₄ nanoparticles determined through depletion of the α2 subunit. “Free” α2 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 α2 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 < 0.05 and ** reflects a p-value < 0.01.

Figure 4

Mass spectrometry analysis of the peptides of each 20S proteasome subunit not engaged in binding with 10.5 nm Fe₃O₄ TEGc nanoparticles. 440 nM 20S proteasome incubated with 20.8 nM (red) and 103.8 nM (blue) nanoparticles for 17 h was trypsin digested in the presence of the nanoparticles. Nanoparticles were then precipitated from the samples, and non-nanoparticle bound peptides were analyzed by mass spectrometry. Protein coverage, the fraction of each full-length amino acid protein sequence determined by mass spectrometry, is altered for majority of the 20S proteasome subunits. Loss of protein coverage is more pronounced with higher nanoparticle concentrations. Standard error of triplicate samples is shown. Statistical significance was assessed with an unpaired t test, where * reflects a p-value < 0.05 and ** reflects a p-value < 0.01.

Figure 5

“Hotspots” of adsorption between 10.5 nm Fe₃O₄ TEGc nanoparticles and the 20S proteasome. 440 nM 20S proteasome was incubated with 103.8 nM 10.5 nm Fe₃O₄ TEGc nanoparticles (nanoparticle:protein ratio of 1:4) for 17 h and digested by either trypsin, chymotrypsin, or Asp-N. The nanoparticles were pelleted with the adsorbed peptides, and “free” peptides were analyzed by mass spectrometry. Peptides present in controls but consistently absent following all three digests were considered the peptides most strongly incorporated into the nanoparticle corona. Their sequences are depicted in red in (a) and yellow in (b) and shown in Table 2 and Supporting Information Figure S6. Positions of these peptides, as well as those detected in control- and 10.5 nm Fe₃O₄ TEGc nanoparticle-treated samples (green), were located using PyMOL software and the crystal structure of the mammalian 20S proteasome. Together, Views 1–4 show the complete 360° surface area of the 20S proteasome. Regions in white represent peptides not covered by digestion of control or experimental samples, as the enzymes were not 100% proteolytically efficient; it remains to be determined whether these regions contain of nanoparticle binding areas of interest. Additional depleted peptides shorter than 7 amino acids, as well as amino acid sequences missing at higher nanoparticle concentrations, are shown in Supporting Information Figure S6, Tables S2, S3. (b) View 1 has been expanded to include the electrostatic surface potential and polarity patches of the 20S proteasome using VMD and APBS software and the PDB2PQR web server.^– The remainder of this information for Views 2–4 can be found in Supporting Information Figure S7.

Figure 6

Fluctuations in 20S proteasome activity following incubation with 10.5 nm Fe₃O₄ TEGc and 10.5 nm Fe₃O₄ PEG600c nanoparticles. 20 ng of 20S proteasome (267 pM) was incubated with 10.5 nM, 6.3 nM, 3.1 nM, 1.6 nM, 0.8 nM, or 0.4 nM nanoparticles (left to right) for 1 or 17 h, as shown. Approximate nanoparticle:protein ratios are indicated above the plots as white numbers superimposed on black polygons. Three distinct 20S proteasome activities were evaluated by cleavage-matching luminogenic substrates; each protease activity is indicated separately: chymotrypsin-like activity associated with the β5 subunit (blue diamonds), trypsin-like activity associated with the β2 subunit (red circles), and caspase-like activity associated with the β1 subunit (green squares). Error bars were calculated as standard deviation of two separate experiments, each performed in triplicate. Statistical significance was assessed with an unpaired t test, where * reflects a p-value < 0.05 and ** reflects a p-value < 0.01.

Figure 7

Fluctuations in 20S proteasome activity following incubation with 4.1 nm Fe₃O₄ TEGc and 4.1 nm Fe₃O₄ PEG600c nanoparticles. 20 ng of 20S proteasome (267 pM) was incubated with 10.5 nM, 6.3 nM, 3.1 nM, 1.6 nM, 0.8 nM, or 0.4 nM nanoparticles (left to right) for 1 or 17 h, as shown. Approximate nanoparticle:protein ratios are indicated above the plots as white numbers superimposed on black polygons. Three distinct 20S proteasome activities were evaluated by cleavage-matching luminogenic substrates; each protease activity is indicated separately: chymotrypsin-like activity associated with the β5 subunit (blue diamonds), trypsin-like activity associated with the β2 subunit (red circles), and caspase-like activity associated with the β1 subunit (green squares). Error bars were calculated as standard deviation of two separate experiments, each performed in triplicate. Statistical significance was assessed with an unpaired t test, where * reflects a p-value < 0.05 and ** reflects a p-value < 0.01.

Figure 8

Fluctuations in 20S proteasome activity following incubation with 20.2 × 3 nm and 5.1 × 2.8 nm TiO₂ nanorods. 20 ng of 20S proteasome (267 pM) was incubated with 405 nM, 40.5 nM, 4.5 nM, and 0.45 nM nanorods (left to right) for 1 or 17 h, as shown. Approximate nanoparticle:protein ratios are indicated above the plots as white numbers superimposed on black polygons. Three distinct 20S proteasome activities were evaluated by cleavage-matching luminogenic substrates; each protease activity is indicated separately: chymotrypsin-like activity associated with the β5 subunit (blue diamonds), trypsin-like activity associated with the β2 subunit (red circles), and caspase-like activity associated with the β1 subunit (green squares). Error bars were calculated as standard deviation of two separate experiments, each performed in triplicate. Statistical significance was assessed with an unpaired t test, where * reflects a p-value < 0.05 and ** reflects a p-value < 0.01.