Bovine Serum Albumin Nanocomposites Inducing the

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Tellurium/Bovine serum albumin nanocomposites induce the formation of stress granules in a protein kinase R dependent manner Yanrong Zhou, Yanli Bai, Huabing Liu, Xiaohan Jiang, Ting Tong, Liurong Fang, Dang Wang, Qiyun Ke, Jiangong Liang, and Shaobo Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09402 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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ACS Applied Materials & Interfaces

Tellurium/Bovine serum albumin nanocomposites induce the formation of stress granules in a protein kinase R dependent manner

Yanrong Zhou,†,# Yanli Bai,†,§ Huabing Liu,†,§ Xiaohan Jiang,†,§ Ting Tong,†,§ Liurong Fang,†,# Dang Wang,†,# Qiyun Ke,†,# Jiangong Liang,*,†,§ and Shaobo Xiao*,†,# †

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural

University, Wuhan 430070, China #

The Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College

of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China §

College of Science, Huazhong Agricultural University, Wuhan 430070, China

Corresponding Authors *E-mail: [email protected] (S. Xiao); [email protected] (J. Liang) *These authors jointly supervised this work.

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ABSTRACT: The effect of nanoparticles (NPs) on cellular stress responses is important to the understanding of nanotoxicities and developing safe therapies. Although the relationship between NPs and cellular stress responses has been preliminarily investigated, stress responses to NPs remain unclear. Here, Tellurium/bovine serum albumin (Te/BSA) nanocomposites were prepared using sodium tellurite, BSA, and glutathione (GSH) as precursors. The as-prepared Te/BSA nanocomposites, with particle size similar to those of many viruses, are found to induce the formation of stress granules (SGs), a kind of cytoplasmic RNA granule formed under various stresses. The SGs in Te/BSA nanocomposite-treated cells are composed of T-cell internal antigen 1 (TIA1), TIA1-related protein (TIAR), and eukaryotic initiation factor 3η (eIF3η). Using chemical inhibitors and siRNA-mediated silencing, protein kinase R (PKR) is identified as the eIF2α-kinase activated upon Te/BSA nanocomposite incubation, which is also the dominant kinase responsible for eIF2α activation under virus infection. Mechanistically, PKR is activated in a heparin-dependent manner. This study reveals a biological effect of Te/BSA nanocomposites on stress responses, providing a preliminary basis for further research on virus-like particles (VLPs) and the application of NPs in biology.

KEYWORDS: Tellurium/Bovine serum albumin (Te/BSA) nanocomposite, stress granules (SGs), protein kinase R (PKR), virus-like particles (VLPs), heparin

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1. INTRODUCTION Stress granules (SGs) are cytoplasmic mRNP granules formed in response to stress-induced translation arrest.1 Since their discovery in 1999, SGs have been found to be assembled under stresses of various stimuli, such as osmotic stress (sorbitol), oxidative stress (arsenite), endoplasmic reticulum stress (thapsigargin), mitochondrial stress (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, [FCCP]), proteasome inhibition (MG132), and abortion of translation initiation (pateamine A).2 In addition to their basic role in RNA metabolism, SGs have also been considered a hub for the regulation of many signaling pathways through sequestration of multiple proteins. For example, RACK1/p38/c-Jun N-terminal kinase (JNK) signaling cascade that regulates apoptosis; rapamycin (TOR) signaling that controls cellular growth and survival; Rho GTPase signaling implicated in Wnt fate; innate immune response; and some mRNA metabolism-related signaling; indicating that SGs are involved in the pathogenesis of diseases such as cancer, viral infection, and neurodegenerative disorders.3 The connection between SGs and cancers has been identified by several groups, in particular the role of SGs in chemotherapeutic treatments of cancer.4– 5

For instance, bortezomib (PS-341/Velcade), an FDA-approved anti-tumor

drug, stimulates the assembly of pro-survival anti-apoptotic SGs in cancer cells, which undergo severe apoptosis when the formation of bortezomib-induced SGs is inhibited.6 Similarly, antimetabolite 5-fluorouracil (5-FU), another chemotherapeutic agent used for treatment of diverse types of cancers,7 also induces the formation of RACK1-positive SGs with pro-survival and 3



anti-apoptotic properties.8 The formation of SGs has also been detected under treatment with some other chemotherapeutic agent analogous to 5-FU, such as 6-thioguanine and 5-azacytidine.8 Collectively, SGs are likely to have a role in the strategies for developing resistance to chemotherapy treatments of cancer cells, suggesting the potential of SGs as targets for anti-cancer therapy. The correlation of SGs with viruses remains controversial, however, growing evidence suggests that SG assembly in host cells is an important antiviral mechanism against many viruses, such as influenza A virus (IAV),9 Japanese encephalitis virus,10 measles virus,11 vesicular stomatitis virus (VSV),12 poliovirus,13 mengovirus, and Theiler’s murine encephalomyelitis virus (TMEV).14 Viral replication has been shown to be suppressed by SGs or components of SGs through the recruitment of some vital antiviral proteins in innate immune response.15 Therefore, the interaction of virus proteins with SG components is utilized by some viruses to counter antiviral SGs and support the propagation of viruses, which has provided a basis for research on antiviral targets.16 In recent years, several groups have studied the relationship between stress response and NPs. Kodiha et al. showed that gold NPs trigger cellular stress response and reduce molecular chaperone hsp70 and proteins modified by O-GlcNAc in the nucleoli and nucleus.17 In addition, zinc oxide NPs were found to induce the formation of SGs in A549 cells.18 Romashchenko et al. found that inorganic NP-associated proteomes significantly overlap with SG components in mammalian 4


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cells, which provided a basis for further research into cellular responses to NPs.19 However, the mechanism by which NPs interact with SGs remains unclear. NPs, including bovine serum albumin (BSA)-based NPs, play a vital role in biomedicine, especially in cancer imaging and therapy.20–21 For example, BSA-Au-MnO2 composite nanoparticles can act as radio-sensitizers, facilitating highly effective radiotherapy of tumors.22 In addition, cationic BSA-conjugated poly lactic-co-glycolic acid NPs, with the ability to circumvent the blood-brain barrier, are used for delivery of methotrexate into brain tumors.23 Hydrophilic drug (HD)-loaded multifunctional BSA nanocapsules with redox-responsive controlled release of HDs, have been shown to be selectively internalized by tumor cells, making them potential vehicles for targeted delivery.24 And Wang et al. found that glycyrrhetinic acid-containing Gd-DOTA derivative (GGD)-BSA nanoparticles (GGD-BSA NPs) showed remarkable sensitivity in noninvasive detection of liver tumors.25 Considering the extensive application of BSA-based NPs in biology and the pivotal position of SGs in diverse cellular pathways, the objectives of this study were to (i) investigate the correlation of NPs with stress response using Te/BSA nanocomposites as a model and (ii) preliminarily explore the related mechanisms for the potential application of NPs in biology.

2. EXPERIMENTAL SECTION 2.1. Cell Culture. MARC-145 cells (a monkey kidney cell line) and HeLa cells (cervical carcinoma cells) were both cultured and maintained in Dulbecco’s Modified 5



Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in a humidified incubator at 37 °C under 5% CO2. 2.2. Reagents and siRNAs. GSK2606414 (516535 PERK Inhibitor I, henceforth referred to as PERK-I, dissolved in dimethyl sulfoxide [DMSO], Merck Millipore) is a highly potent PERK inhibitor.26 C16 (an imidazolo-oxindole compound, Merck Millipore) and 2-aminopurine (2-AP, a nucleotide analogue solubilized in DMSO, Sigma-Aldrich) act as potent PKR phosphorylation inhibitors.27–28 To assess the role of PKR and PERK, MARC-145 cells were pretreated separately with PERK-I (2.0 μM, 4.0 μM), C16 (0.50 μM, 5.0 μM), 2-AP (2.0 mM, 4.0 mM), or solvent control (DMSO) for 2 h and then incubated or mock-incubated with Te/BSA nanocomposites for 4 h. Confocal microscopy analysis and western blot analysis were then carried out. To investigate the effect of HRI and GCN2, three pairs of HRI- or GCN2-specific small interfering RNA (siRNA) sequences or negative control (NC) siRNA (Invitrogen) were designed and separately transfected at a final concentration of 50.0 nM using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions. The most efficient pair of siRNA was then selected for subsequent experiments through RT-qPCR and western blot assays. At 36 h post transfection, MARC-145 cells were treated with Te/BSA nanocomposites for 4 h, followed by indirect immunofluorescence assay. The siRNA sequences used are listed in Supplementary Table 1. 2.3. Western Blot Analysis. MARC-145 cells in 6-cm2 culture dishes were rinsed three times with PBS, followed by addition of 300 μL/dish of lysis buffer (LBA) (4% 6


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sodium dodecyl sulfate [SDS], 3% DL-dithiothreitol [DTT], 65 mM Tris-HCl [pH 6.8], 30% glycerin) supplemented with protease inhibitor (PMSF). The protein concentrations in the cell lysates were measured and equal amounts of each sample were denatured in 5×SDS loading buffer by boiling at 95 °C for 10 min for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore), then the membranes were blocked with 10% (w/v) bovine serum albumin (BSA) in tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) for 4 h at room temperature. The membranes were then incubated with rabbit antibodies against P-eIF2α (Ser51), eIF2α, GCN2 (Cell Signaling Technology, Inc), HRI (Boster Biological Technology), or PACT (ABclonal Biotechnology Co., Ltd) for 4 h at room temperature. Next, the membranes were washed with TBST and then exposed to horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Beyotime) for 1 h at room temperature. The β-actin was detected with an anti-β-actin mouse mAb (Beyotime) as a loading control. Protein signals were visualized using the Clarity Enhanced Chemiluminescence (ECL) reagent (Bio-Rad). The ratio of P-eIF2α/eIF2α was analyzed using ImageJ Software. 2.4. Indirect Immunofluoresence Assay. MARC-145 cells or HeLa cells seeded on circular glass coverslips (NEST Biotechnology) in 24-well plates were fixed with 4% paraformaldehyde (pre-cooled to 4°C) for 15 min and immediately permeabilized with methanol (pre-cooled to −20 °C) for 10 min. The cells were blocked with 5% (w/v) BSA in phosphate buffered saline (PBS) for 45 min, followed by incubation for 7



1 h with goat polyclonal antibodies (pAb) against TIA1, TIAR, or eIF3η, or rabbit pAb against eIF4G (Santa Cruz Biotechnology, Inc). The cells were then incubated for 1 h with Alexa Fluor 594-conjugated donkey anti-goat IgG or Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Inc), followed by counterstaining of the cell nuclei with 0.05% 4ʹ,6-diamidino-2-phenylindole (DAPI, Beyotime) for 15 min. All incubation steps were performed at room temperature. The resulting fluorescence images were obtained using an Olympus FV10 laser scanning confocal microscope (Olympus) or N-structured illumination microscope (N-SIM) (Nikon). 2.5. Quantitative Real-time PCR (RT-qPCR). Total RNA of MARC-145 cells transfected with three pairs of HRI- or GCN2-specific siRNAs or NC siRNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. 0.50 µg of each sample was subsequently reverse transcribed to cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. The resulting cDNA was then used as the template in a SYBR green qPCR assay (10-µl RT reaction mixture containing 0.50 µl cDNA, 5.0 µl SYBR Select Master Mix, and 100 nM of each primer) with an Applied Biosystems ViiA 7 real-time RT-PCR system (Applied Biosystems). The thermal cycling conditions were 10 min at 95 °C and 40 cycles of 15 s at 95 °C, 30 s at 56 °C, and 40 s at 72 °C. Each cDNA sample was assayed three times and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The primers used are shown in Supplementary Table 2. 8


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3. RESULTS AND DISCUSSION 3.1. Characterization of Te/BSA Nanocomposites. Te/BSA nanocomposites were prepared as described in the Supporting Information. The morphology of the synthesized Te/BSA nanocomposites was characterized by transmission electron microscopy (TEM) (Figure 1a). It can be seen that the Te/BSA nanocomposites were well dispersed and essentially spherical in shape, with an average size of 85.8 ± 23.6 nm. The high resolution TEM (HRTEM) images in Figure 1b and 1c show the high crystallinity of the Te/BSA nanocomposites with a lattice spacing of 0.33 nm, which is concordant with that of trigonal Te nanorods.29 Energy dispersive X-ray spectroscopy (EDX) of the Te/BSA nanocomposites revealed that C and Te were the main elements present (Figure 1d).

Thermogravimetric analysis

(TGA) of

the Te/BSA

nanocomposites was performed as previously described with slight modification.30 As shown in Figure S1, the Te/BSA nanocomposites exhibited a weight loss of ~80%, which is likely to correspond to loss of BSA from the nanocomposites.

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Figure 1. TEM (a) and HRTEM (b and c) images of the Te/BSA nanocomposites. (d) EDX spectrum of Te/BSA nanocomposites.

The elemental composition and chemical structure of the obtained Te/BSA nanocomposites were further investigated using X-ray photoelectron spectroscopy (XPS) measurements. In Figure 2a, the XPS survey spectrum displays bands attributed to C1s (284.9 eV), O1s (531.1 eV), N1s (399.6 eV), and Te3d (573.9 eV). The deconvolution of the C1s peak (Figure 2b) indicates the presence of C=O (287.8 eV), C–O (286.0 eV), C–N (284.9 eV), and C=C/C–C (284.4 eV).31–33 As illustrated in Figure 2c, the N1s peaks at 400.8, 399.6, and 399.3 eV were attributed to (C)3–N, C–N–C, and C=N–C, respectively.34–36 The Te3d core level spectra are shown in Figure 2d, with the peaks at binding energies of 584.3 and 573.9 eV corresponding to Te3d3/2 and Te3d5/2, respectively.37–38

Figure 2. XPS survey spectrum of Te/BSA nanocomposites (a) and high resolution XPS spectra of C1s (b), N1s (c), and Te3d (d). 10


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The UV-vis absorbance spectra of the Te/BSA nanocomposites showed strong optical absorption in the UV region below 276 nm, with a tail extending to the visible range (Figure S2), which may be ascribed to the n–π* transition of the C=O band.39 In addition, the zeta-potential of the Te/BSA nanocomposites was measured to be 15.7 ± 0.62 mV, indicating the presence of positively charged surface groups, which may facilitate electrostatic interaction with negatively charged cell surface receptors and promote the internalization of Te/BSA nanocomposites into cytoplasm. The X-ray diffraction (XRD) patterns of the Te/BSA nanocomposites (Figure S3a) show a broad diffraction peak centered at around 23°, indicating the existence of (100) facets of Te, which is in agreement with the standard literature values (JCPDF no. 36-1452).40 3.2. Formation of SGs Under Te/BSA Nanocomposite Treatment. To our knowledge, no observations of BSA-based NP-induced formation of SGs in mammalian cells have been reported. To determine the ability of Te/BSA nanocomposites to assemble SGs, we first evaluated their cytotoxicity with an MTT assay (Figure S4a). MARC-145 cells were then incubated or mock-incubated with Te/BSA nanocomposites, followed by immunostaining with TIA1, a robust SG marker, to detect SG formation at the indicated time points. In unstressed eukaryotes, TIA1 is located in both the cytoplasm and nucleus. When exposed to environmental stresses, TIA1 and some other related proteins are recruited to nucleate SG assembly in the cytoplasm.41 Our confocal microscopy analysis revealed that SG formation occurred rapidly post Te/BSA nanocomposite treatment in a time-dependent manner within 4 h 11



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(beginning at 1 h and reaching a maximum at 4 h, Figure S5a). Dose-dependent SG induction was also observed in the Te/BSA nanocomposite-treated cells (Figure S5b). Moreover, quantification of these results demonstrated that cells exposed to Te/BSA nanocomposites at a concentration of 0.10 mg/L for 4 h yielded the most robust SGs (Figure S5c), which was also clearly observed under structured illumination microscopy

(SIM)

(Movie

S1).

Furthermore,

the

formation

of

Te/BSA

nanocomposite-induced SGs visualized by TIA1 was also observed in HeLa cells, suggesting that the findings are not restricted to MARC-145 cells (Figure S6). 3.3. Preliminary Exploration of Potential Te/BSA Nanocomposite-Induced SG Formation Mechanism. We then explored the underlying mechanisms behind the formation of SGs in Te/BSA nanocomposite-treated cells. First, the concentration of dissolved tellurium in the Te/BSA nanocomposite-treated cells was tested based on a previously reported method with some modifications.42 The results of ICP-AES showed that the total concentration of intracellular and extracellular tellurium was 5.8 mg/L for the Te/BSA nanocomposite-treated group. Cells were then treated with Na2TeO3 at the same concentration of tellurium and the ability of tellurium to induce SG assembly was tested. The results showed that no SGs formed in Na2TeO3-treated cells (Figure S7), indicating that dissolved tellurium did not contribute to the formation of SGs in Te/BSA nanocomposite-treated cells. Additionally, the effects of the other two precursors, BSA and GSH, on the formation of SGs were also investigated. The data indicated that neither BSA nor GSH induced SG formation (Figure S7). Therefore, Te/BSA nanocomposites, but not their precursors contribute 12


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to SG assembly. A previous study reported that oxidative stress could induce SG formation,43 therefore the role of ROS in the induction of SG was explored. We tested the relative level of ROS in cells treated with Te/BSA nanocomposites using a previously reported method with moderate modification.44 As shown in Figure S8a, arsenite, used as a positive control, increased ROS levels in comparison with the negative control group (Mock) as previously reported,45 while Te/BSA nanocomposites did not upregulate the level of ROS. In fact, we observed that over long periods of exposure Te/BSA nanocomposites reduced ROS levels to some extent (Figure S8b), indicating that ROS generation was not the reason for SG formation. To investigate whether SG assembly was associated with Te/BSA nanocomposite size, L-Te/BSA nanocomposites (L-TeNPs, Te/BSA nanocomposites with larger size) were synthesized and characterized (Figure S1–4, S9–10). After treating MARC-145 cells with L-TeNPs, SG formation was also observed, but no significant changes were detected in the assembly of SGs in cells exposed to L-TeNPs at different time points (data not shown). The frequency of SG formation induced by L-TeNPs was found to be much lower than that resulting from Te/BSA nanocomposites at the same concentration (Figure 3a and S11). We hypothesize that the larger average size of L-TeNPs compared with Te/BSA nanocomposites may lead to differences in the efficiency of their uptake into cells, and subsequently a variation in stress intensity. To test this hypothesis, we conjugated FITC to both L-TeNPs and Te/BSA nanocomposites (L-TeNPs-FITC and Te/BSA nanocomposites-FITC conjugates) to 13



make them more visible and relatively quantifiable by confocal microscopy (Supporting Information). After treating MARC-145 cells with the same concentration (0.10 mg/L) of L-TeNPs-FITC or Te/BSA nanocomposites-FITC for 4 h, a confocal microscopy analysis was carried out. As illustrated in Figure 3b, both L-TeNPs-FITC and Te/BSA nanocomposites-FITC were generally localized in the cytoplasm, however significantly higher fluorescence intensity was observed in Te/BSA nanocomposite-FITC-treated cells, indicating the penetration of more Te/BSA nanocomposites into the cytoplasm and higher stress, which resulted in a higher frequency of SG induction.

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Figure 3. (a) The formation of SGs in MARC-145 cells treated with L-TeNPs (first row) and Te/BSA nanocomposites (second row). (b) The location of L-TeNPs (first row) and Te/BSA nanocomposites (second row) in MARC-145 cells.

To explore whether appropriate NP size of is sufficient to induce SG formation, polystyrene spheres, with a similar size to that of the Te/BSA nanocomposites (Figure S12), were used for preliminary evaluation of the physical effects of NPs in inducing SGs. First, the cytotoxicity of polystyrene spheres was tested by MTT assay. As shown in Figure S13, the cell viability was more than 90% when treated with polystyrene spheres at a concentration of 16 g/L for 4 h, therefore this concentration was chosen for subsequent experiments. The results of indirect immunofluoresence assays revealed that TIA1, TIAR, and eIF3η did not assemble into granules in polystyrene sphere-treated cells (Figure S14), indicating that polystyrene spheres are not able to induce SG formation. These results show that not all NPs with similar size have the ability to induce SG formation. 3.4. Components of Te/BSA Nanocomposite-induced SGs. More than 120 proteins were found in SGs, primarily eukaryotic initiation factors (eIF) and RNA binding proteins (RBPs), several of which, such as TIA1 (T-cell internal antigen 1) and TIAR (TIA1-related protein), are recruited to nucleate SG assembly based on their aggregation-prone protein regions.1 Previous studies suggest that SGs are cytoplasmic RNA granules with stress-specific differences in composition.46 For example, vesicular stomatitis virus (VSV) induces the formation of cytoplasmic RNA 15



granules containing TIA1, but not eukaryotic initiation factor 3 (eIF3) or eIF4A.12 Additionally, PABP (poly-A binding protein), TIA1-related protein (TIAR), eIF4G, eIF4E, and eIF4A, but not eIF3b, are recruited to nucleate selenite-induced SGs.47 To further establish the composition of Te/BSA nanocomposite-induced SGs, we attempted to visualize several common SG components such as eIF3η, eIF4G, and TIAR under Te/BSA nanocomposite treatment. As shown in Figure 4a and 4b, cytoplasmic granules positive for eIF3η and TIAR were observed in Te/BSA nanocomposite-treated cells. In contrast, eIF4G, one of the bona fide SG markers,48 was selectively excluded from Te/BSA nanocomposite-induced SGs (Figure 4c). eIF4G, a component of eIF4F complex (eIF4A and eIF4E, eIF4G), constitutes typical SGs with eIF3 and 40S ribosomal subunits (Figure S15).49 In this work, eIF4G was found to be absent in the Te/BSA nanocomposite-induced SGs, implying the possible lack of the other two eIF4F components (eIF4A and eIF4E) in these SGs. Arsenite-induced SG assembly strongly relies on individual eIF3 subunits, therefore the presence of eIF3η in the Te/BSA nanocomposite-induced SGs may also be essential for SG formation.49 Overall, SGs formed in mammalian cells vary in composition with Te/BSA nanocomposites used as stimuli.48 3.5. Stress Kinase/s Involved in SG Formation. Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) is the main trigger of SG formation.50 eIF2α is a component of the eIF2-GTP-tRNAMet ternary complex required for 43S pre-initiation complex assembly and subsequent translation initiation. In cells exposed to various stress conditions, eIF2α is phosphorylated at serine 51 16


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separately by stress-induced activation of stress kinases PKR (protein kinase R, activated in response to UV exposure, viral infections, and heat shock), PERK (protein kinase R-like endoplasmic reticulum kinase, sensing endoplasmic reticulum stress), GCN2 (general control nonderepressible 2 kinase, which senses amino acid levels and is activated by amino acid deprivation), and HRI (heme-regulated inhibitor kinase, which monitors oxidative stress/ROS levels and is activated by heme deprivation). Phosphorylation of eIF2α leads to the assembly inhibition of 43S pre-initiation complexes, and consequently results in translation initiation repression, which is the most common mechanism responsible for the formation of SGs.51–52

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Figure 4. Te/BSA nanocomposite-induced SGs including eIF3η (a) and TIAR (b), but not eIF4G (c).

To investigate the stress kinase/s contributing to the formation of SGs induced by Te/BSA nanocomposites, MARC-145 cells were treated with specific siRNAs or inhibitors against them. Given the lack of availability of HRI- or GCN2-specific inhibitors, specific siRNAs of these two proteins were designed and used. The knockdown efficiency of HRI- or GCN2-specific siRNAs was examined by RT-qPCR assay and the most efficient one chosen for each protein was able to reduce the endogenous mRNA levels of HRI or GCN2 by approximately 90% relative to the negative control (NC) siRNA (Figure 5a and 5b). The knockdown efficiency of these two siRNAs was then further demonstrated through western blot assay (Figure 5c and 5d). As shown in Figure 5e, the immunostaining analysis indicated that there was no obvious difference between the HRI-knockdown cells/the GCN2-knockdown cells and the NC-treated cells in the percentage of Te/BSA nanocomposite-induced SG-positive cells, suggesting that neither HRI nor GCN2 was involved in the Te/BSA nanocomposite-induced stress response. Furthermore, the potential influences of the other stress kinases (PKR and PERK) on the Te/BSA nanocomposite-induced SG assembly were investigated. C16 and 2-AP for PKR, and PERK-I for PERK have been widely used as chemical inhibitors in previous studies.26,

53–55

MTT assays revealed no appreciable cytotoxicity in

MARC-145 cells at concentrations of 0.05–5.0 µM for C16, 1.0–4.0 mM for 2-AP, 18


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and 1.0–4.0 µM for PERK-I (Figure S16). The immunofluorescence microscopy results showed that the frequency of SG assembly induced by Te/BSA nanocomposites in PERK-I-treated cells was equivalent to that in DMSO-treated cells at the concentrations of PERK-I sufficient to inhibit PERK activation according to previous studies (Figure 6b).53, 56 However, compared with the control, 2-AP and C16 substantially dissolved Te/BSA nanocomposite-induced SGs in a dose-dependent manner (Figure 6a). These data reveal the essential role of PKR, rather than PERK, in the SG formation in response to Te/BSA nanocomposite treatment.

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Figure 5. Assessment of the silencing efficiency of GCN2- or HRI-specific siRNAs using an RT-qPCR assay (a and b) or western blot assay (c and d). Values are the mean ± SD of three independent tests. (e) GCN2- or HRI-specific siRNA-mediated depletion did not suppress the SG formation triggered by Te/BSA nanocomposites.

The phosphorylation of eIF2α is regulated by different aforementioned kinases during various environmental insults, and contributes to the formation of SGs. To further investigate the pathway of SG induction by Te/BSA nanocomposite treatment, the levels of phosphorylated eIF2α (P-eIF2α) in MARC-145 cells treated with specific inhibitors targeting PKR or PERK, were analyzed using a western blot assay. As shown in Figure 6c, Te/BSA nanocomposite treatment (Lane 2) strongly increased the eIF2α phosphorylation level when compared with the group without Te/BSA nanocomposite treatment (Lane 1). As expected, Te/BSA nanocomposite-induced eIF2α phosphorylation was reduced by 2-AP and C16 (Lane 4 and 5), but remained unchanged when treated with PERK-I (Lane 3). Moreover, there was no significant difference in the level of eIF2α among all of the groups. These findings indicate that Te/BSA nanocomposites induce the formation of SGs through the phosphorylation of eIF2α in a PKR dependent manner, which differs from arsenite, a canonical inducer of SG assembly by phosphorylation of eIF2α through HRI.57 Selenite has also been found to trigger eIF2α phosphorylation, but without identification of the stress kinase/s involved in selenite-induced SG assembly.47 Phosphorylation of eIF2α has been reported to provide cytoprotective functions by inducing survival pathways for 20


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cells suffering from stress insults, which implies a novel target for drug design and the potential use of Te/BSA nanocomposites.47, 58

Figure 6. Assembly of SGs induced by Te/BSA nanocomposites via PKR. (a) PKR phosphorylation inhibitor (C16 and 2-AP) disassembled SG formation induced by Te/BSA nanocomposites in a dose-dependent manner. (b) PERK inhibitor (PERK-I) did not block the SG assembly triggered by Te/BSA nanocomposites. (c) Te/BSA nanocomposite-induced phosphorylated eIF2α (P-eIF2α) expression levels were down-regulated by PKR phosphorylation inhibitor (C16 and 2-AP), but not by DMSO and PERK-I. The concentrations for these experiments were 4.0 mM (2-AP), 5.0 µM (C16), and 4.0 µM (PERK-I).

Subsequently, the pathway through which PKR was activated was investigated. It is well known that dsRNA, heparin, and PACT are three activators of PKR.59 As dsRNA primarily activates PKR during viral infection, heparin and PACT were considered the 21



potential factors in induction of PKR activation during treatment with Te/BSA nanocomposites. First, MARC-145 cells were incubated with Te/BSA nanocomposites for 4 h and then the protein level of PACT was detected by western blot assay. The result demonstrated that Te/BSA nanocomposite treatment did not increase the PACT expression level (Figure S17), suggesting that PACT is not the activator of PKR in Te/BSA nanocomposite-treated cells. MARC-145 cells were then treated with FITC-heparin (H7482; Invitrogen) using a previously described method with some modifications.60 The fluorescence images indicated that the amount of heparin entering cells significantly increased under treatment with Te/BSA nanocomposites (Figure S18), indicating the possibility that Te/BSA nanocomposites activate PKR in a heparin-dependent manner. Given that (i) the majority of viruses or virus-like particles (VLPs) have the ability to regulate SG assembly via PKR in most cases,61 and (ii) the diameter of Te/BSA nanocomposites is analogous to that of most viruses,62 we speculate that Te/BSA nanocomposites may penetrate cells via a mechanism similar to that of virions. Engineered VLPs, which are non-infectious particles composed of capsid proteins without the viral genome, have potential applications as an attractive therapy delivery platform based on mimicking viral characteristics for better internalization by cells with longer systemic residence.63–65 For example, rabies virus-mimetic silica-coated gold nanorods, which mimic the rabies virus in shape, size, in vivo behavior, and surface glycoprotein properties, have been introduced to treat brain tumors.64 A virus-mimetic nanostructure system for efficient tumor-targeted gene delivery shows 22


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enhanced stability and long-term efficacy for systemic p53 gene therapy of human prostate cancer.66 In addition, an engineered polymer for siRNA delivery with structural features similar to those of viral vectors has been developed.67–68 Multiple materials have been selected for modification of VLPs to give uniform and reproducible nanostructures.69–70 Considering their described biological properties, Te/BSA nanocomposites can serve as candidate NPs for VLP engineering.

4. CONCLUSIONS In this study Te/BSA nanocomposites were synthesized using sodium tellurite, GSH, and BSA as precursors, and the as-prepared nanocomposites were characterized by TEM, EDX, UV-vis absorption, XPS, XRD, and DLS. Confocal microscopy analysis revealed that Te/BSA nanocomposites assemble SGs that selectively accumulate TIA1, TIAR, and eIF3η, but not eIF4G. Furthermore, a dose- and time-dependent increase of SGs per cell was observed when using TIA1 as a marker protein in Te/BSA nanocomposite-treated cells. Interestingly, cell penetration of Te/BSA nanocomposites varies with their size, which might contribute to the regulation of their ability to assemble SGs. Furthermore, Te/BSA nanocomposites were found to trigger SG formation through a heparin-mediated PKR-dependent pathway. The similarities between Te/BSA nanocomposites and viruses in size and ability to induce SGs via the PKR pathway, could facilitate the potential application of Te/BSA nanocomposites in VLP engineering, a potent strategy for future therapy.

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ASSOCIATED CONTENT Supporting Information. Thermogravimetric analysis (TGA), UV-vis absorption spectra, XRD patterns, and cytotoxicity of Te/BSA nanocomposites and L-TeNPs. TEM, HRTEM images, EDX spectrum and XPS survey spectrum of the L-TeNPs. Confocal microscopy analysis of SG assembly in MARC-145 cells or HeLa cells treated with Te/BSA nanocomposites, precursors of Te/BSA nanocomposites, polystyrene spheres, or arsenite. The level of ROS and PACT in Te/BSA nanocomposite-treated cells. The TEM images and cytotoxicity of polystyrene spheres. Cytotoxicity of stress kinases. The effect of Te/BSA nanocomposites on heparin. The formation of SGs observed with SIM. The sequences of siRNAs and primers used in this study.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (S. Xiao) *E-mail: [email protected] (J. Liang) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS This research was supported by the National Natural Sciences Foundation of China (31490602, 31372439), the National Basic Research Program (973) of China (2014CB542700), and the Special Project for Technology Innovation of Hubei Province (2017ABA138).

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Te/BSA nanocomposites induce the phosphorylation of eIF2α depending on PKR, resulting in the translation arrest and subsequent formation of SGs, which consist of TIA1, TIAR and eIF3η. 32x12mm (600 x 600 DPI)


Bovine Serum Albumin Nanocomposites Inducing the

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