extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine Chemistry

text:
Doxycycline reverses epithelial-to-mesenchymal transition and suppresses the proliferation and metastasis of lung cancer cells

The gelatinase inhibitor doxycycline is the prototypical antitumor antibiotic. We investigated the effects of doxycycline on the migration, invasion, and metastasis of human lung cancer cell lines and in a mouse model. We also measured the effect of doxycycline on the transcription of epithelial-mesenchymal transition (EMT) markers, and used immunohistochemistry to determine whether EMT reversal was associated with doxycycline inhibition. Doxycycline dose-dependently inhibited proliferation, migration, and invasion of NCI-H446 human small cell lung cancer cells. It also suppressed tumor growth from NCI-H446 and A549 lung cancer cell xenografts without altering body weight, inhibited Lewis lung carcinoma cell migration, and prolonged survival. The activities of the transcription factors Twist1/2, SNAI1/2, AP1, NF-κB, and Stat3 were suppressed by doxycycline, which reversed EMT and inhibited signal transduction, thereby suppressing tumor growth and metastasis. Our data demonstrate functional targeting of transcription factors by doxycycline to reverse EMT and suppress tumor proliferation and metastasis. Thus, doxycycline selectively targets malignant tumors and reduces its metastatic potential with less cytotoxicity in lung cancer patients.

INTRODUCTION
Antibiotics that target mitochondria have great potential as anticancer drugs. [1]. For example, tetracyclines are cytotoxic to some tumor cells [2] and have been shown to induce apoptosis in osteosarcoma, prostatic cancer cells, and lymphocytes [4,5]. Doxycycline, a semi-synthetic tetracycline, inhibits matrix metalloproteinase (MMP) activation and cell proliferation [3] and can also interfere with tumor-related protein synthesis in mammalian cells [6][7][8]. The selective permeability of different mammalian cells to doxycycline led to the hypothesis that it could be used to arrest cell proliferation and treat malignancies. Thus, doxycycline has been used in combination with targeted drugs in clinical trials with patients with advanced cancer.
Lung cancer is the most common cause of cancer mortality worldwide [9]. Tumor recurrence and metastasis are common in lung cancer despite various lines of standard therapy and the introduction of targeted agents. The vast majority of patients with lung cancer fail to respond to tyrosine-kinase inhibitors against the epidermal growth factor receptor (EGFR) [10], and no effective drug is available for small cell lung cancer [11,12]. Therefore, the development of alternative treatments is urgently needed to improve outcome.
To investigate the therapeutic efficacy and the mechanism of action of doxycycline in lung cancer, we investigated the effects of doxycycline on migration, invasion, and metastasis of various lung cancer cell lines. We also examined its functional interference in EMT. In www.impactjournals.com/oncotarget addition, we used a tumor-bearing mouse xenograft model to investigate doxycycline inhibition of tumor growth in vivo.

Doxycycline reduces cell viability and alters cell cycle dynamics in human lung cancer cell lines
Using MTT assay, we determined the effect of 48 h treatment with doxycycline on cell viability of various cancer cell lines. As shown in Fig. 1A, lung cancer cells were more sensitive to doxycycline than most of the other cell lines. NCI-H446 and A549 cells showed sensitivity to doxycycline with IC 50 values of 1.7043 ± 0.1241 and 1.0638 ± 0.1266 μM, respectively. Proliferation of NCI-H446 (Fig. 1B) and A549 cells (Fig. 1C) was inhibited by doxycycline in a dose-dependent manner.
We also investigated whether doxycycline interferes with the cell cycle. NCI-H446 and A549 cells were treated with different doses of doxycycline for 24 h, followed by cell cycle analysis using flow cytometry. Cells treated with doxycycline started to arrest at G0/G1 phase after treatment for 24 h (Fig. 1D & 1E). After treatment, NCI-H446 cells at G0/G1 accounted for approximately 24% of the total cell population, and A549 cells in G0/G1 population accounted for approximately 44%.

Doxycycline inhibits lung cancer cell invasion and migration in vitro
To study whether doxycycline inhibits NCI-H446 and A549 cell invasion we used Matrigel-coated transwell chambers. For both cell lines, when compared with the control group, the number of cell invasions through the Matrigel-coated filter was dose-dependently reduced by doxycycline ( Fig. 2A & 2B). Notably, the inhibitory effects of doxycycline on cell invasion were due to its cytotoxic effects, given that cell viability was also decreased at the concentration ranges tested.
Next we assessed the ability of doxycycline to inhibit the migration of NCI-H446 and A549 cells using a wound-healing assay. Confluent cells were scraped with a sterile pipette tip, and the remaining cells were allowed to migrate into the gap created in the absence or presence of doxycycline. Remarkably, after 24 and 48 h treatment, the wound gap of both cell types was wider in the doxycycline-treated groups than in the untreated groups ( Fig. 2C & 2D), indicating that doxycycline inhibits motility of both NCI-H446 and A549 cells. The cell growth curves of NCI-H446 and A549 were shown in Fig. S1 The degradation of the extracellular matrix (ECM) and basement membrane are crucial steps in cancer invasion and metastasis and the proteolytic enzymes MMP-2 and MMP-9 are involved in this process. We next measured the secretion of MMP-2 and MMP-9 from NCI-H446 and A549 cells with or without doxycycline treatment. As shown in Fig. 2E, doxycycline inhibited MMP-2 and MMP-9 secretion into the medium in a dosedependent manner. This finding suggests that doxycycline may reduce lung cancer metastasis by inhibiting the degradation of the ECM and basement membrane.

Doxycycline inhibits the expression of epithelial markers and changes cellular morphology
Vimentin and E-cadherin regulate the expression of proteins involved in ECM degradation. Thus, we used immunofluorescent staining to measure the effect of doxycycline on vimentin and E-cadherin levels. NCI-H446 and A549 cells were treated with different doses of doxycycline for 24 h,. In response to doxycycline treatment, vimentin expression decreased, whereas E-cadherin expression increased in both cell lines (Fig. 3A & 3B). We also tested the effect of doxycycline on the cellular morphology of NCI-H446 and A549 by HCS. In response to doxycycline treatment, the perimeter-toarea ratio decreased, whereas pyknosis increased in both NCI-H446 and A549 cells ( Fig. 3C & 3F). The relative area of nucleus also increased, whereas the DNA content in cells decreased in both NCI-H446 and A549 cells ( Fig. 3D & 3E). As shown in Fig. 3, the results of DNA reduction were comparisons of all cells, not only S stage cells. For A549 cells, as shown in Fig. 1E, the number of S stage cells did not differ between groups.

Doxycycline inhibits the activity of EMT transcription factors
NCI-H446 and A549 cells transfected with luciferase reporter plasmids for various gene promoters or response elements were treated with doxycycline for 48 h. In NCI-H446 cells, doxycycline suppressed the expression of Twist1, Twist2, SNAI1, STAT-3, and NF-κB and promoted the expression of AP-1. However, no change in SNAI2 expression was observed (Fig. 4A). In A549 cells, doxycycline suppressed the expression of Twist1, Twist2, SNAI1, SNAI2, STAT-3, and NF-κB and promoted the expression of AP-1 (Fig. 4B). Multidimensional liquid chromatography-tandem mass spectrometry was performed to evaluate differentially expressed proteins in NCI-H446 cells after doxycycline treatment. 36 proteins were upregulated by doxycycline and 42 were downregulated. After doxycycline treatment in cancer cell lines, RPLs were directly or indirectly regulated. Some RPLs were inhibited by doxycycline, such as RPL23A, RPL10A, RPL12, RPL38 and RPL13. On the contrary, RPL3 was compensatorily increased to maintain basic protein synthesis. (Fig. 4C-4G). These proteins are involved in cell migration, cytoskeletal maintenance, cell proliferation, adhesion, and differentiation (Fig. 4H). www.impactjournals.com/oncotarget

Doxycycline has an antitumor effect in a mouse xenograft model
We next examined the effects of doxycycline on spontaneous lung metastasis using NCI-H446 human lung cancer and Lewis lung carcinoma (LLC) xenografts in nude mice. Doxycycline treatment inhibited tumor growth in a dose-dependent manner. In particular, the NCI-H446 xenografts in the cyclophosphamide group died out before the end of the experiment (Fig. 5A & 5C, 5E & 5F). Body weight increased in the doxycycline-treated group (Fig. 5B  & 5D). The median survival time of the control groups of LLC xenografts was 15.5 days. When compared with the control group, the median survival times of high-dose, middle-dose, and low-dose doxycycline treatment groups were increased by 235%, 197%, and 184%, respectively, and the median survival time of the cyclophosphamide treatment group was increased by 171% (Fig. 5G). The number of tumors that shifted stoves was decreased from 22.4 ± 7.96 to 4.20 ± 1.79 in the lungs of nude mice with LLC xenografts treated with doxycycline, (Fig. 5H). These results strongly suggest that doxycycline inhibits tumor growth and metastasis.

Doxycycline alters the expression of EMT markers in cancer tissues
Immunohistochemical staining for E-cadherin, vimentin, MMP-2, and MMP-9 showed their expression to be correlated with doxycycline treatment. E-cadherin exhibited low expression, whereas vimentin, MMP-2, and MMP-9 exhibited high expression in tumor cells treated with doxycycline compared with the untreated group in both the membrane and cytoplasm (Fig. 6A & 6B).

DISCUSSION
By preventing and suppressing tumor invasion and metastasis it may be possible to decrease the mortality rates of patients with malignant tumors [13]. Despite efforts to develop alternative treatments for lung cancer [14,15], in many cases the cancers are refractory and there is still no effective therapy [16,17,18,19].
Doxycycline has been shown to have high antimetastatic activity and low cytotoxicity in melanoma and breast carcinoma [20,21]. In clinical trials with patients with pleural effusion, satisfactory results were achieved when doxycycline was used and there was inhibition of the activity of the suspended tumor cells. The current study focuses on the mechanism and anti-metastatic effects of doxycycline. We used several human cancer cell lines to investigate the effect of doxycycline on cell viability and the cell cycle. Our results showed that doxycycline inhibited the proliferation of NCI-H446 and A549 cells more than the other cell lines. Doxycycline has a strong inhibitory effect on malignancies originating from mesenchyme and neuroectoderm, including melanomas, endothelial cell tumors, and sarcomas [20,22,23]. All three kinds of lung cancer cells used in this study have mesodermal characteristics and readily undergo EMT [24][25][26]. NCI-H446 is a small cell lung cancer cell originating from neuroectoderm, while A549 cells are thought to have already undergone EMT. By contrast, lung squamous cell carcinoma is not very sensitive to doxycycline (unpublished data).
Doxycycline has a strong inhibitory effect on cells with mesenchymal characters. Early studies showed that doxycycline influences focal adhesion kinase (FAK) and cell adhesion and causes cells to lose anchors during migration [20]. Our current study does not examine whether doxycycline directly inhibits chemotaxis or indirectly inhibits chemotaxis by the inhibition of cell adhesion. It is possible that increased invasion and migration might due to increased generation of chemokines from tumor cells. Doxycycline also has other targets, including MMPs and E-cadherin, ultimately inhibiting metabolism and metastasis [27]. Two members of the MMP family, MMP-2 and MMP-9, are necessary for tumor invasion and metastasis [28,29] [30]. Therefore, decreasing MMP activity could inhibit cancer cell invasion and metastasis [31,32]. We examined the regulation of MMP activity after doxycycline treatment. Our data show that doxycycline inhibits MMP-2 and MMP-9 activation, which is consistent with its inhibitory effects on metastasis.
EMT initiates tumor metastasis and alters cellcell adhesion [33][34][35]. E-cadherin and vimentin, two biomarkers of EMT, mediate cell adhesion. Downregulation of E-cadherin expression is both necessary and sufficient to confer metastatic ability to lung cancer cells that are otherwise non-metastatic [36,37]. Vimentin is an intermediate filament protein that, along with microtubules and actin filaments, forms the cytoskeleton. In some carcinomas, including prostatic carcinoma, vimentin is overexpressed and is therefore a marker for invasiveness and metastasis [38]. In this study, we found that doxycycline increased E-cadherin levels and decreased vimentin protein expression. This finding suggests that doxycycline suppresses EMT in lung cancer. In addition, studies of promoter activation and proteomics after doxycycline treatment revealed that doxycycline inhibits EMT-related transcription factor activity and that doxycycline exerts its antitumor effect by interfering with tumor cell EMT. These findings show that doxycycline acts upstream of EMT-related signal transduction to inhibit a wide range of cellular functions. The inhibitory effects of doxycycline on bacteria is derived from its interference with rhibosomes, which result in the inhibition of protein synthesis. After doxycycline treatment in cancer cell lines, RPLs were directly or indirectly regulated. Some RPLs were inhibited by doxycycline, such as RPL23A, RPL10A, RPL12, RPL38 and RPL13. On the contrary,  RPL3 was compensatorily increased to maintain basic protein synthesis. As shown in Table S1, many molecular and biological functions of these RPLs are similar even the same between Eukaryotes and prokaryotes. In summary, doxycycline may be an effective alternative treatment for persistent carcinoma, as well as a new candidate for adjuvant chemoradiotherapy.

Cell viability assay
Cell viability was determined by MTT assay. Cells (5 × 10 3 cells/mL) were seeded in 96-well culture plates. After overnight incubation, cells were treated with various concentrations of doxycycline hydrochloride. After 48 h incubation, cell viability was measured after the addition of 20 μL MTT at 37°C for 4 h. Afterward, 150 μL dimethyl sulfoxide was added to dissolve the formazan crystals. Optical density was measured at 570 nm with a microplate reader (Multiskan™ FC, Thermo Scientific, Waltham, MA, USA).

Wound-healing assay
NCI-H446 and A549 cells were grown on a 35 mm dish to 100% confluence and then scratched to form a 100-μm wound using sterile pipette tips. The cells were then cultured in the presence or absence of doxycycline hydrochloride in serum-free media for 24 h. Images of the cells were taken at 24 and 48 h using a light microscope (Nikon, Japan).

Invasion assays
Cell invasion assays were performed using a transwell chamber inserted with a polyethylene terephthalate filter membrane containing 8.0 μm pores in 24-well plates (Corning, USA). For cell invasion assays, the filter membranes were coated with Matrigel. Cells (1 × 10 5 cells/mL) suspended in 200 μL of serumfree medium were seeded onto the upper compartment of the transwell chamber. The lower chamber was filled with medium containing 10% fetal bovine serum (as chemoattractant for migrated and invaded cancer cells) and various concentrations of doxycycline hydrochloride. After incubation for 24 h, the medium in the upper chamber was removed, and the filters were fixed with 10% methanol for 20 min. The cells remaining on the upper surface of the filter membrane were then completely removed by wiping with a cotton swab, and the cells on the opposite surface of the filter membrane were stained with 0.1% crystal violet for 10 min. The invading cells were then visualized and counted from six randomly selected fields using an inverted microscope at 100 × magnification.

Fluorescence-activated cell sorting (FACS) analysis
NCI-H446 and A549 cells were harvested separately and washed twice with phosphate buffered saline (PBS). The cells were then fixed with cold 70% ethanol for 12 h at 4°C. Afterward, the cells were washed with 1 mL PBS. The cells were treated with 500 μL PBS containing 100 μg/mL ribonuclease and 50 μg/mL PI. The cells were analyzed by flow cytometry (Millipore guava easyCyte™).

Immunofluorescent staining
NCI-H446 and A549 cells (4 × 10 3 cells/mL) were seeded in 96-well culture plates. After overnight incubation, the cells were treated with various concentrations of doxycycline hydrochloride. After incubation for 24 h, cells were washed twice in PBS, fixed with 10% formalin in PBS, permeabilized, and blocked with PBS containing NP-40 (0.1%) and bovine serum albumin (3%). The cells were then incubated in the same solution containing primary antibodies specific for either E-cadherin antibody (1:50 dilution) or vimentin antibody (1:50 dilution) for 1 h at room temperature (25°C). Cells were washed four times in PBS and incubated in secondary antibody (1:200 dilution) for 30 min at room temperature. Cells were then washed four times in PBS and covered with Hoechst 33342 dye for 30 min at room temperature. Cells were washed four additional times in PBS then proteins were visualized with high-content screening (HCS) systems.

Gelatin zymography assay
NCI-H446 and A549 cells (1 × 10 6 cells/well) were plated in 12-well plates and incubated in serumfree RPMI 1640 medium in the presence of doxycycline hydrochloride for 24 h. At the end of incubation, the conditioned medium was harvested, placed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel containing 0.2% gelatin (Sigma-Aldrich Corp), and then separated by electrophoresis. The gels were soaked in 2.5% Triton X-100 in deionized water twice for 60 min at 25°C to remove SDS. Gels were incubated at 37°C with substrate buffer (50 mM Tris HCl, 5 mM CaCl 2 , 0.02% NaN 3 , and 1% Triton X-100, pH 8.0) for 18 h. The gel was stained using 0.2% Coomassie blue for 1 h and destained in water containing 10% acetic acid and 50% methanol. Bands corresponding to the activity of MMP-2 and MMP-9 were quantified with ImageJ (National Institutes of Health).

Dual-luciferase assay
NCI-H446 and A549 cells were transfected with dual-reporter constructs using transfection reagents. After changing to fresh medium 24 h after transfection, cells were treated with various concentrations of doxycycline hydrochloride. After 48 h, the culture medium was collected into a 96-well white plate and luminescence measured with a luminometer. Details are shown in Table S3 and S4.

Multidimensional liquid chromatographytandem mass spectrometry
NCI-H446 and A549 cells (4 × 10 3 cells/mL) were seeded in a 100 mm dish to 70-80% confluence. The cells were then cultured in the presence (3.4 μM) or absence of doxycycline hydrochloride for 24 h. After cell lysis, samples were tested with multidimensional liquid chromatography-tandem mass spectrometry.

Animal studies
Male BAlB/c nu/nu mice, 5-6 weeks old, were maintained in a specific pathogen-free animal care facility according to institutional guidelines. Xenografts of tumors were established by subcutaneous injection of 1 × 10 7 cells (suspended in PBS) into the flank. One day after tumor cell inoculation, the mice were randomly divided into 5 groups (n = 10/group). After the tumors reached an approximate volume of 100 mm 3 (approximately six weeks after injection), the mice were treated with 60, 30, or 15 mg/kg of doxycycline; 20 mg/kg of cyclophosphamide; or saline by oral gavage once a day. Body weights were measured at different time points after tumor cell inoculation. Tumor diameters were measured every day, and tumor volumes were calculated according to the formula V = ab 2 /2 (a = length of tumor, b = width of tumor). Seven weeks after treatment, all mice were euthanized and both xenografts and lungs were resected and measured. Lung tissue was harvested for histologic examination and the nodes in lungs were observed using a stereoscopic microscope. Metastases from xenograft to lungs were measured after HE staining.
Another 50 mice were allocated randomly to 5 groups as described above (10 mice per group), in order to measure survival rates. Each mouse was injected 1 × 10 7 cells (suspended in PBS) in the caudal vein. The survival time of every mouse were recorded.

Immunohistochemical analysis
Fresh tissues from mice were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm thick slices, and placed on slides. The tissues were deparaffinized with xylene, dehydrated in decreasing concentrations of ethanol, and subsequently incubated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. For antigen retrieval, tissues were treated with citrate buffered saline (pH 6.0) for 15 min at 95°C. Tissues were incubated with normal goat serum for 20 min at room temperature to block unspecific labeling and then incubated with the following primary antibodies in a humidified chamber overnight at 4°C: rabbit polyclonal anti-E-cadherin (Zhongshan, ready-to-use), goat polyclonal anti-vimentin (Affinity, dilution 1:50), and rabbit polyclonal anti-MMP-9 (Zhongshan, ready-to-use). Diaminobenzidine and hematoxylin were used for color development and as counterstain, respectively. Expression of E-cadherin and vimentin were independently evaluated by two investigators. Tumor cells with brown staining of the cytoplasm, nucleus or membrane were considered positive and then scored based on four classes: none (0), weak brown (1+), moderate brown (2+), and strong brown (3+). The percentage of stained tumor cells was divided into five classes: 0 for negative cells, 1 for 1-25%, 2 for 25-50%, 3 for 50-75%, and 4 for > 75%.

Statistical analyses
All data are expressed as means ± standard deviation. Comparisons between groups were performed by one-way analysis of variance followed by Bonferroni post hoc test (SPSS software package version 17.0, SPSS Inc., Chicago, IL, USA). The level of significance was set at P < 0.05.


extfieldsofstudy: Chemistry Medicine

text:
Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona composition.

Background
Magnetic nanoparticles (MNP) represent perfectly suitable materials for a variety of biomedical and biotechnological applications. In many cases, MNP have to penetrate into different cell types of living tissue. This tissue and cellular uptake is strongly influenced by the particle size, as well as its surface chemistry and modification with functional groups or biomolecules. A detailed investigation and clarification of the interactions between surface chemistry of the particles and living tissue is a key to understand and control cellular uptake mechanisms [1].
Upon application of nanoparticles into biological media (e.g., whole-blood or plasma), the formation of a protein "corona" around the particles takes place immediately. This corona is a completely closed protein monolayer of a few nanometers on the surface of the nanoparticles [2] and can be divided into a "soft" and a "hard" corona [3]. In the soft corona, a permanent exchange of macromolecules from the surrounding medium and the particle surface takes place and this leads to a fast and persistent variation of the structure of the soft corona. The hard corona consists of macromolecules rather fixed to the particle surface and shows a more or less temporal constant composition. The volatility of the proteins in the soft corona aggravates a detailed investigation of the influence of the unstable part of the corona and thus most work has been spent on the investigation of the hard corona [4]. It was found that the corona is much more complex than previously considered. The influence of different particle parameters on corona formation has been already investigated. Lundquist and coworkers [5] found that for a fixed type of material, the biologically active proteins in the corona are strongly determined by the size as well as the zeta potential of the particles. Furthermore, it is well-known that the adsorption of blood serum proteins to particles is time-dependent [6]. Proteins with the highest mobility are bound to the surface first and later they will be replaced by less motile proteins which show a higher affinity to the surface. This process can take several hours. Casals et al. [7] confirmed that a soft corona loosely attached to the particles surface changes to an irreversibly attached hard corona over time. Comprehensive review articles about the influence of different nanoparticle parameters (e.g., composition, size, shape, crystallinity, surface area, surface defects, charge, roughness, transfer capability, and hydrophobicity/hydrophilicity) on the corona composition were published in the past years by several authors [4,[8][9][10][11][12][13].
Other less investigated but very important factors influencing the corona composition are the temperature at which the incubation of particles and protein source takes place as well as the duration of the incubation. For the clarification of this issue, the major aim of this study was the investigation of the influence of incubation temperature and time on the composition of the corona. For this, magnetic iron oxide nanoparticles with different coatings and, thus, different zeta potential were incubated with fetal calf serum (FCS) at different temperatures and incubation times. Magnetic nanoparticles enable an effective magnetic washing and separation which is very advantageous for the handling of small amounts of sample. In this study, the incubation was carried out at a homogeneous temperature in the whole sample in a water bath. The formed corona and the agglomeration behavior of the incubated particles were investigated as a function of incubating temperature and time by different methods.

Preparation of MNP
The superparamagnetic iron oxide nanoparticles used in this paper were prepared similar to the well-known wet chemical precipitation method [14] but using another alkaline medium [15]. For this, a 1.17-M NaHCO 3 solution was directly added to a FeCl 2 /FeCl 3 solution with a Fe 2+ /Fe 3+ ratio of 1:1.7, and a brownish precipitate occurred. After the addition of distilled water, the particles were boiled for 5 min at 100°C. In this way, single-core MNP were formed under the release of CO 2 , and the color of the solution turned black. Afterwards, the obtained MNP suspension was washed twice by magnetic separation with distilled water using a high-performance permanent magnet to remove excess educts.
For the investigation of the influence of particle surface charge on the formation of the protein corona, MNP were coated with different materials (dextran (DEX), carboxymethyl-dextran (CMD), and diethylaminoethyldextran (DEAE)). These materials have a neutral dextran backbone but different substitution patterns and thus enable a variation of surface charge (DEX-neutral, CMD-negative, DEAE-positive). For coating the MNP with dextran and its derivatives, the nanoparticles were dispersed by ultra-sonic treatment (Sonopuls GM200, BAN-DELIN electronic, Berlin, Germany) for 30 s. HCl was added to adjust the pH value at 2 to 3, and the suspension was tempered at 45°C in a water bath and stirred. At the same time, the coating agents were dissolved in distilled water in a mass-ratio (coating/core) of 1:1. The so-prepared coating solution was steadily added to the nanoparticle suspension and stirred for 1 h at 45°C. Afterwards, the suspension was treated with ultrasound for 30 s again, washed magnetically two times with distilled water to remove coating material excess, and the desired concentration was adjusted by adding distilled water.
Due to limited stability against agglomeration of dextran-based coatings, MNP with a coating of poly(tert-butoxycarbonyl acrylic acid) (PtBAA) were used for some investigations [16]. This particle system shows high stability against agglomeration and thus allows the minimization of the influence of agglomeration on corona formation. For coating of MNP with PtBAA, 40 mg of PtBAA were dissolved in 40 mL MilliQ water at pH = 12. To this solution, 40 mL of a dispersion of MNP (1 g/L) was added. The mixture was stirred at 50°C for 1 h, the dispersion was centrifuged with 8000 rpm for 30 min, and the supernatant was removed. The particles were redispersed in MilliQ water using ultrasonication. This procedure was repeated five times.
All prepared nanoparticle suspensions show a stability against sedimentation of several months as described in previous investigations [17,18].

Serum Incubation of the MNP
For producing a protein corona around MNP, the particles have to be incubated in a natural protein source which leads to an accumulation of proteins on the surface of the MNP. For our studies, FCS was used as natural protein source. FCS incubation of MNP was performed by water bath heating resulting in a homogeneous temperature distribution throughout the sample. In the following sections, uncoated and coated magnetic nanoparticles are referred as MNP and serum-incubated MNP are referred as MNP@Corona.
For water bath incubation, FCS was tempered at defined temperatures (incubation temperature) in a water bath. Fifteen milligrams of coated MNP from previously prepared suspensions were filled up with 2 ml of tempered FCS and kept at the same temperature (incubation temperature) for a certain time (incubation time). Incubation time starts with the application of FCS. During the time of incubation, ultra-sonic treatment at a given temperature was carried out (S100H, Elmasonic, Germany) to re-disperse possible agglomerates. At defined incubation time points (1, 5, 10, and 20 min), the suspensions were taken out of the water bath and put on a magnet for magnetic separation, excess FCS was withdrawn and distilled water was added.
The washed incubated nanoparticle suspensions were kept at 4°C for short-term storage or at −80°C for long-time storage.

Thermogravimetric Analysis
A suitable way to determine the mass of the corona bound to the particle surface is thermogravimetric analysis (TGA). Therefore, uncoated MNP were incubated (25°C/10 min in FCS), and resulting fluids were freeze dried to obtain fine dry powders for TGA experiments. These samples were heated (STA409, Netzsch, Selb, Germany) from room temperature up to 330°C, and the corresponding mass loss was continuously determined. The obtained curves for MNP@Corona were normalized to curves for uncoated MNP.

Structural and Magnetic Characterization
Magnetic core size was determined by X-ray diffraction (XRD, X'Pert PRO, PANalytical, The Netherlands) and using the Scherrer formula as well as by transmission electron microscopy (TEM; 200 kV FEI Tecnai G 2 20, equipped with a 4k × 4k Eagle HS CCD and a 1k × 1k Olympus MegaView camera for overview images).
The magnetic properties were measured by vibrating sample magnetometry (VSM; Micromag TM 3900, Princeton Measurement Systems, USA). Measurement was performed on liquid samples or dried powders. The concentration of MNP within the liquid samples and the amount of proteins bound to particle surface were calculated from the obtained saturation magnetization. The overall magnetic behavior of the samples was derived from coercivity and relative remanence.

Magnetorelaxometry
Magnetorelaxometry (MRX) was performed to investigate the Brownian relaxation behavior [19] of the MNP and the MNP@Corona hybrids. The relaxation curves describe the decay of an initial magnetization (after a magnetization pulse from a coil) due to Brownian and Néel relaxation of the particles within a fluid. From these relaxation curves, the size and size distribution of the particles was calculated by fitting the so-called cluster moment superposition model (CMSM) to the relaxation data [20]. The distribution of the hydrodynamic diameters d h or cluster diameters d c is assumed to be a lognormal one. Previous investigations showed a good agreement with hydrodynamic diameters obtained by dynamic light scattering (DLS) [21].
In the study presented here, we applied two different setups for MRX. A setup measuring the magnetic relaxation by means of highly sensitive low-T c -SQUID sensors at a distance of 10 mm above the sample (SQUID-MRX) [22] was used to investigate the agglomeration behavior of different coated MNP and resulting MNP@Corona in detail. For the measurement of the kinetics of corona formation around MNP, a setup which utilizes fluxgate sensors (FG-MRX) for measuring the magnetization decay was used [23].
In SQUID-MRX, samples were magnetized for 1 s with a magnetic field of 2 mT and relaxation was measured in a time window of 450 μs to 0.5 s after magnetization pulse. For FG-MRX investigations, magnetic moments of MNP were aligned in a field of 2 mT for 2 s duration and relaxation of the sample net magnetic moment was measured over a time period of 1.5 s after the magnetization pulse.
Since superparamagnetic nanoparticles show no thermally blocked magnetism, they relax predominantly via Néel relaxation. To observe a Brownian relaxation in MRX, larger ferrimagnetic nanoparticles of about 50 nm [17,24] were used here in the presented MRX studies. Of course, this results in higher absolute particle and agglomerate sizes than for superparamagnetic cores, but we suppose that the overall behavior of corona formation and agglomeration, investigated on ferrimagnetic cores, is similar to that of superparamagnetic cores.

Measurement of the Surface Charge
To determine the surface charge of the MNP and MNP@Corona hybrids, the zeta potential is a valid and widely used parameter. For this measurement, a Zetasizer (Nano ZS, Malvern, UK) and appropriate software (Zetasizer ver. 6.20) were used. Before the measurement, samples were diluted in the ratio 1:30 with distilled water and treated in an ultrasonic bath. The medium viscosity and dielectric constant were taken from water at 25°C with 0.8 cP and 0.8872, respectively. Measurements were performed in three consecutive runs and obtained values were averaged.

Gel Electrophoresis
The determination of the composition of the protein corona on the surface of MNP@Corona hybrids was carried out by means of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). For this, 2 × Laemmli sample buffer (Bio-Rad, Munich, Germany) supplemented with 2-mercaptoethanol (final concentration 355 mM) was added to the samples in the first step and heated up to 95°C to crack secondary and tertiary structure of proteins. Then, the denatured proteins were separated by molecular weight with PAGE on a 4-12 % Bis-Tris gel (Bio-Rad, Munich, Germany). After the run, the proteins were visualized by highly sensitive silver staining (SilverXpress Silver Staining Kit (Invitrogen, Heidelberg)). Gel images were processed by ImageJ (National Institutes of Health, Bethesda, USA) [25].
As references, a molecular weight standard protein collection Kaleidoscope marker (Bio-Rad, Munich, Germany) and untreated FCS were used.

Results and Discussion
The core diameter of MNP was determined by means of XRD and TEM to be around 10 nm (Fig. 1). Analysis of the diffractogram confirmed a spinel structure of the prepared particles with maghemite (γ-Fe 2 O 3 ) as dominant magnetic phase.
These data are confirmed by measurements of the static magnetization-versus-magnetic-field curves (Fig. 2). The saturation magnetization (M S ) of freeze-dried samples of uncoated MNP is 68.2 Am 2 /kg, which is a typical value for maghemite. Resulting from a coercivity (H C ) of less than 0.2 kA/m and a relative remanence (M R /M S ) of about 0.005 at room temperature, the used particles show superparamagnetic behavior. Estimation of magnetic core size following the Chantrell method [26] provides a mean core size of 9.6 nm. This value is in good accordance with results from XRD and TEM [18].
For the investigation of the amount of proteins bound to the particle surface, uncoated MNP were incubated in FCS for 10 min at 25°C and obtained MNP@Corona were freeze dried to a powder after magnetic washing of the sample to remove the excess FCS. This MNP@Corona powder shows an M s of 60.8 Am 2 /kg. Taking into account a nonmagnetic behavior of the protein coating, this decrease in saturation magnetization corresponds to a fraction of about 10 % by mass of proteins included in the hybrid particles. Although within this study we cannot prove whether serum incubation of MNP leads to a homogeneous layer of proteins on the particle surface or rather to protein bundles containing some MNP, we use the term "coated" for the serum incubated particles throughout the manuscript.
This composition of the freeze-dried MNP@Corona was confirmed by TGA measurements (Fig. 3). For temperatures up to 330°C, TGA measurements of the native particles show a weight loss of about 1 % which can be attributed to the evaporation of adsorbed water and a possible phase transformation of a small amount of impurity phases (hydrated oxides and hydroxides of iron) to hematite. Compared to this, TGA investigation of protein corona-coated MNP provides a mass loss of about  9 % in two steps. Again, the first step corresponds to the evaporation of adsorbed water and impurity phase change to hematite, whereas in the second step, the surface proteins decompose. Normalizing this curve to the losses in pure particles, a corona mass of 8 % is obtained. This value is slightly below the value from VSM. Although this difference probably is within the error of both TGA and VSM, another reason might be incomplete decomposition/evaporation of proteins during heating up to 330°C.
The results regarding the occurrence of a nonmagnetic layer on the particle surface after FCS incubation of MNP confirm the formation of a protein corona around MNP. A further evidence for a successful protein corona formation is given by changes in surface charge of particles after serum incubation. Figure 4 shows the zeta potential of pure MNP and for MNP coated with DEAEdextran, dextran, and CM-dextran before and after serum incubation.
It is clearly demonstrated that serum incubation significantly changes the surface charge of particles. Independent of the surface charge of particles before incubation, serum-treated particles reveal a negatively charged surface showing a zeta potential in the range from −32 to −41 mV. Since proteins and their subunits as well as other serum components (e.g., lipids) have a negative charge at pH 7, this fact can be interpreted as a confirmation of the formation of a protein corona around magnetic nanoparticles. Furthermore, a certain influence of the initial surface charge on the resulting zeta potential after incubation can be seen. This behavior has been already described by [5] and has to be investigated in more detail in further studies.
In measurements after different storage times, it was found that the formed protein corona is stable for about 9 days at room temperature and then starts to decay. To prevent the wash off of the protein corona during storage, serum-incubated samples are stored in a slight excess FCS.
Cell toxicity investigations (CellTiter-Glo and Presto-Blue) revealed no toxic effect of bare cores, coated cores, as well as pure coating materials (DEX, CMD, DEAE, and PtBAA) on tested cell lines (human brain microvascular endothelial cells) [18]. Another major factor for the suitability of nanoparticles for in vivo applications is their agglomeration behavior. Agglomerates larger than a few micrometers bear the risk to occlude blood vessels (especially capillaries) which may lead to an embolization and thus serious side effects for patients. In most cases, agglomeration cannot be prevented  completely and thus it has to be tolerated as long as agglomerates are well below 1 μm [27]. To exclude any risk from agglomerates, the agglomeration behavior of the samples has to be investigated.
Applying dynamic light scattering (DLS) methods, agglomeration behavior of particle suspensions can be assessed by detecting an increase in hydrodynamic diameter. However, the interpretation of the polydispersity index (PDI) in terms of the width of the size distribution is challenging, in particular because of the strong weighting of larger objects (∝V 2 , V-volume of the scattering objects). Because of its valuable information for the interpretation of aggregation, the size distribution of aggregates or clusters was evaluated by SQUID magnetorelaxometry (SQUID-MRX). The size distribution is assumed to be of lognormal form and is expressed in the diameter of mean cluster volume, d vc , i.e., the mean volume equivalent cluster diameter, the mean of volume weighted size distribution, d wvc , and the geometric dispersion parameter σ c (Table 1), derived from the CMSM fit. It was shown earlier that σ c correlates well with visually observable precipitation and that the "z average" diameter of DLS ranges between d vc and d wvc , depending on σ c [20].
In the following, we quantitatively discuss the changes in the size distribution caused by dispersion in FCS compared to the original aqueous MNP dispersion. Incubation of neutrally charged dextran-coated MNP with FCS reduces σ c slightly and increases the mean diameter d vc by 16 nm (while σ c was fixed at the reference value, Table 1). This behavior might be attributed to the growth of an additional layer of 8 nm thickness onto the MNP shell.
In case of the transfer of positively charged DEAEcoated MNP into FCS, the dispersion parameter σ c grows dramatically. Such a behavior might refer to agglutination as a possible mechanism of aggregation [28].
Accordingly, the comparison of mean diameters is hard to interpret. After fixing σ c at the reference value while fitting the CMSM to the data of MNP in FCS, again an increase of d vc was found, here by 19 nm. However, if MNP really agglutinate, it cannot be derived from the present data whether the MNP got homogeneously covered by an opsonisation layer or not.
Also in case of a negatively charged CMD shell, the dispersion parameter increases during exposure to FCS. In contrast to the DEAE sample, the mean diameters, obtained while fixing σ c , decrease (Table 1). Again, significant broadening of the size distribution points to the aggregation or transformation of existing aggregates. Thus, quantitative answers about opsonisation cannot be made.
To get an additional impression of agglomeration behavior, relaxation curves were qualitatively analyzed (Fig. 5). In case of neutrally charged dextran-coated MNP (Fig. 5a), serum incubation of these particles leads to a slight increase of relaxation time in comparison to the undiluted original sample which probably is caused by an increasing particle size due to the growth of a protein corona on the particle surface. Also, a slight agglomeration might explain the observed effect, as discussed later for DEAE-coated MNP. However, the decrease of σ c (Table 1) is a clear indicator for an increase of the particle diameter, and we regard it as rather unlikely to originate from aggregation. Aging of the samples for 4 days leads to a minor increase in relaxation time which might be caused most probably by further growth of the opsonisation layer or possibly by crosslinking between surface proteins [29,30].
MNP with DEAE-dextran (Fig. 5b) showed similar tendencies like dextran-coated particles but stronger variances in relaxation behavior of investigated samples. Serum incubation of DEAE-dextran-coated MNP led to distinctly higher relaxation times and, thus, a larger amount of protein corona on particle surface can be supposed. This behavior is even more pronounced after aging for 4 days, possibly due to further growth of corona or due to protein cross-linking. A possible explanation might be that there is a stronger affinity between negatively charged proteins and highly positively charged surface of DEAE-dextran-coated particles than for particles with pure dextran or CM-dextran coating. From this, a higher protein load on the surface may be caused which results in a larger effective particles size and thus a higher relaxation time. This hypothesis has to be verified in further studies by means of measurements, providing data for the amount of proteins bound to particles.
For CMD-coated MNP, also significant changes in relaxation behavior for different samples were found (Fig. 5c). In contrast to dextran and DEAE-dextran for Table 1 Parameters of the distribution of volume equivalent hydrodynamic diameters of MNP before and after incubation in FCS obtained by fitting of CMSM to MRX data. Alternatively, σ c was fixed with fitting indicated by "(fix)," in order to render the mean diameters comparable CMD-coated MNP, the relaxation gets faster after FCS incubation. However, as mentioned above, the increase of σ c indicates some aggregation or even disaggregation of already present aggregates. Note that the obtained d v around 100 nm is significantly larger than the core diameter of 10 nm (Fig. 1). After aging the sample for 4 days, the difference in relaxation behavior vanishes and a relaxation curve similar to that before serum incubation is found, possibly also due to a further growth of a corona or due to cross-linking between particles. By means of MRX, a clear change of the cluster size distribution due to the FCS incubation was shown. But it cannot be distinguished whether an opsonisation or an aggregation is responsible for the observations. So one may speculate that no protein corona is formed during serum incubation of these samples since there was no significant change in zeta potential (Fig. 4) observed, and CMD as well as most proteins in FCS is negatively charged. At least partially, opsonisation is supported by experimental evidence of surface proteins by means of gel electrophoresis investigations for FCS-incubated CMD-coated MNP as shown in own previous investigations [31].
Since corona formation on DEAE-coated MNP leads to a significant effect on relaxation time, this MNP type was used to investigate the protein corona formation dynamics by means of fluxgate MRX. For this, MRX measurements were performed immediately after adding FCS to a MNP suspension at room temperature. Figure 6a depicts the temporal evolution of the MRX signal. The first unaveraged measurement was recorded 15 s after adding FCS to the ferrofluid, and then measurements were repeated every 15 s. As can be seen, no changes are discernable. The decay of magnetization after the addition of FCS is slower compared to the aqueous suspension (same volume H 2 O was added to MNP suspension). This increase of the relaxation time constant is mainly caused by an increase of the effective hydrodynamic size by agglutination [28] since the viscosity of FCS is with 1.56 mPa·s only about 50 % higher than that of water.
From the relaxation curves, it can be seen that the formation of a protein corona occurs immediately within seconds, and no clearly visible changes are detected over the observed time period (up to 285 s). This conclusion is valid only for the thickness of the corona and does not reflect any changes in corona composition. However, on a longer time scale (Fig. 6b), a further change of relaxation time was observed for 1 and 4 days of incubation. There are two possible reasons for such a behavior. First, a further growth of the protein corona over a long time might occur, which is relatively unlikely. The more probable reason for the continuous decrease of relaxation time might be attributed to cross-linking of surface proteins as discussed above which results in larger agglomerates [29,30].
Altogether, it can be stated from MRX measurements that the formation of a protein corona occurs immediately after mixing MNP and FCS and that the resulting corona has (depending on the underlying coating material) an effect on particle agglomeration in aqueous MNP@corona hybrid particle suspensions. Agglomerates with sizes below 250 nm in diameter, as observed here, can be tolerated for medical application of investigated particles.
In order to study the impact of incubation time and temperature in more detail, we used a MNP system with high stability against agglomeration and sedimentation to exclude any influence of particle agglomeration on the corona formation. For this, we used particles coated with poly(tert-butoxycarbonyl acrylic acid) (PtBAA), a negatively charged polyelectrolyte [16]. PtBAA-coated MNP show a negative surface charge, similar to earlier observations after coating MNP with CMD. These MNP were incubated with FCS for up to 20 min within a temperature range from 25 to 70°C (Fig. 7). The different temperatures were realized by heating in a water bath.
Zeta potential measurements of PtBAA-MNP@Corona as function of incubation time and temperature reveal that the formation of the protein corona does not alter the overall net charge of these materials (Fig. 7). Serum proteins are negatively charged at pH 7.2 in fetal calf serum. Therefore, the overall negative charge of the nanoparticles remains constant between −40 and −30 mV in all cases.
Nevertheless, when trying to derive a model for the influence of incubation time and temperature on the composition of the resulting corona, the zeta potential is not a suitable measure to determine details (since no clear correlations between incubation parameters and resulting surface charge can be found) but rather a raw indicator for changes in the structure of the protein corona.
To get more profound information about the protein load and composition of the formed protein corona, PtBAA samples were subjected to SDS-PAGE. With this approach, components of the corona were denatured and separated according to their molecular weight (Fig. 8a).
At 25 and 37°C, an increase of protein content with time is visible, whereas at 50°C, a more or less steady distribution is observed. Please note that the overall protein distribution reflects the situation in untreated FCS. Figure 8b shows a quantitative analysis of the protein distribution. It can be clearly seen that only weak heating times of 20 min, a significant increase in bound protein mass occurs. Since this effect was found for 25 and 37°C incubation series as well as in MRX investigations (1-day curve), we exclude an experimental artifact. At 70°C, a higher protein yield is detected independent of the incubation time. Almost all of the serum proteins are denatured above 65°C and thus misfolded polypeptides and protein agglomerates are formed during incubation and attach to the surface of PtBAAcoated MNP. Some of these clusters could not be resolved by conventional lysis conditions and cause an accumulation of polypeptides in the range between 25 and 75 kDa. The loss of high-molecular weight proteins might be due to the temperature-related structural changes, too. Unspecific intramolecular bounds lead to more globular shapes which exhibit a higher electrophoretic mobility. This observation needs more detailed analysis of the distinct proteins which are involved in corona formation.
Additionally, Fig. 8b provides an impression on the composition of protein corona. It becomes obvious that heating time and temperature have an influence on corona composition. Since SDS-PAGE analysis and zeta potential investigations provide a global overview on corona composition, other methods have to be utilized for a detailed clarification of protein corona composition on polypeptide level. A promising method for this task is matrix-assisted laser desorption/ionization (MALDI) in combination with a mass spectrometer which is tested in ongoing studies.

Conclusions
Our MRX investigations show that immediately after contact of MNP with a protein source (FCS), a protein corona is formed on the particle surface. This leads to an increase of particles size and, depending on any previously applied MNP coating, to a slight agglomeration of the MNP during the first minutes of incubation. Longer incubation (from hours to days) leads to stronger agglomeration of corona-coated MNP, probably due to cross-linking of the surface proteins. We quantified the amount of proteins bound to MNP under these conditions by a combination of magnetic measurements and thermogravimetry to about 10 %.
Independent of the used polymer shells herein used for MNP (DEAE, CMD, dextran, PtBAA), zeta potentials from −30 to −40 mV were found after serum incubation. Slight variations in the zeta potential of the serumincubated MNP are a first hint towards differences in composition of the formed protein corona at different incubation times and temperatures, possibly as also the coating material play a role. Quantitative SDS-PAGE analysis of serum-incubated particles revealed, as already found by MRX, a relatively constant amount of bound proteins during the first minutes of serum incubation. After longer incubation (20 min), a considerably higher amount of surface proteins was determined for incubation temperatures of 25 and 37°C. For incubation temperatures of 50 and 70°C, the incubation time did not seem to play a major role, which might be attributed to denaturation of proteins during incubation.
The analysis of the molecular weight of proteins found in the corona showed a clear influence of incubation time and temperature on corona composition which has to be investigated in more detail in future studies by means of MALDI. Furthermore, magnetic nanoparticles can be used in prospective investigation for magnetic heating by means of reversal losses in an alternating magnetic field [32,33] during the incubation to control the composition of the corona by using a temperature gradient from the particle surface to the surrounding protein source.


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A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter

Cellular immunity against viral infection and tumor cells depends on antigen presentation by the major histocompatibility complex class 1 molecules (MHC I). Intracellular antigenic peptides are transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I, which are exported to the cell surface and present peptides to the immune system1. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumor development. To escape immune surveillance, some viruses have evolved strategies to either down-regulate TAP expression or directly inhibit TAP activity. To date neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. In this study we describe the cryo-electron microscopy (cryo-EM) structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. We show that the twelve transmembrane helices and two cytosolic nucleotide-binding domains (NBDs) of the transporter adopt an inward-facing conformation with the two NBDs separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and also prevents NBD closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the ER, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.

Inside our body, every nucleated cell has surface "barcodes" that are surveyed by the immune system. These barcodes are peptides derived from intracellular proteins, presented on the surface by MHC I molecules to indicate whether the cell is healthy (reviewed in ref. 1). Peptides generated from normal cellular proteins are ignored by cytotoxic T cells, whereas viral-derived or malignant peptides will trigger an adaptive immune response, resulting in elimination of the infected or tumor cells. The peptide repertoire is generated in the cytoplasm, mainly by the proteasome, but also in part by cytosolic peptidases (Fig 1a). Peptide uploading onto MHC I molecules takes place inside the ER and is orchestrated by a macromolecular assembly collectively called the MHC class I peptide-loading complex (PLC). Cytosolic peptides are delivered across the ER membrane by the ATP-binding cassette (ABC) transporter TAP. The chaperones calnexin and calreticulin stabilize nascent MHC I molecules awaiting peptides. The tapasin/ERp57 heterodimer brings MHC I molecules and TAP within close proximity and catalyzes peptide loading. Peptide-loaded MHC I molecules are then released from the ER and transported to the cell surface for antigen presentation.
As the MHC I antigen presentation pathway plays a crucial role in eradicating intracellular pathogens, it is not surprising that some viruses have evolved the ability to interfere with this process (reviewed in ref. 2). The peptide transporter TAP in particular is a primary target for viral evasion (reviewed in ref. 3). TAP is a heterodimeric ABC transporter that contains two subunits, TAP1 and TAP2, which share 37% sequence identity and are predicted to have similar structures. Each subunit contains an N-terminal transmembrane region (TMD0) that interacts with tapasin, followed by six transmembrane (TM) helices that form the peptide translocation pathway and a canonical nucleotide-binding domain (NBD) that hydrolyzes ATP (Fig 1b) 4 . The core TAP, devoid of the TMD0s, is necessary and sufficient for peptide transport 4 . So far, five viral proteins have been identified as TAP inhibitors. Four are encoded by members of the herpes virus family and one by cowpox virus 3 . These viral inhibitors are valuable tools for selective immune suppression and for understanding the fundamental mechanism of antigen presentation.
Here we focus our study on a TAP inhibitor encoded by herpes simplex virus (HSV). Both types of HSV, HSV-1 (oral herpes) and HSV-2 (genital herpes), somehow elude the human immune system and lead to a lifelong infection. The first clue as to how HSV bypasses the immune system came from observations that cells infected by HSV have reduced surface expression of MHC I molecules 5 and are resistant to cytotoxic T cells 6 . Since this resistance develops within three hours of HSV infection, researchers narrowed their search for the responsible gene to those few expressed in the early stage of infection 7 , 8 . Out of these, an 88-residue protein, ICP47, was found to bind to TAP and prevent peptide translocation into the ER 7 , 8 . Consequently, empty MHC I molecules were retained in the ER and viral peptide presentation was suppressed. Subsequent studies have shown that ICP47 interacts with TAP from the cytosolic side of the membrane and somehow prevents peptide binding 9 , 10 . The functional domain of ICP47 has been mapped to the N-terminal 35 residues 11 , 12 , which form an extended helix-loop-helix structure in lipid bilayers 13 .
In this study, we pursued structural determination of a TAP/ICP47 complex using cryoelectron microscopy (cryo-EM). The small size of the complex (166 kDa total) and the predicted pseudo two-fold symmetry between TAP1 and TAP2 make it extremely challenging to accurately align particles for 3D reconstruction. To maximize the difference between the two TAP subunits, we used a shorter allele of TAP2 that lacks the last 17 amino acids in NBD2 (Fig 1b) 14 . Co-expression of TAP1 and TAP2 in Pichia pastoris (Extended Data Fig 1) produced a heterodimer that dissociates readily in detergents. However, by incubating the TAP-enriched membranes with ICP47 before detergent solubilization, the complex consisting of TAP1, TAP2, and ICP47 becomes more stable (Fig 1c and 1d). Cryo-EM analysis of this complex (Extended Data Fig 2 and Fig 1d) produced a density map ( Fig  2) of an overall resolution of 6.5 Å, determined by the gold-standard refinement procedure (Extended Data Fig 3) 15 . In this reconstruction the TM helices and the connectivity between the helices are clearly resolved. The density corresponding to one NBD is significantly smaller than that for the other, allowing us to confidently differentiate TAP1 from TAP2 ( Fig  2b). Most importantly, we observe strong density corresponding to the functional region of ICP47, which reveals how this viral protein inhibits peptide translocation (Fig 2b).
The core region of TAP adopts an inverted "V"-shaped structure, with the two TMDs making close contact on the side corresponding to the ER lumen and the NBDs separated from each other inside the cytosol (Fig 2c). Domain swapping of TM helices 4 and 5 across the TAP1/TAP2 interface is a prominent structural feature (Fig 2c). Indeed, the overall structure of TAP is very similar to that of other ABC exporters, including the lipid flippase MsbA from Gram-negative bacteria 16 , the protein transporter PCAT1 from Gram-positive bacteria 17 , and the multidrug transporter P-glycoprotein in eukaryotes 18 , 19 . Although these transporters recognize very different substrates, they must share a common evolutionary origin and a common mechanism for coupling ATP hydrolysis to substrate translocation.
No density was observed for the N-terminal TMD0 domain of both TAP1 and TAP2 subunits (Fig 2). Studies have shown that the TMD0s are essential to the assembly of the large peptide-loading complex 4 , 20 but dispensable in peptide translocation 4 . ICP47 inhibits both full-length TAP and the core construct 4 . Our results indicate that in the absence of tapasin the two TMD0s are flexibly tethered to the core region of TAP.
Biochemical data and homology modeling suggest that the peptide translocation pathway lies at the interface of the two TMDs 21 , 22 . Inside this pathway, we observe strong density consistent with the helix-loop-helix structure of ICP47 (Fig 2b and 3a). Guided by the NMR structure and secondary structure prediction 13 , we built residues 3-16 into the shorter helical density and residues 22-40 into the longer density (Fig 3). Additional density is packed along the cytosolic region of TAP2, into which we modeled residues 41-50. The C-terminal region, neither required for TAP inhibition nor conserved between HSV-1 and HSV-2 ( Fig  3c), is not resolved in the EM map, suggesting high mobility.
On the basis of this model, the N-terminal half of ICP47 forms a hairpin-like structure pinned against the inner surface of TAP2 TM helices 2, 3, 6 and TAP1 TM helix 4 (Fig 3b).
The two helices of ICP47 run anti-parallel to each other, connected by a sharp turn at the top of the TM cavity. The extensive packing between TAP and ICP47 is consistent with the nanomolar affinity of ICP47, orders of magnitude higher than those of the substrate peptides 9 , 12 .
The potency of ICP47 appears to come from its helical hairpin structure, which provides a greater interface with TAP than a typical substrate. To test whether the flexibility of the connecting loop is important, we constructed a "turn-to-helix" mutant by replacing residues 16-22 with alanine, which has the highest propensity to form an α-helix and thus would oppose (but not preclude) the formation of a turn. ICP47 activity was measured in human epithelial cells, in which cytosolic expression of ICP47 inhibits endogenous TAP and reduces the amount of MHC I molecules expressed on the cell surface. A GFP tag was fused to the C-terminus of ICP47 as a marker to select for cells expressing similar amounts of ICP47. Consistent with the EM structure and previous mutagenesis data 11 , the "turn-tohelix" mutant is much less potent than the wt construct. Specifically, mutant ICP47 reduced surface MHC I expression by only 5-fold as opposed to a 20-fold reduction by wt ICP47 (Fig 3d).
Although ICP47 competes for the same binding site, we do not believe it mimics the substrate binding process. Unlike substrates, ICP47 inhibits rather than stimulates ATP hydrolysis 23 . Furthermore, in contrast to ICP47, which separates the two NBDs, substrate binding induces partial closure of the NBDs 24 . More recently, electron paramagnetic resonance (EPR) studies showed that TAP binds its substrates in their extended conformation, comparable to how MHC I molecules present peptides 25 .
Comparison of our structure with the NMR structure 13 suggests that ICP47 undergoes major conformational changes upon association with TAP (Fig 4). In isolation, the N-terminal two helices of ICP47 are flexibly linked and bind to the surface of the membrane at a slight tilt 13 .
In the complex with TAP, ICP47 forms a straight hairpin and inserts perpendicularly into the membrane. Although exact determinations of the amino acid register cannot be made at the current resolution, the overall structure readily explains how ICP47 inhibits peptide transport into the ER. By plugging a long helical hairpin into the translocation pathway, ICP47 directly blocks substrates from binding. Furthermore, because ICP47 is too large to be transported by TAP, its high-affinity binding traps TAP in an inactive conformation. Like other ABC transporters, TAP functions by alternating between two major conformations, each exposing the translocation pathway to one side of the membrane (Fig 4). Binding of ICP47 stabilizes the inward-facing conformation, and thus prevents TAP from transitioning to an outward-facing state in which the NBDs form a closed dimer and the translocation pathway orientates towards the ER lumen (Fig 4).
In addition to viral inhibition discussed in this study, the structural basis of two cellular regulatory mechanisms has been elucidated 26 -28 ; both are relevant to nutrient uptake in bacteria. As a classic example of carbon catabolite repression, when a preferred carbon source is available, bacteria suppress the uptake of maltose through direct binding of a regulatory protein to the maltose transporter 26 . Methionine and molybdate transporters offer another example 27 , 28 . In both cases, at high intracellular concentration, the substrate binds and inhibits the corresponding transporter, a feedback mechanism that limits the amount of import into the cell 27 , 28 . Unlike viral inhibition, both cellular inhibitions are allosteric and reversible, regulated by the metabolic state of the cells. One common theme among all these inhibition mechanisms is that the inhibitor binds and stabilizes the transporter in the inwardfacing state, a conformation unable to hydrolyze ATP. We speculate that this strategy may be advantageous in preserving cellular energy sources. It is also possible that for most ABC transporters, the inward-facing state is most common and thus naturally targeted by regulators.

Expression and purification of ICP47
The gene encoding human herpes simplex virus 1 (HSV-1) ICP47 was synthesized (BioBasic) and subcloned into the ligation-independent cloning vector pMCSG20 (Amp R ), which contains an N-terminal glutathione S-transferase (GST) affinity tag preceding a tobacco etch virus (TEV) protease cleavage site. BL21(DE3)RIL E. coli cells containing the pMCSG20-ICP47 vector were grown to mid-exponential phase at 37°C in LB medium and expression was induced with 200 μM isopropyl β-D-thiogalactoside (IPTG) at 20°C for 24 h. The cells were harvested via centrifugation (4,000 xg for 12 min at 4°C) and broken by two passes through a high-pressure homogenizer (EmulsiFlex-C3; Avestin). Cell lysate supernatants were loaded onto glutathione sepharose 4B resin (GE Healthcare) equilibrated with PBS buffer, pH 7.4, containing 5 mM D,L-dithiothreitol (DTT). To remove the GST tag from ICP47, the column was equilibrated into a TEV protease cleavage buffer containing 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5 mM EDTA, and 5 mM DTT and incubated with TEV protease overnight. Untagged ICP47 was eluted and further purified by Superdex 75 gel-filtration chromatography (GE Healthcare) in TEV-cleavage buffer. Fractions containing the protein were pooled, concentrated to 3 mg/ml, and flash-frozen in liquid nitrogen.

Co-expression of TAP1/TAP2
Synthetic human TAP1 and TAP2 genes were codon-optimized for expression in Pichia pastoris (BioBasic) and subcloned into pPICZ-C-XE-Protein A (Zeo R ) and pPICZ-C-XE vectors (Zeo R ), respectively. In order to ensure co-expression at a 1:1 molar ratio, we took advantage of the compatible cohesive ends of BamHI and Bgl II restriction sites to generate an expression cassette as outlined in Extended Data Figure 1. The resultant vector was linearized by PmeI digestion and transformed into a HIS+ strain of SMD1163 by electroporation (BioRad Gene Pulser II). Transformants were selected on yeast extract peptone dextrose sorbitol (YPDS) agar containing 800 μg/ml zeocin. Colonies were grown in yeast extract peptone dextrose (YPD) cultures at 28°C until they reached an OD 600 of 4 to seed flasks containing minimal glycerol medium (MGY), 13.4% yeast nitrogen base and 1% glycerol at a starting OD 600 of 0.5. The MGY cultures were grown at 28°C for 24 h until they reached an OD 600 of 20, at which point they were harvested by centrifugation (1,500 xg for 15 min at 4°C) and used to seed flasks containing minimal methanol medium (MMY), 13.4% yeast nitrogen base and 0.5% methanol at a starting OD 600 of 10. These MMY cultures were grown at 28°C for 24 h before harvesting (1,500 xg for 15 min at 4°C). Cell pellets were fractionated and flash-frozen in liquid nitrogen.

Purification of the TAP/ICP47 complex
Cells expressing TAP1-Protein A/TAP2 were lysed using a mixer mill (Retsch Mixer Mill 400) and incubated with purified ICP47 in a buffer containing 50 mM Tris-HCl, pH 8, 500 mM NaCl, 15% glycerol, DNase I, protease inhibitors, and 2 mM TCEP for 30 min. Cells were then solubilized with 1.5% n-dodecyl-β-D-maltoside (DDM; Anatrace) for 2 h. The solubilized fraction was isolated by centrifugation (70,000 xg for 40 min at 4°C). The TAP1-Protein A/TAP2/ICP47 complex was isolated using the Protein A affinity tag on TAP1 by lgG Sepharose 6 Fast Flow (GE Healthcare). After extensive washing, PreScission protease (GE Healthcare) was added to the column and incubated overnight to remove the Protein A tag. The complex was eluted with additional buffer and further purified using a Superose 6 column (GE Healthcare) equilibrated with 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM TCEP, 1 mM DDM, and 1 mM octaethylene glycol monododecyl ether (C12E8; Anatrace). The peak fraction was used to prepare cryo-EM grids.

Initial cryo-electron microscopy imaging and generation of an initial model
Vitrified specimens of TAP/ICP47 complex were prepared on glow-discharged Quantifoil holey carbon grids by plunge-freezing into liquid ethane using a Vitrobot (FEI). Cryo-EM data were collected at liquid-nitrogen temperature using a K2 Summit direct electron detector camera (Gatan Inc.) on a Tecnai F20 electron microscope (FEI) operating at 200 keV. Dose-fractionated image stacks were recorded with UCSF Image 4 29 in superresolution counting mode at a calibrated magnification of 40410x (nominal magnification of 29,000x) with a dose rate of 8 electrons/pixel/s (5.2 electrons/Å 2 /s). Frames were read out every 200 ms and 30 frames were collected, resulting in an exposure time of 6 s and a total dose of 31.2 electrons/Å 2 . Dose-fractionated image stacks were 2x binned and motioncorrected, as described 29 . The defocus was determined with CTER 30 . BOXER was used to interactively pick 28,813 particles from ~750 images 31 . The particle images were subjected to the iterative stable alignment and clustering (ISAC) procedure 32 implemented in SPARX 33 . Four ISAC generations specifying 100 particles-per-group and a pixel error threshold of 0.7 yielded 324 averages. Of these, 270 averages were used to calculate an initial 3D density map with the validation of individual parameter reproducibility (VIPER) procedure in SPARX.

Electron microscopy sample preparation and imaging for the final 3D reconstruction
Cryo-EM grids were prepared by pipetting 3 μl freshly purified TAP/ICP47 (2 mg/ml) onto glow-discharged C-flat holey carbon CF-1.2/1.3-4C grids (Protochips) and letting the sample adsorb for 20 s. The grids were blotted for 4 s at 90% humidity using a Vitrobot Mark IV (FEI) and immediately plunge-frozen in liquid nitrogen-cooled liquid ethane. The grids were imaged using a FEI Titan Krios electron microscope operating at an acceleration voltage of 300 keV. Images were recorded using a K2 Summit direct electron detector (Gatan Inc.) set to super-resolution counting mode with a super-resolution pixel size of 0.675 Å using the program SerialEM 34 . In addition, a Gatan Imaging filter with a slit width of 20 eV was used to remove inelastically scattered electrons. Movie frames were recorded with an exposure time of 200 ms using a dose rate of 10 electrons/pixel/s or 5.5 electrons/Å 2 /s (1.35 Å at the image plane). Three datasets were recorded using different total doses and defocus ranges (Extended Data Table 1 and Extended Data Figure 2).

Image processing
Movie frames were corrected for gain reference and binned by a factor of 2, giving a pixel size of 1.35 Å. Drift correction was performed using the program Unblur 35 , 36 . Next, the drift-corrected frames were summed into single micrographs, which were used to estimate the Contrast Transfer Function (CTF) using CTFFIND4 37 . The program Summovie was used to recalculate the summed images, first with a low-pass filter for autopicking and then with the noise power restored after filtering for particle extraction 38 . Autopicking, particle extraction, 2D classification, 3D classification, and initial 3D refining were all performed in Relion 39 . In order to achieve a more robust classification of the extracted particles, 2D classification was performed with a particle mask diameter of 145 Å while ignoring the effects of the CTF until the first zero transition. 3D classification was performed on particles from selected 2D classes using the initial model calculated in SPARX as a reference map. Particles from selected 3D classes from both datasets were combined for 3D refinement.
Using the orientation parameters determined by Relion, 3D refinement in FREALIGN was also performed 40 . The final map, reconstructed from 139,293 particles, had a resolution of 6.5 Å as determined by Fourier shell correlation (FSC) of independently refined halfdatasets using the 0.143 cut-off criterion (Extended Data Figure 3).

Model building
We used a model of the TAP1 nucleotide-binding domain (NBD) from a previously reported structure of the isolated domain (PDBcode 1JJ7) 41 to generate a homology model for the TAP2 NBD using the program Modeller 42 . We also generated a homology model of the TAP1 and TAP2 transmembrane domains (TMDs) using the half-transporter subunit of human ABCB10 (PDBcode 4AYT) as the source structure 43 . We manually docked these poly-alanine models into our final cryo-EM map and rebuilt each model in Coot 44 .

Flow Cytometry analysis of MHC class I surface expression
MHC I surface expression was analyzed using the phycoerythrin-coupled antibody W6/32 (Abcam), which recognizes a monomorphic epitope shared among MHC class I molecules.
Genes encoding HSV-1 wild-type ICP47 and the "turn-to-helix" (TtH) mutant were cloned into a modified version of the pCDNA 3.1 vector (Life Technologies) that adds a C-terminal EGFP tag. HeLa cells (ATCC ® CCL-2™) were seeded in 6-well plates at a density of 5 × 10 5 cells/well and transfected with wt ICP47, the TtH mutant, or the empty vector. Cells were detached from the plate using trypsin-EDTA (0.05%) at 72 h post-transfection and washed in ice-cold FACS buffer (Ca ++ /Mg ++ free phosphate buffer, 10% FCS, 1% sodium azide) and centrifuged at 400 ×g for 5 min at 4°C. Nonspecific binding was blocked by incubating the cells with phosphate buffer containing 5% (w/v) bovine serum albumin (BSA) for 15 min on ice. Antibody was added at 5 μg/ml and incubated for 30 min at 4°C in the dark. Subsequently, the cells were washed 3 times in FACS buffer, resuspended at a density of 3 × 10 6 cells/ml, and counted using a BD LSR II Flow Cytometer (BD Biosciences). The cells were analyzed at wavelengths 405 nm for DAPI nuclear stain, 488 nm for GFP fluorescence, and 561 nm for phycoerythrin fluorescence. Only live, single cells with the same levels of GFP fluorescence were used in phycoerythrin gating to compare MHC class I expression. The flow cytometry data were analyzed using FlowJo 10.1 single cell analysis software (Tree Star). All experiments were repeated three times. The cell line was tested for mycoplasma contamination by PCR using the Universal Mycoplasma Detection Kit (ATCC® 30-1012K™)      TAP functions via alternating access, cycling between two major conformations. In the absence of substrates, the transporter rests in an inward-facing state in which the two NBDs are separated and the translocation pathway is exposed to the cytosol. Upon association of substrates and ATP, the transporter undergoes a conformational change that reorients the TMDs and positions ATP at a closed NBD dimer interface for hydrolysis. ATP hydrolysis releases the substrate and resets the transporter to the resting state. ICP47 binds TAP stabilizes the inward-facing conformation.