extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.

Introduction
To understand how protein folding occurs in living cells we need to be able to describe protein folding pathways in the presence of cellular folding factors, and to characterize interactions between these factors and their unfolded and partly-folded protein substrates in thermodynamic, kinetic and structural terms. Such studies are not yet well-developed. In this paper we have selected unfolded, partly-folded and folded species corresponding to stages on a well-defined oxidative protein folding pathway and characterized in physico-chemical terms their interaction with PDI, the major catalyst of oxidative protein folding in the endoplasmic reticulum of eukaryotic cells.
Oxidative protein folding is a special case of protein folding in which the process of 'conformational folding' is accompanied by the formation of disulfide bonds (disulfide generation). The process occurs in several cellular compartments [1], but has been most extensively studied in relation to proteins secreted by eukaryotic cells, whose oxidative folding occurs primarily in the endoplasmic reticulum [2].
For over 50 years, studies in vitro on the refolding of purified proteins from the reduced, unfolded state have played a significant role in defining ideas about protein folding in general [3,4]. For several small proteins, oxidative protein folding pathways have now been defined, in terms of the identity of disulfide-bonded intermediates and their conformations [5][6][7][8][9]. In every case, generation of native disulfides involves both disulfide formation and disulfide isomerization steps; rate-determining steps frequently involve the concomitant formation of 'correct' buried disulfide bonds and of the native conformation [3,10].
Enzymatic catalysis of oxidative folding was first described in 1963 [11,12] and since then the key catalyst, protein disulfideisomerase (PDI), has been extensively characterized [13]. PDI catalyzes oxidative folding of many substrates including proteins with well-defined oxidative folding pathways such as ribonuclease [14,15], lysozyme [16] and basic pancreatic trypsin inhibitor (BPTI) [17][18][19]. In each case, PDI catalyzes formation and isomerization of disulfides, both in early unstructured intermediates, and in later rate-determining steps in which relatively structured intermediates undergo disulfide isomerization to form stable conformation and stable native disulfides. These ratedetermining steps require partial unfolding of structured intermediates, so their catalysis by PDI implies that it interacts with substrate transition states which are less folded than either 'structured intermediate' reactants or fully folded products (consistent with PDI having 'chaperone' activity independent of its ability to catalyze thiol:disulfide interchanges [13]).
Studies on PDI catalysis of oxidative protein folding have focused on defining the enzyme-catalyzed disulfide-generation pathway of the substrate protein but have not analyzed interactions between the substrate and catalyst to provide understanding of how this interaction changes through the folding pathway. In this paper, we describe studies on the interaction between PDI and the small disulfide-bonded protein BPTI, using derivatives of BPTI selected to model stages on its oxidative folding pathway.
Native BPTI contains two buried disulfides (linking residues 30-51 and 5-55) and one exposed disulfide (linking residues 14-38); its disulfide regeneration pathway has been the subject of considerable work and some contention ( Figure 1). Pioneering work in the 1970s [20,21] established that it was a restricted pathway with few defined intermediates; the 30-51 disulfide was predominant in species containing a single disulfide and the ratedetermining step on the oxidative folding pathway was the formation of the two-disulfide intermediate containing both buried native disulfides (30-51+5-55) but lacking the exposed disulfide. Intriguingly, this species could not be formed directly from either of the one-disulfide intermediates containing a single native disulfide, but only via disulfide rearrangement of alternative twodisulfide intermediates. Later work [22] re-quantified the levels of the intermediates, leading to a revised pathway focusing on the quantitatively predominant two-disulfide intermediates, those containing two 'native' disulfide bonds ((30-51+14-38) and (5-55+14-38). However, the rate-determining steps remained disulfide isomerizations at the two-disulfide stage leading to formation of the (30-51+5-55) species. Species containing a non-native disulfide are crucial transient intermediates on the disulfide regeneration pathway, as originally proposed [23,24].
The two-disulfide BPTI species containing the 'native' 30-51 disulfide and the 'non-native' 5-14 disulfide is one of these transient intermediates in the disulfide isomerization pathway; it can be formed by isomerization from the predominant twodisulfide intermediates and can isomerize in a single step to yield the final intermediate. A stable analogue of this species can be generated by mutation or modification of Cys38 and Cys55. Previous work on such a double mutant (C38S/C55S) indicated that it had a partly-folded conformation [6]. In this study, we have extended this earlier work and selected this protein as a model of a partly-folded intermediate on the BPTI oxidative folding pathway. We have studied its interaction with PDI and compared this interaction with those of native, folded BPTI and of reduced/ alkylated BPTI (selected as a model of the fully unfolded protein). Hence we have compared the interactions of PDI with a series of proteins representing stages along an oxidative protein folding pathway.

Recombinant Constructs, Mutagenesis and Expression
Recombinant proteins were expressed in E. coli BL21 (DE3) pLysS strains from a vector derived from pET23b (Novagen). BPTI constructs were expressed with a leading methionine, used as a start codon, but the wild-type was otherwise identical to mature BPTI. The wild-type BPTI construct, and details of expression, isolation and solubilisation of inclusion bodies were described previously [25]. To generate Cys-to-Ser mutations, PCR was performed using PfuUltra High-Fidelity DNA Polymerase (Agilent) to amplify the entire vector, following manufacturers' guidelines. PDI constructs included a His-tag sequence (MHHHHHHM) to facilitate purification and were expressed and purified as described previously [26]. To produce 15 N-labeled proteins, cells were grown in minimal medium containing 1 g/l of 15 N ammonium sulfate.

Preparation of BPTI and derivatives
Solubilized BPTI inclusion bodies were centrifuged, supernatant was collected and reduced BPTI was isolated by desalting through 465 ml HiTrap columns (GE Healthcare), attached to an Ä KTApurifier 100 FPLC, eluting with 10 mM HCl.
Both wild-type and mutant (C38S/C55S) BPTI were subjected to oxidative refolding, by dilution with 10 mM HCl and gradual addition (with stirring) of refolding buffer to a final protein concentration of 50 mM (0.3 mg/ml). The refolding buffer was 0.1 M Tris-HCl, 1 mM EDTA, pH 8.7 with 0.5 mM glutathione disulfide (GSSG) and 2 mM glutathione (GSH). The mix was incubated with gentle stirring overnight at 4uC before centrifugation at 75,0006 g. To generate reduced/alkylated BPTI, reduced wild-type protein (50 mM) was incubated with 50 mM Figure 1. Disulfide generation pathway for oxidative folding of reduced BPTI. The figure represents species and steps on the pathway from reduced BPTI (R, circled) to generate native BPTI (N, square box). Intermediates are identified in terms of the disulfide bonds that are present with colour intensity reflecting the extent of native conformation. The intermediates in hexagonal boxes contain a non-native disulfide and occur transiently in the pathway between the predominant 2-disulfide intermediates and the final 2-disulfide intermediate. Other intermediates (in rounded boxes) contain only native disulfide bonds (either one or two). Solid arrows represent steps that can occur via a single thiol:disulfide interchange reaction (isomerization or oxidoreduction with an exogenous thiol/disulfide reagent). Dashed arrows represent hypothetical steps in which one protein disulfide is lost and a different one is formed, requiring a minimum of two thiol:disulfide interchanges. doi:10.1371/journal.pone.0082511.g001 iodoacetamide in 100 mM sodium phosphate pH 7.0 at room temperature for 20 min.
All BPTI proteins were concentrated and reagents removed by solid phase extraction (SPE) using pre-packed 3 ml Grace Vydac SPE columns, containing reverse phase C-18 bonded to 13 mm silica beads with a 300 Å pore diameter. Protein samples were prepared following the manufacturer's instructions and applied to equilibrated columns by gravity flow overnight at 4uC. Acetonitrile was evaporated from the eluted protein in a vacuum desiccator overnight. The protein sample was then lyophilized for 4-6 hours and stored at 220uC.
Authentic BPTI (from bovine pancreas) was sourced from Sigma-Aldrich.

Mass spectrometry
Masses of various forms of wild-type and mutant BPTI were determined by electrospray ionization mass spectrometry on either a Bruker microTOF or a Synapt G2 Q-Tof instrument. Oxidatively-refolded and reduced/alkylated species were generated as described above. Refolded/alkylated material was generated by reaction of the refolded protein (50 mM) with 5 mM iodoacetamide in 20 mM phosphate buffer, pH 7.3 containing 8 M urea, for 20 mins in the dark at room temperature before quenching with 20% acetonitrile/2% trifluoroacetic acid..

Surface Plasmon Resonance
Surface plasmon resonance (SPR) was performed using a Biacore 2000 instrument (GE Healthcare). Proteins for immobilization were prepared at 50 mg/ml in HEPES-buffered saline containing EDTA and surfactant P20 (HBS-EP). A CM5 sensor chip was used, onto which proteins were immobilized by amine coupling, with pH scouting determining an optimal pH of 5.0. The standard protocols were followed using the manufacturer's instructions for both amine coupling and binding analysis. Analytes for binding analysis were prepared in HBS-EP running buffer to a volume of 370 ml for 2 replications. Analyte was injected at a flow rate of 50 ml/min. BIAevaluation version 3.1 was used to process and analyze the raw sensorgram data. Sensorgrams were baseline corrected using the Y-transform function. All curve fitting used simultaneous k a /k d calculations and the 1:1 (Langmuir) binding model was deemed the most appropriate model after parameter optimization. Analyses assuming a heterogeneous ligand provided two dissociation constants per ligand, but the K D values generated were so close as to suggest that the simpler binding model with a single dissociation constant was more appropriate.

NMR Spectroscopy
Unless otherwise stated, the NMR buffer used was 25 mM sodium phosphate, 100 mM NaCl, 10% D 2 O, pH 6.5. All BPTI experiments were performed on a Bruker AV II 700 MHz spectrometer, using a TCI cryoprobe with Z gradients (Bruker). For PDI bb'x samples, HSQC spectra were acquired at 298 K using a four channel Varian Unitylnova 600 MHz NMR spectrometer, equipped with a 5 mm HCN z-pulse field gradient probe. Both 2D TOCSY and 2D NOESY experiments were performed using 4096 data points in the F2 dimension and 512 data points in the F1 dimension, with mixing times of 60 ms (TOCSY) and 100 ms (NOESY). Unless otherwise stated, HSQC experiments were performed using 2048 data points in the F2 dimension ( 1 H) and 128 data points in the F1 dimension ( 15 N). For PDI titrations into BPTI, 15 N-labelled BPTI was used with molar ratios ranging from 200:1 BPTI:PDI to 1:1 BPTI:PDI. For wildtype and mutant BPTI, HSQC spectra were obtained using 0.4 mM samples. Due to its limited solubility, HSQC spectra of reduced/alkylated BPTI were acquired using 0.1 mM samples. To compensate for its lesser solubility, the number of scans was increased 5-fold for reduced/alkylated BPTI. BPTI titrations into PDI bb'x were obtained using 0.25 mM bb'x. HSQC spectra using wild-type BPTI and mutant BPTI used bb'x:BPTI ratios of 25:1, 5:1 and 1:1. Due to the limited solubility of reduced/ alkylated BPTI, HSQC spectra were obtained using 0.25 mM bb'x at bb'x:BPTI ratios of 25:1 and 8.3:1.
For hydrogen/deuterium exchange, the NMR buffer used 100% D 2 O as the solvent (25 mM sodium phosphate, 100 mM NaCl, 100% D 2 O, pH 6.5). All experiments were performed at 278 K. To allow a greater frequency of collection of HSQC spectra within the first hour of the time-course, the acquisition time was decreased by halving the number of data points per scan in the F1 dimension.
Data analyses were carried out using CcpNMR Analysis version 2 [27]. NMR assignments from Biological Magnetic Resonance Data Bank (BMRB) entry 5359 [28] were used to help assign the wild-type BPTI spectrum. Likewise, a previous study of PDI bb'x [26] provided assignments for bb'x control spectrum (BMRB entry 15974). To assign the backbone resonances for the mutant BPTI, 3D 15 N NOESY-HSQC and 3D 15 N TOCSY-HSQC experiments were performed.

A mutant BPTI mimics a partly-folded intermediate in the oxidative folding pathway
We expressed mutant (C38S/C55S) BPTI in E.coli, recovered it from inclusion bodies and refolded it ( [25], see Methods). The product was homogeneous and the mass was 6613 Da (Table 1, Figure S1), consistent with the presence of two CysRSer mutations and the formation of two disulfide bonds between the remaining Cys residues. This was confirmed by the fact that there was no change in mass when the refolded mutant was subjected to alkylation in denaturing conditions, whereas alkylation of the reduced mutant led to incorporation of 4 carboxamidomethyl Masses were determined by electrospray ionization mass spectrometry. Data marked * are means of 3 independent determinations all falling within +/2 1 Da; the remaining data are single determinations. Example deconvoluted spectra for the refolded species are shown in Figure S1. The observed mass of the refolded wild-type corresponds to the theoretical mass of fully-oxidised wild-type BPTI containing 3 disulfide bonds (6643 Da), while that of the refolded double mutant corresponds to the mass expected for the species containing 2 disulfide bonds (6613 Da). The mass differences between the refolded and reduced species confirm the formation of 3 and 2 disulfide bonds respectively. The mass of a carboxamidomethyl group (-CH 2 -CO-NH 2 ) is 58 Da, hence the masses observed for reduced/alkylated wild-type and mutant are as expected for alkylation of 6 and 4 Cys residues respectively. Alkylations of the refolded species were carried out in presence of 8M urea. No alkylated species were detected and hence any protein molecules containing free -SH groups must represent ,1-2% of the total refolded material. doi:10.1371/journal.pone.0082511.t001 groups ( Table 1). Hence the refolded mutant protein contained no buried -SH groups. For NMR studies, the protein was expressed in minimal medium and biosynthetically-labeled prior to oxidative refolding. 1 H-15 N HSQC spectra were collected at several temperatures. The HSQC spectrum at 36uC (Figure 2A) shows very poor dispersion and only a few well-resolved resonances, indicating little stable structure; at 20uC ( Figure 2B) greater signal dispersion is evident and the spectrum at 5uC ( Figure 2C) is well-dispersed with many well-resolved peaks, indicating the presence of a well-defined conformation. However, comparison between this HSQC spectrum and that of wild-type BPTI at the same temperature ( Figure  S2) shows extensive differences, so it was impossible to assign the mutant spectrum directly. To perform de novo assignment on the mutant, 2D 1 H-1 H TOCSY and 1 H-1 H NOESY spectra were collected at 5uC, and 3D 15 N-edited NOESY/TOCSY experiments were performed to resolve ambiguities. This allowed assignment of all the well-resolved backbone amide resonances in the 1 H-15 N HSQC spectrum of mutant BPTI collected at 5uC ( Figure 2C).
Interestingly, the amide resonances of residues 1-14 are not well-defined in this spectrum and could not be assigned (apart from Gly12), indicating that the N-terminal region is in intermediate exchange and not in a defined conformation even at 5uC, whereas the remainder of the molecule is well-folded. Hence the mutant has a partly-folded structure and will be referred to henceforward as 'partly-folded BPTI'. The result is consistent with earlier work (at non-physiological pH 4.6), which showed that the NMR spectrum of this mutant was highly temperature-dependent and that it had a partly-folded conformation at 22uC which was lost at higher temperatures [6]. The NMR properties of the mutant are also very similar to that of a quadruple mutant (C5S/C14S/C38S/C55S) which contains only the native 30-51 disulfide bond [5], confirming that our mutant contains the native 30-51 plus the non-native 5-14 disulfide. Hence our 'partly-folded BPTI' corresponds to a transient intermediate on the oxidative folding pathway of the wild-type protein (cf. Figure 1).

Comparison with native and unfolded BPTI
Recombinant wild-type BPTI was prepared similarly and the reduced protein was either re-oxidized or alkylated to block all six Cys residues (see Methods). Mass spectrometry (Table 1, Figure  S1) and CD spectroscopy (not shown), and comparison with authentic BPTI from a commercial source, indicated the formation of fully-oxidized, folded BPTI and of alkylated, unfolded BPTI respectively.
No fully-oxidized form of BPTI containing non-native disulfides has ever been identified. However, to confirm beyond doubt that our oxidized refolded wild-type material was identical to native authentic BPTI, the 2D 1 H-1 H NMR spectra of reoxidized recombinant wild-type BPTI were recorded and compared with those of authentic non-recombinant BPTI. 1 H-1 H TOCSY and 1 H-1 H NOESY spectra were also used to derive resonance assignments. The assignments for the authentic and reoxidized recombinant proteins are shown overlaid in Figure S3. The close correspondence between the resonances recorded for authentic and reoxidized recombinant proteins (including for all Cys residues and their neighbours) provides confirmation -at the level of atomic resolution -that the recombinant material is fully oxidized and refolded.
Recombinant wild-type BPTI was then biosynthetically 15 Nlabeled and prepared in both the oxidized and reduced/alkylated states for 15 N-1 H NMR analysis The 15 N-1 H HSQC spectrum of oxidized BPTI at 36uC ( Figure 3A) shows sharp well-dispersed peaks characteristic of a native protein, confirming that the protein is folded under these conditions. The 15 N-1 H HSQC spectrum of reduced/alkylated BPTI at 5uC (Figure 3B), shows few, poorlydispersed resonances with chemical shifts characteristic of random coil conformation; this material is essentially unfolded. Neither the oxidized/refolded nor the reduced/alkylated BPTI showed a significant temperature-dependence in their 1 H-15 N HSQC spectra over the range 5-36uC (data not shown), the former being folded and the latter unfolded throughout this range. These products will be referred to henceforward as 'folded' and 'unfolded' BPTI respectively. Figure S2 directly compares the 15 N-1 H HSQC spectra of folded, partly-folded and unfolded BPTI species at 5uC.
The dynamics of the proteins were compared using H/D exchange at 5uC. For mutant partly-folded BPTI, almost complete exchange of backbone amide protons was observed after 5 minutes, in contrast to the wild-type folded protein where approximately 50% of backbone amide protons did not exchange rapidly (Figure 4). This indicates that the partly-folded conformation of the mutant is highly dynamic and lacks a stably-folded core. By contrast, the amides of 24 residues of the refolded wild-type BPTI are well-protected in these conditions and indicate the presence of a stable, folded core. The protected amides are found in regions that form secondary structure elements in the native protein (see cartoon of secondary structure beneath each panel in Figure 4) and the slowly-exchanging amides observed in this experiment at pH 6.5 correspond very closely to those that showed slow exchange in classic work conducted at pH 4.5 [29].

Interaction of BPTI proteins with PDI
BPTI is small (,7 kD) compared to full-length PDI (.50 kD), so when unlabeled PDI is added to 15 N-labeled BPTI species, their binding will result in a broadening and loss of intensity of the ligand resonances in heteronuclear NMR spectra. The interaction of 15 N-labeled partly-folded BPTI (0.4 mM) with unlabeled PDI was studied at 5uC. In 1 H-15 N HSQC spectra, loss of ligand signal could be detected even at very low PDI concentrations (4 mM = 100-fold excess of BPTI, data not shown) and this effect became more pronounced as the PDI concentration was increased, so that no signal at all was detectable in presence of one molar equivalent of PDI (data not shown). Spectra of partlyfolded BPTI in 50-fold excess ( Figure S4A) and 10-fold excess ( Figure 5A) confirm that loss of signal due to line broadening increases with PDI concentration. Furthermore, the significant effects observed even when the ligand is in considerable molar excess over PDI indicate that there is very rapid exchange between bound and free ligand. This result is unexpected in view of the size of the ligand.
To test if PDI is able to bind a fully-folded protein, labeled folded BPTI was used in a comparable experiment. There is very obvious loss of signal at the 1:1 ligand:PDI ratio ( Figure S4B), but there is also some effect at the 10:1 ratio ( Figure 5B), which becomes clearer when signal intensities are quantified. Figure 5C shows quantitatively the signal intensities from partly-folded and folded BPTI when present in a 10-fold excess over unlabeled PDI compared to in absence of PDI. In both cases there is loss of signal on interaction with PDI but this is considerably more marked for the partly-folded BPTI.
Comparable experiments were performed by titrating PDI into the less soluble unfolded BPTI at 0.1 mM. Spectra at 50:1 and 5:1 excess of ligand over PDI ( Figure S4C, S4D) may be compared with the spectrum in absence of PDI ( Figure 3B); there is considerable loss of signal even at a 50-fold excess of ligand, consistent with a large fraction of unfolded BPTI binding to and dissociating from PDI on the timescale of the NMR experiment. The very significant loss of signal from all forms of BPTI when in presence of much lower concentrations of PDI implies rapid association and dissociation rates on a sub-millisecond timescale.
Clearly all forms of BPTI, even native BPTI, are recognized as ligands by PDI and the effects on signal intensity at different ligand:PDI ratios show qualitatively that the affinity of PDI increases with increased protein ligand unfolding. To provide more quantitative data, PDI and a ligand-binding fragment of PDI (the bb'x domains [26]) were immobilized onto separate channels of a sensor chip for analysis of ligand-binding by surface plasmon resonance. The various forms of BPTI were applied at several temperatures; binding and dissociation curves were fitted simultaneously and analysed assuming the interactions can be described by 1:1 binding stoichiometry with a single dissociation constant (see Methods). Table 2 presents the dissociation constants from these analyses which indicate clearly that affinity increases with increased ligand unfolding (i.e. K D values increase from unfolded to partly-folded to native, folded BPTI) and that affinity of PDI for these ligands is greater at 36uC than at 5uC. Furthermore, affinities for these ligands are very similar for full-length PDI and for the  bb'x fragment, reinforcing the conclusion that this fragment comprises the principal ligand binding site for peptides and unfolded proteins [30][31][32]. Reproducibility of the data is good, except for binding of folded BPTI at 5uC where the affinities are so low as to be difficult to determine with precision. Unfolded BPTI shows some non-specific affinity for an immobilized control protein (ovalbumin) but the affinities of partly-folded and folded BPTI for this control protein at 36uC are barely measurable, (K D values 200-300 fold greater than for PDI) (data not shown).

The protein ligand interaction site on PDI
Previous work identified residues within the b' domain of PDI that form the binding site for peptides and unfolded proteins [26,[33][34][35], but no study has been done with a partly-folded intermediate on an oxidative protein folding pathway. 15 N-labelled PDI bb'x was expressed and purified [26] and its interaction with unlabeled partly-folded BPTI was analyzed by collecting 1 H-15 N HSQC spectra of bb'x in absence and presence of mutant C38S/ C55S BPTI (Figures 6A, 6B). Although many resonances are identical in the two spectra, others are either shifted or broadened. For example, the indole side-chain N-H proton resonance of W347 (boxed) shows a change in 1 H shift from 10.4 ppm to 10.1 ppm. This change was seen previously on addition of a peptide ligand and results from displacement of the W347 sidechain from a hydrophobic site due to ligand binding [26,33]. These spectra were analyzed by the 'minimum-shift' method [26,36] (Figures 6C, 6D), showing that the effect of ligand-binding is focused on specific regions within the b' domain, with little effect on the b domain. Figure 6E represents the data structurally, showing that the binding site for partly-folded BPTI comprises mainly core b-sheet residues of the b' domain, corresponding to residues previously identified as the binding site for peptides and unfolded proteins [26,34].

PDI discriminates between substrates based on their degree of folding
The three proteins studied here showed contrasting conformational and dynamic properties. Reoxidized wild-type BPTI was folded at all temperatures and showed backbone amide H/D exchanges occurring over a range of timescales, with a core of well-protected amides. Reduced/alkylated BPTI behaved as an unfolded protein with an NMR spectrum showing poor dispersion at all temperatures. Oxidized mutant C38S/C55S BPTI, containing one native and one non-native disulfide, showed considerable temperature-dependence in its conformational properties, giving a well-defined assignable NMR spectrum only at 5uC or below. The N-terminal region of this species was not folded, even at low temperatures, and all backbone amides exchanged on a time-scale of 10 2 seconds at pH 6.5; so this protein is only partlyfolded and lacks a stably-folded core. Hence, the three proteins provide good models for species at different stages along an oxidative protein folding pathway.
NMR and SPR studies showed that all three proteins interact with PDI but with different affinities; the folded protein bound least tightly and the unfolded protein most tightly. Hence PDI's ability to discriminate between 'substrates' and 'products' arises from quantitative differences in its affinity for unfolded, partlyfolded and fully-folded proteins. We infer that PDI selects and binds to unfolded regions in all substrates in a manner analogous to its binding of peptide ligands [26,32,33]. Each protein's conformation is dynamic and regions of folded protein transiently unfold and refold on timescales that reflect the stability of the folded state. Our data suggest that regions of unfolded polypeptide are selected preferentially as ligands by PDI and that the observed quantitative differences in affinity of PDI for the three substrates reflect their tendencies to local unfolding. It is possible that PDI recognizes the partly-folded ligand exclusively through its 'unfolded' region (residues 1-14), but the fact that native BPTI is also bound (albeit more weakly) suggests that transiently unfolded regions are recognized. This interpretation is supported by the fact that the affinities determined by SPR for all three proteins increase with temperature from 5uC to 36uC, consistent with the view that PDI binds unfolded and dynamic regions of its protein ligands, which become increasingly available as the temperature increases.
The SPR experiments also allowed a direct comparison of binding by full-length PDI and the bb'x fragment; there was no difference in the affinities observed, implying that any interactions made by these protein ligands with other regions of PDI -the a, a' and c domains -do not contribute significantly to the binding. In summary, we have shown that PDI makes similar reversible noncovalent interactions with a set of proteins that model an oxidative folding pathway; dissociation constants lie in the range between 10 23 -10 26 M and dissociation rate constants are .10 3 s 21 .

Interactions between PDI and ligands in the cell
How well does the system studied here represent PDI/substrate interactions which occur in cellulo? Complex disulfide isomerizations occur in the oxidative folding pathways of several proteins in cellulo [37,38] but detailed studies of interactions with folding catalysts have not been attempted. We have studied these interactions in a model system and mimicked some of the conditions that hold in vivo. All NMR and SPR experiments were  performed at approximately physiological pH and at temperatures from 36uC down to 5uC. Protein concentrations were in the 0.1-1 mM range (comparable to the concentration of PDI within the ER lumen) but pure proteins were used and no agents were added to mimic the overall crowding that occurs within the ER [39].
Crowding would lead to further folding of partly-folded species, so perhaps the partly-folded model protein used here would retain some folding at physiological temperature. Crowding would also increase the affinity of PDI for ligands in general [40], though it would not be expected to change the relative order of affinities. A further issue relates to oxidants for protein disulfide formation in the ER. The non-catalyzed disulfide generation pathway of BPTI described above (Figure 1) was determined from experiments using small molecule disulfides as oxidants. The complexity of the disulfide isomerizations on the oxidative folding pathway of BPTI has been ascribed to the difficulty such disulfide oxidants have in accessing and oxidizing buried thiol groups. Recently, the oxidative folding pathway of BPTI was studied using H 2 O 2 as oxidant and evidence was presented suggesting that this small highly reactive oxidant can effectively oxidize buried thiols, simplifying the disulfide generation pathway [41]. Furthermore, there is now a much clearer picture of the various routes by which oxidizing equivalents can flow via PDI to reduced proteins within the ER [42]. Enzymatic machinery exists within the ER to use both O 2 and H 2 O 2 as ultimate oxidants and to transfer oxidizing equivalents from them to PDI. Although various parts of this machinery have been reconstituted in vitro [43,44], there has been no reinvestigation of disulfide regeneration pathways of protein substrates under these circumstances.
These issues bear on our observation that the same site on PDI is involved in binding peptides, unfolded proteins and partlyfolded proteins. Can a substrate protein stay bound to PDI through the complete oxidative folding process, undergoing a series of disulfide formation and isomerization steps? Apparently this may depend on the oxidant. In the situation where the oxidizing equivalents are derived from O 2 via the oxidase Ero1, substrate dissociation must occur between each oxidative step, since Ero1 binds to PDI via the ligand-binding b' domain [43] and may compete directly with other ligands for the same site [45]. Hence cycles of substrate and partner protein binding and release must occur in PDI turnover and these will be facilitated by the rapid dissociation rates that can be inferred from our observations. In other work [46], we are characterizing the flexibility and mobility of PDI and we observe extensive relative motion of its domains which may facilitate its ability to interact with a wide range of different substrate and partner proteins.
In conclusion, PDI is distinctive compared to thioredoxin or DsbA, with which it shares capabilities in thiol/disulfide redox chemistry [31,47]. The key features enabling PDI to act as an effective catalyst of oxidative protein folding are i) its ability to bind to transiently-and permanently-unfolded regions of substrate proteins, through its promiscuous ligand binding site on the b' domain, ii) its capacity to distinguish folded, partly-folded and unfolded proteins through quantitative differences in binding affinity, iii) its extensive inter-domain flexibility allowing it to accommodate substrate and partner proteins of different shapes and sizes at the same promiscuous binding site, and iv) its ability to bind and release ligands on a sub-millisecond timescale, enabling it to change partners rapidly.  . Identification of the site on PDI that interacts with partly-folded BPTI. 1 H-15 N HSQC spectra of 0.25 mM 15 N-labeled bb'x fragment of PDI recorded at 25uC, A) in absence of any ligand, and B) in presence of 50 mM partly-folded BPTI. Peaks for the indole NH resonance of Trp 347 are highlighted within a dashed box. Chemical shift perturbations were measured from peaks in the spectrum in absence of ligand to the nearest peak found in the spectrum recorded in presence of ligand, and the net chemical shift perturbation was calculated as D 15 N/6+D 1 H. Net chemical shift perturbations were plotted versus sequence position in PDI, C) for data in presence of 50 mM ligand (panel B above) and D) for data in presence of 0.25 mM ligand (spectrum not shown). Blue bars represent shifts of peaks identified in the target spectrum, maroon bars represent shifts from peaks that could not be identified in the target spectrum, presumed to be due to line broadening. The cartoon below each graph indicates the position of each domain in the bb'x construct. The chemical shift perturbations seen in the presence of 0. 25


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

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KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.

Introduction A significant step in breast cancer progression is activation of oncogenes via gene amplification and overexpression [1]. The chromosome 8p11-p12 amplicon, containing approximately 55 genes, is present in 12% to 15% of breast cancers and is correlated with poor prognosis in primary breast tumors. Amplification of 8p11-p12 is also correlated with histologic grade, increased Ki-67 proliferation index, and decreased 5-year metastasis-free survival [2][3][4][5][6][7]. Due to its relevance in breast cancer, many studies have been aimed at characterizing this amplification and identifying the driving oncogenes in this region. Over the past several years, our laboratory and several others have analyzed the 8p11-p12 amplicon to identify possible driver oncogenes and determine the clinical relevance of these gene amplification events in breast cancer. These studies have resulted in the identification of a number of genes that play a role in breast cancer when the amplicon is present, including LSM-1, C8orf4 (TC-1), RAB11FIP1, WHSC1L1, ERLIN2, PROSC, PPAPDC, DDHD2 and others [2,6,[8][9][10][11][12][13]. Gelsi-Boyer et al. demonstrated that the amplicon can be sub-divided into four distinct regions that can be amplified independently of each www.neoplasia.com Volume 16 Number 8 August 2014 pp. 644-655 644 other, and this partly explains the large number of candidate oncogenes identified to date from this region [3]. Recently, our lab has taken a genome-scale shRNA screening strategy to identify additional driving oncogenes in a panel of SUM breast cancer cell lines, with and without 8p11-p12 amplification, for which copy number and expression data are available. KAT6A was found to be a consistent hit in the shRNA screen and is significantly amplified and overexpressed in SUM-52 cells.
KAT6A, a gene commonly found in the 8p11-p12 amplicon, was first identified in 1996 as part of a chromosomal translocation t(8;16)(p11;p13) with CREB-binding protein in a subtype of acute myeloid leukemia (AML) [14]. Since then, additional translocations involving KAT6A in AML have been discovered, including translocations with the HATs p300 and TIF2 [15]. The KAT6A-TIF2 fusion is capable of immortalizing myeloid progenitor cells and inducing acute myeloid leukemia upon injection into irradiated mice [16]. Characterization of KAT6A in these gene rearrangements provides compelling evidence that KAT6A is an oncogene in leukemogenesis, much like c-myc, which is well documented in various chromosomal translocations promoting several forms of leukemia, and is also amplified in breast cancer.
In this study, we provide evidence that KAT6A functions as a novel oncogene in luminal breast cancers bearing the 8p11-p12 amplicon. We identified KAT6A as a top shRNA screen hit among genes both amplified and overexpressed in the SUM-52 breast cancer cell line. The influence of KAT6A knockdown on proliferation and viability of SUM-52 cells, and other cell lines with the 8p11-p12 amplicon was validated in separate experiments involving several short hairpin RNAs (shRNAs). Finally, data obtained with primary human breast cancers coupled with TCGA data support a role for KAT6A as a breast cancer oncogene.

Reagents
All chemicals were purchased from Sigma Aldrich unless otherwise noted. Antibodies against Actin and anti-acetyl Histone 3 were purchased from Millipore. The total-Histone 3 antibody was purchased from Abcam, and the KAT6A antibody from Novus Biologicals. The FGFR inhibitor PD170374 was purchased from Selleckchem. Expression Arrest GIPZ lentiviral KAT6A shRNA constructs and packaging mix were purchased from Open Biosystems. Primers used for RT-PCR reactions were ordered from IDT. The SUM breast cancer cell lines were maintained in Hams F-12 cell culture medium as described previously [17][18][19].

Cellecta Screening Method
Virus pools expressing shRNA constructs were prepared according to the Cellecta Pooled Lentiviral shRNA Libraries User Manual protocol (www.cellecta.com). HEK 293 T cells were transfected with each of the three Cellecta library plasmid DNA pools (Human Modules 1-3) and the Cellecta Ready-to-Use Packaging Mix (Cat #CPCP-K2A). For each module, virus was titered and used to transduce 5 × 10 7 SUM-52 cells at an MOI of 0.5 in the presence of 5 μg/ml polybrene. Following transduction, cells were cultured for 3 days to allow expression of the resistance marker. Non-transduced cells were eliminated from the culture by the addition of 2 μg/ml puromycin to the growth media. Three days after the addition of puromycin, cells were trypsinized and one-half of the total population was harvested for genomic DNA preparation. This DNA served as the reference time point DNA. The remaining cells were plated and grown for approximately 7 population doublings before being harvested for genomic DNA preparation. Genomic DNA was prepared by phenol:chloroform extraction according to the Cellecta Pooled Lentiviral shRNA Libraries User Manual protocol.
See Supplementary Methods for detailed descriptions of the shRNA screen and statistical analyses.

RT-PCR
For RT-PCR reactions, total RNA was isolated using the Qiagen RNeasy Plus Mini Kit. RNA was converted into cDNA using the iScript Advanced cDNA Synthesis Kit (BioRad). Following cDNA conversion, RT-PCR reactions were performed in 96-well whitewalled plates using FastStart Universal SYBR Green Master mix (Roche). Reactions were performed on an Eppendorf Mastercycler Realplex2 under the following parameters: 1 cycle for 10 minutes at 95°C, 1 cycle for 15 seconds at 95°C, and 40 cycles at 53°C for 30 seconds. Primer sets included a control GAPDH primer set and a primer set specific to the KAT6A transcript.

Growth and Colony-Forming Assays
For growth assays, cells were plated in triplicate in 6-well plates at a density of 35,000 cells/well. Nuclei were harvested and cell counts obtained using a Beckman Coulter Counter as described previously [7].
For colony-forming assays, cells were plated in triplicate in 6-well plates at varying cell densities. Colonies formed for two weeks and then were fixed with 1 mL/well of 3.7% paraformaldehyde for 20 minutes at RT. Colonies were stained with 1 mL/well of 0.2% crystal violet for 15 minutes at RT and then de-stained with dH 2 O. For colony-forming assays including treatment with the FGFR inhibitor PD170374, cells were plated as stated above and treated with 0.1 μM of drug for 3 consecutive days. Colonies formed for an additional 10-14 days following drug treatment and were stained with crystal violet as described. Colony counts were generated using the GelCount System from Oxford Optronix.

Mammosphere Formation
Cells were plated in triplicate for primary mammosphere formation in low-attachment 6-well plates. Spheres formed for one week, at which point two wells were transferred to standard 6-well plates in media supplemented with FBS. Attached spheres were fixed with 1 mL/well of 3.7% paraformaldehyde for 20 minutes at RT and stained with 1 mL/well of 0.2% crystal violet for 15 minutes at RT. For secondary mammosphere formation, spheres from one well of the primary mammosphere assay were harvested and centrifuged at 2500 RPM for 5 minutes. Spheres were re-suspended in 1 mL of trypsin and incubated at RT for 5 minutes. The trypsin was diluted with media and centrifuged as before. The cell pellet was washed with 5 mL of PBS and centrifuged, and the final cell pellet was resuspended in 1 mL of 1 × Hanks buffer solution. Cells were re-plated in triplicate in 6-well low attachment plates. Spheres were allowed to form for 2 weeks and then transferred and stained with 0.2% crystal violet as described above. Images of primary and secondary spheres were taken using an EVOS Imaging System (Life Technologies). Mammosphere counts for both primary and secondary assays were generated using the GelCount System from Oxford Optronix. RNA sequencing on an Illumina HiScanSQ. RNA integrity was verified on an Agilent 2100 TapeStation (Agilent Technologies, Palo Alto, CA). 100-200 ng of total RNA was used to prepare RNA-Seq libraries using the TruSeq RNA Sample Prep kit following the protocol described by the manufacturer (Illumina, San Diego, CA). Sequencing was performed on an Illumina HiScanSQ. Samples were demultiplexed using CASAVA (Illumina, San Diego, CA). Fastq files were used to map reads to the human genome (hg19, UCSC) utilizing Tophat2 with default settings. Bam files were imported into Partek Genomics Suite (St. Louis, MO) for analysis. Samples were filtered for ≥ 10 reads. Differential gene expression was performed by comparison of biological duplicate samples in Genomics Suite using advanced analysis of variance (ANOVA). Differential gene express values were then filtered by ± 1.5 fold change and by unadjusted P ≤ .05.

Rationale for Studying the Oncogenic Potential of KAT6A
In an effort to comprehensively identify genes that control cell proliferation and survival in breast cancer cells, our laboratory is performing genome-scale, RNAi-based negative selection screens in our panel of SUM breast cancer cell lines. In this screen, the SUM-52 breast cancer cell line was transduced with a library of 82,000 lentiviral vectors that express shRNAs targeting 15,377 cellular genes with a minimum of 4 shRNAs per gene. Cells transduced with this library were harvested at either day 3 after infection or after approximately 7 population doublings, and the abundance of shRNAs at both time points was determined by PCR amplification, purification and sequencing of shRNA-associated barcode sequences. Fold depletion values were calculated by comparing the abundance of each shRNA at the final time point with the abundance at the initial time point. Fold depletion scores following statistical analysis for all of the shRNAs targeting each gene were used to identify genes that are necessary for the growth/survival of the SUM-52 breast cancer cell line. Integration of this list with the list of genes that are significantly copy number amplified in this cell line identified a small number of genes that are amplified, overexpressed and necessary for growth/survival of the cell line. KAT6A was found to be a consistent hit in this screen, as well as an additional screen using an early generation lower complexity library, and is significantly amplified and overexpressed in SUM-52 cells ( Figure 1A).
Based on the RNAi screen data, we performed microarray analysis to examine the expression level of KAT6A in a small panel of primary breast cancers available in our lab that potentially carry the 8p11-p12 amplicon. As seen in Figure 1B, several primary tumor samples from this panel exhibited high levels of KAT6A gene expression. Additionally, tumors with the highest expression of KAT6A also had a KAT6A gene amplification. Likewise, a series of SUM breast cancer cell lines were analyzed by microarray, and KAT6A expression was highest in cells harboring the 8p11-p12 amplicon, particularly SUM-52 cells ( Figure 1C). The SUM-52 cell line was generated from a patient with luminal B breast cancer who presented with an ER-positive primary tumor. The data in Figure 1D shows that KAT6A protein expression was highest in SUM-52 cells compared to normal MCF10A cells, SUM-149 and SUM-225 cell lines. Whereas SUM-225 cells have two small regions of 8p11 amplification, KAT6A is not amplified in these cells, and SUM-149 cells do not harbor the 8p11-p12 amplicon. As expected, KAT6A protein expression in SUM-225 cells was barely detectable; however, SUM-149 cells, although lacking the 8p11-p12 amplicon, express KAT6A protein at levels similar to SUM-52 cells. Because KAT6A is known as a histone acetyltransferase (HAT) with HAT activity on histone 3, we examined the acetylation status of histone 3 in cells with amplification of KAT6A. Total histone 3 acetylation was highest in SUM-52 cells compared to MCF10A, SUM-225, and SUM-149 cells ( Figure 1D); however, the difference in histone 3 acetylation was most interesting between SUM-52 and SUM-149 cells. Although both cell lines express comparable levels of KAT6A protein, histone 3 acetylation was only increased in SUM-52 cells.
To explore further the relevance of KAT6A in breast cancer, we examined TCGA data derived from 1,000 primary breast cancers, and found that KAT6A expression is altered in 22% of breast cancer cases. Additionally, there is a strong correlation between copy number and expression at the mRNA level ( Figure 1E). Kaplan-Meier analysis of more than 4000 breast cancer patients (http://kmplot.com/analysis/) showed that high level KAT6A expression (highest quartile) was associated with a statistically significant reduction in overall survival ( Figure 1F). Interestingly, the Kaplan-Meier curves diverge after approximately 100 months of follow-up, suggesting that KAT6A plays a role in late recurrences, an important clinical problem in luminal breast cancer. These data support the hypothesis that KAT6A functions as a driving oncogene in breast cancer, and warrant further investigation to validate its activity as a transforming oncogene.

KAT6A Knockdown Reduces Growth in 8p11-p12 Amplicon-Bearing Breast Cancer Cells
To validate the shRNA screen results, KAT6A knockdown studies were performed with several cell lines. Lentiviral shRNA vectors were prepared by transfection of 293FT cells with five shRNA plasmids targeting different KAT6A sequences. Initially, only SUM-52 and MCF10A cells were transduced with the lentiviral shRNAs. Following lentiviral infection and puromycin selection, RNA was harvested to confirm knockdown of KAT6A by RT-PCR. The results showed that two shRNAs yielded the best knockdown, sh1-566 (sh1) and sh3-895 (sh3), compared to a non-silencing shRNA sequence, (NS) (Supplementary Figure 1). To measure the effects of reduced KAT6A expression on the growth of the SUM-52 and SUM-225 cell lines, the cells were plated in monolayer in 6-well plates, and their growth was assessed by cell counts on days 1, 4, and 7. Knockdown of KAT6A at the message and protein level in SUM-52 cells (Figure 2A) resulted in 50% reduction in cell growth ( Figure 2C). KAT6A is not part of the 8p11-p12 amplicon in SUM-225 cells, and only a limited growth response to reduced KAT6A expression was observed ( Figure 2D). Infection of SUM-149 cells, which lack the 8p11-p12 amplicon but still express the protein, did not affect proliferation ( Figure 2E). Additionally, knockdown of KAT6A in MCF10A cells ( Figure 2B) showed no effect on proliferation ( Figure 2F). These data suggest that the effects of KAT6A on breast cancer cell proliferation are restricted to cells in which the gene is amplified and overexpressed.

KAT6A Reduces the Clonogenic Capacity of SUM-52 Cells
Since reduction of KAT6A expression yielded growth inhibition in SUM-52 cells, we next addressed the colony-forming efficiency of SUM-52 cells after knockdown of KAT6A. As shown in Figure 3A, SUM-52 cells with depleted KAT6A levels exhibited a dramatic reduction in colony-forming ability compared to control cells. Moreover, colonies that did grow after KAT6A knockdown were not only smaller in size, but exhibited an abnormal morphology ( Figure 3A and B). Cells with KAT6A knockdown produced small, scattered colonies composed of cells showing morphological signs of pyknosis. By contrast, non-silencing control cells produced tightlypacked colonies with cells displaying a normal appearance. Figure 3C shows the size distribution of colonies derived from KAT6A knockdown cells. The largest colonies from the knockdown cells were approximately 300 μm in diameter, whereas colonies from control cells were greater than 1000 μm in diameter. The Figure 1. Rationale for studying the oncogenic potential of KAT6A. A. Fold depletion scores from the genome-wide shRNA screen were used to generate probability density plots. The black plot was generated using depletion scores for all genes, the blue plot was generated using depletion scores for commonly essential genes, the turquoise plot was generated using depletion scores for the negative control gene set, and the green plot is a normal distribution whose mean was determined by the median of the fold depletion scores for all genes. The vertical red line indicates the 95th percentile of the normal distribution that was used as a cutoff for determining hits in the screen. colony-forming efficiency of MCF10A cells, however, was unaffected by knockdown of KAT6A ( Figure 3D and E). In a separate experiment, we examined the role of KAT6A knockdown in anchorageindependent colony formation. KAT6A knockdown also inhibited the soft agar colony-forming ability of SUM-52 cells, as KAT6A knockdown cells formed fewer colonies compared to the nonsilencing control ( Supplementary Figure 2A and B). To extend these observations to other 8p11-p12 harboring cancer cell lines in which KAT6A is amplified and overexpressed, the ovarian cancer cell lines OAW28 and 59 M were assayed for colony-forming efficiency following KAT6A knockdown (Supplementary Figure 3A and B). These additional cell lines were identified using the TCGA Bioportal and the Broad Institute's Cancer Cell Line Encyclopedia, which indicated a GISTIC copy number score of 1.563 for 59 M cells and  Neoplasia Vol. 16, No. 8, 2014 KAT6A is a Candidate Oncogene in Luminal Breast Cancer Turner-Ivey et al.
1.665 for OAW28 cells. As seen in Figure 3F and G, both OAW28 and 59 M cells, respectively, responded dramatically to knockdown of KAT6A, with both cell lines displaying a reduction in colony forming ability. Based on these results, KAT6A functions in regulating the clonogenic capacity of not only SUM-52 cells, but also of other cancer cell lines with the 8p11-p12 amplicon and KAT6A amplification/overexpression.

KAT6A Plays a Role in Breast Cancer Stem Cell Activity and Differentiation
In light of the effects of KAT6A knockdown on the growth and colony-forming efficiency of SUM-52 cells, the role of KAT6A in breast cancer stem cell activity and self-renewal was examined in a primary mammosphere assay. As shown in Figure 4A, approximately 2% of non-silencing control SUM-52 cells formed mammospheres. By contrast, less than 1% of KAT6A knockdown cells formed spheres.
To confirm and extend these results, a secondary mammosphere assay was performed. The non-silencing control cells formed 4-fold more mammospheres than in the primary assay, suggesting that the cells plated for secondary mammosphere assay contained a higher percentage of cancer-initiating stem cells. Consistent with results in the primary mammosphere assay, non-silencing control cells again formed more mammospheres than KAT6A knockdown cells in the secondary mammosphere assay ( Figure 4B). Images of mammospheres revealed striking differences not only between non-silencing and KAT6A knockdown spheres, but also between primary and secondary mammosphere assays. Mammospheres derived from control cells displayed classical sphere morphology. Although KAT6A knockdown spheres appeared similar, they were smaller in size compared to the control spheres ( Figure 4C). Allowing secondary mammosphere cultures to grow over an extended period revealed a remarkable difference in morphology between the non-silencing and KAT6A knockdown mammospheres. Mammospheres derived from non-silencing control cells began to form three-dimensional duct-like structures after two weeks in culture ( Figure 4D), which were never observed in mammosphere cultures derived from KAT6A knockdown cells. These results indicate that SUM-52 cells can form complex three dimensional structures after extended mammosphere culture, and that this capacity is lost upon KAT6A knockdown.
In many breast cancer cell types, expression of surface markers is a measure of stemness and self-renewal potential. In particular, the CD44 + /CD24 -/low expression pattern is associated with the breast cancer stem cell phenotype in some cell lines and tumors. Because knockdown of KAT6A resulted in reduced mammosphere formation, we investigated the expression levels of CD24 and CD44 in KAT6A knockdown and control cells by flow cytometry. Knockdown of KAT6A resulted in an increase in cells positive for both CD24 and CD44 to a level approximately three times above the non-silencing control. Perhaps more interesting was the increase in CD44 expression seen in KAT6A knockdown cells, as evidence now suggests that CD44 positivity is associated with differentiation [20]. Only 3.1% of non-silencing control cells were positive for CD44, Figure 4. KAT6A plays a role in breast cancer stem cell activity. A. SUM-52 control and KAT6A knockdown cells were plated in triplicate in 6-well low-attachment plates for a primary mammosphere assay. After one week, mammospheres were transferred to standard 6-well plates overnight, stained with 0.2% crystal violet and counted. B. Cells from the primary mammosphere assay were harvested, trypsinized, and re-seeded in a secondary mammosphere assay at 3,000 cells/well. After one week, spheres were processed as above for staining. C. Images of primary mammospheres following one week of growth. D. Images of secondary mammospheres following two weeks of growth.

Influence of KAT6A on Gene Expression in SUM-52 Cells
To gain insight into the role of KAT6A as a breast cancer oncogene, we performed RNA sequencing in SUM-52 KAT6A knockdown and control cells. The heat map in Figure 5A was generated from four replicate experiments using the two different shRNAs described above. Since the replicates from each shRNA clustered together, we pooled the data resulting in four data sets for the bioinformatic analysis of the effect of KAT6A knock-down on gene expression. As can be seen from the figure, KAT6A knockdown had a significant effect on gene expression. We used NIH DAVID to analyze the differentially expressed genes that resulted from KAT6A knockdown, and identified several biological processes, molecular functions, and KEGG pathways associated with this gene set. The biological processes affected by KAT6A knockdown are listed in Table 1, with genes involved in Response to Hormone Stimulus and Gland Development processes as top hits. From this analysis, several well-known genes involved in regulating growth were highlighted. Some of the significantly down-regulated genes in KAT6A knockdown cells included ESR1, IRS1, IGF1R, and ERBB4 (Table 2 and Supplementary Table 1, 2). Interestingly, not only was the adenylate cyclase gene ADCY7 significantly down-regulated in response to KAT6A knockdown, but so were its two regulatory subunits, PRKAR1B and PRKAR2B. A number of interesting transcription factors were also identified using NIH DAVID analysis. As shown in Table 3 (and Supplementary Table 3), GATA3, TBX3, and MYC were all significantly down-regulated upon KAT6A knockdown. KEGG pathway analysis yielded additional insight into the role of KAT6A, showing signaling pathways effected by knockdown of KAT6A. As seen in Table 5, some of the major pathways enriched following KAT6A knockdown included Wnt, PI3K-Akt, Insulin/IGF-1, and ErbB signaling. Finally, merging the list of significantly regulated genes identified by RNA sequencing in KAT6A knockdown cells with hits from the genome-wide shRNA screen in SUM-52 cells identified several overlapping genes, including IGF1R, IRS1, IGF2BP3, and GATA3 ( Table 5). The identification of genes that are both regulated in expression by KAT6A and hits in the shRNA screen provides functional evidence for the importance of these genes and pathways in breast cancer cells with KAT6A amplification. (See Table 4.)

KAT6A Interacts With FGFR2 to Enhance the Tumorigenicity of SUM-52 Cells
Previous work from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. We have observed that while FGFR inhibition can inhibit the growth of these cells, this growth arrest is reversible, as washout of the drug results in prompt resumption of cell growth and no significant loss of colony forming ability (unpublished   data and Figure 6A). To address the potential interaction of FGFR2 and KAT6A in SUM-52 cells, the colony-forming ability of SUM-52 non-silencing control cells or SUM-52 KAT6A knockdown cells was examined following treatment with the FGFR inhibitor PD170374. As shown in Figure 6A, exposure of non-silencing control cells to FGFR inhibition did not significantly affect the colony-forming ability of the cells after washout of the drug. As observed previously, the colony-forming efficiency of SUM-52 KAT6A knockdown cells was reduced compared to controls. However, unlike the control cells, the colony-forming efficiency of the KAT6A knockdown cells was decreased further following 72 hours of exposure to PD173074, and this decrease was statistically significant (P b .001). Images of colonies with KAT6A knockdown revealed a striking morphological difference compared to control colonies. As seen in Figure 6B, non-silencing control colonies appeared healthy in size and morphology after treatment and washout of PD170374; however, the KAT6A knockdown colonies that did grow after drug treatment were much smaller in size, with cells showing signs of pyknosis. MCF10A cells, however, were not negatively affected by either KAT6A knockdown or PD170374 treatment (seen in Figure 6C). These results suggest that the KAT6A oncogene interacts with the FGFR2 oncogene, a clear driver of transformed phenotypes in SUM-52 cells, and has an influence on the response of cells to a targeted receptor tyrosine kinase inhibitor.

Discussion
Elucidation of novel candidate oncogenes from the 8p11-p12 amplicon has been the subject of on-going studies in our laboratory and a number of other laboratories over many years. The 8p11-p12 amplicon occurs in approximately 15% of breast cancers, the majority of which are luminal cancers. An interesting feature of this amplicon is the large number of candidate oncogenes already identified in this the region, and the consistent finding that many of these genes are coordinately amplified and overexpressed in breast cancer and lung cancer [2][3][4]8,9,12,13,21]. In the present study, we demonstrate that KAT6A is another candidate oncogene from this region. Gelsi-Boyer et al. provided compelling evidence that the 8p11-p12 amplicon can be sub-divided into four distinct regions, each of which can be amplified independently of the others [3]. We have reported previously on candidate oncogenes from region III of this amplicon, which contains genes such as WHSC1L1, DDHD2, and FGFR1 [5][6][7]10,11,22]. The KAT6A oncogene is in region IV of the amplicon, which is closest to the centromere. KAT6A is co-amplified with region III oncogenes in approximately 7% of cases and amplified independently of this region in approximately 3% of breast cancer cases according to TCGA data. We found KAT6A to be a consistent hit in an RNAi-based negative selection screen in SUM-52 breast cancer cells. Since KAT6A is both amplified and overexpressed in SUM-52 cells, the identification of KAT6A as a positive screen hit was suggestive of its importance as an oncogene in this cell line. Validation experiments using several KAT6A-specific shRNAs confirmed the screen result and demonstrated a significantly reduced growth rate and reduction in clonogenic potential in knockdown cells compared to non-silencing controls. Similar results were obtained using ovarian cancer cell lines that have KAT6A amplification, but normal mammary epithelial cells, or other breast cancer cells without the 8p11-p12 amplicon were not affected by KAT6A knockdown. Analysis of greater than 1000 primary breast tumors in the TCGA data base showed that KAT6A is amplified in 8.7% of invasive breast cancers, and this amplification is associated with overexpression at the mRNA level. Furthermore, overexpression of KAT6A was found to be associated with late recurrence of breast cancer. Thus, both functional genomics data obtained in our lab and data from the TCGA point to KAT6A as an important breast cancer oncogene.
Cancer cells are heterogeneous, and within any population of cancer cells in a cell line, only a relatively small fraction are considered clonogens based on their ability to form continuously expanding colonies when grown at clonal density, either in monolayer or in soft-agar. In the work reported here, we demonstrate a greater than 10-fold decrease in the clonogenic capacity of SUM-52 cells following knockdown of KAT6A. Colony size distribution shows that the majority of KAT6A knockdown colonies were smaller than the non-silencing control colonies. We interpret these results to indicate that reduction of KAT6A levels not only reduced the clonogenic fraction of SUM52 cells, but also reduced the growth rate of colonies that did form from the remaining clonogens.
We were intrigued by the relationship between the dramatic decrease in colony and mammosphere formation in SUM-52 cells upon knockdown of KAT6A, and the genes regulated by KAT6A. IGF1R, IRS1, and ERBB4 were among the genes reduced in expression as a result of KAT6A knockdown, which is consistent with a decrease in proliferation and colony formation by these cells. Interestingly, a recent publication by Heiser et al. showing the efficacy of 77 compounds in more than 50 breast cancer cell lines showed that the SUM-52 cell line was among the most sensitive to IGF1R inhibition [23], a result consistent with our association between KAT6A overexpression and the IGF1R.
Several interesting transcription factors were also down-regulated following knockdown of KAT6A, including MYC, TBX3, and P value indicates the probability of obtaining the number of screen hits in the pathway by chance alone. FDR indicates the global probability corrected for false discovery rate of obtaining the indicated number of pathway hits by chance alone. GATA3. It is known that gene rearrangements involving KAT6A that occur in leukemia results in an inability of cells to differentiate, which is in keeping with the known roles of KAT6A and other MYST family genes in regulation of stem cell self-renewal and differentiation in hematopoiesis [24][25][26]. Thus, one mechanism by which KAT6A may affect breast tumorigenesis is by altering self-renewal capacity and differentiation of cells of the luminal mammary lineage. Several of our results are consistent with this hypothesis. Mammosphere formation in SUM-52 KAT6A knockdown cells was not only significantly reduced, but so too was the ability of the cells to form large, branching structures when cultured for extended periods in low-attachment plates in stem cell medium. The down-regulation of key transcription factors associated with mammary gland development in KAT6A knockdown cells is consistent with this observation. C-MYC is a well-known breast cancer oncogene and mechanisms that drive MYC expression in breast cancer cells play a key role in the growth and survival of breast cancer cells [27][28][29]. GATA3 is a key transcription factor in mammary gland development and plays a role in determining luminal cell fate [30,31], and as we and others have shown, KAT6A and other oncogenes of the 8p11 amplicon are over expressed in luminal breast cancers like the SUM-52 cells. Finally, the finding of KAT6A regulation of TBX3 provides a link between breast cancer biology and leukemogenesis induced by this oncogene. KAT6A regulates expression of TBX1 in hematopoietic progenitor cells [26], while TBX3 has been shown to play an essential role in mammary gland development and ductal branching. Furthermore, TBX3 has been shown to be over expressed in a fraction of breast cancers, notably estrogen receptor positive, luminal breast cancers [32]. TBX3 has also been found to be an estrogen responsive gene in MCF-7 cells, and our results show that KAT6A regulates expression of ESR1 in SUM-52 cells. Thus, the genes regulated by KAT6A in SUM-52 cells are all key genes in the development of breast cancers of the luminal lineage, which further implicates KAT6A as a key driver of luminal breast cancer. KAT6A, also known as monocytic leukemia zinc finger protein (MOZ) or MYST3, belongs to the highly conserved MYST family of histone acetyltransferases, important in post-translational modification of histones and chromatin structure. Currently, the MYST family includes Tip60, MOF, HBO1, MOZ (MYST3), and MORF [22]. The MYST family is known to acetylate lysine residues on histones H4, H3, H2A and H2A variants, and recent evidence suggests that MYST proteins regulate themselves by acetylation. Specifically, KAT6A has acetyltransferase activity towards lysine residues on histones H2B, H3 (K14) and H4 (K5, K8, K12, and K16) [22,26]. Acetylation of histone H3K9 by KAT6A has been shown in vivo [33]. Although KAT6A is widely-known as an acetyltransferase, some HAT-independent functions have been reported. For example, KAT6A interacts with RUNX1 [34,35], leading to transcription of hematopoietic genes. KAT6A also interacts with RUNX2 and plays a role in osteogenesis [36,37] and these functions are independent of HAT activity. KAT6A also interacts with PU.1, important in the development of lymphoid and myeloid lineages, and with MLL, a histone methyl transferase that induces di-and tri-methylation of Histone H3 on lysine 4 [26]. ASH2L, another amplified and over expressed gene from the 8p11 amplicon has been shown to be the histone methylase in the MLL complex. The H3K4 di-and tri-methyl marks are associated with active gene expression, and KAT6A-MLL interactions have been associated with expression of homeobox genes in hematopoietic progenitor cells. Interestingly, two homeobox genes, HOXA11 and HOXA13 were regulated by KAT6A in SUM-52 cells. Thus, in breast cancer cells with the 8p11 amplicon, KAT6A may cooperate with other epigenome modifying oncogenes to aberrantly drive expression of a number of important cancer genes.
The significance of KAT6A in breast cancer is perhaps best reflected in the Kaplan-Meier plot shown in Figure 1F, which demonstrates a significant reduction in overall survival of patients with ER positive breast cancer and KAT6A amplification and overexpression. Not only is there a statistically significant divergence in mortality among patients with KAT6A overexpression, but this affect occurs following approximately 100 months of survival. This suggests that KAT6A plays a role in late recurrence of breast cancer, an issue of unique importance in ER-positive breast cancer. The results reported here suggest that amplification/overexpression of KAT6A could be a useful biomarker for predicting the risk of late recurrence, which could aid in the clinical decisions regarding the duration of anti-estrogen therapy. It is well-accepted that cancer is a disease driven by genetic abnormalities; however, it is becoming clear that alterations in epigenetic pathways play a role in oncogenesis. The identification of KAT6A as a driving oncogene in breast cancers with 8p11-p12 amplification is consistent with the emerging field of cancer epigenetics. Future studies will explore the role of KAT6A in breast cancer self-renewal and mechanisms governing clonogenicity. While there are other putative oncogenes in the amplicon, KAT6A has emerged as an oncogene that plays an important role in the selection of this amplicon in luminal breast cancers.


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Qiangzhi Decoction (羌跖汤) protects mice from influenza A pneumonia through inhibition of inflammatory cytokine storm

Objective To investigate the preventive effects of Qiangzhi Decoction (羌跖汤, QZD) on influenza A pneumonia through inhibition of inflammatory cytokine storm in vivo and in vitro. Methods One hundred ICR mice were randomly divided into the virus control, the Tamiflu control and the QZD high-, medium-, and low-dose groups. Mice were infected intranasally with influenza virus (H1N1) at 10 median lethal dose (LD50). QZD and Tamiflu were administered intragastrically twice daily from day 0 to day 7 after infection. The virus control group was treated with distilled water alone under the same condition. The number of surviving mice was recorded daily for 14 days after viral infection. The histological damage and viral replication and the expression of inflammatory cytokines were monitored. Additionally, the suppression capacity on the secretion of regulated on activation normal T cells expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α) in epithelial and macrophage cell-lines were evaluated. Results Compared with the virus control group, the survival rate of the QZD groups signifificantly improved in a dose-dependent manner (P<0.05), the viral titers in lung tissue was inhibited (P<0.05), and the production of inflammatory cytokines interferon-γ (IFN-γ), interleukin-6 (IL-6), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) were suppressed (P<0.05). Meanwhile, the secretion of RANTETS and TNF-α by epithelial and macrophage cell-lines was inhibited with the treatment of QZD respectively in vitro (p<0.05) Conclusions The preventive effects of QZD on influenza virus infection might be due to its unique cytokine inhibition mechanism. QZD may have significant therapeutic potential in combination with antiviral drugs.

from aqueous extract of four medicinal plants including Notopterygiumincisum Ting ex H. T. Chang, Isatisindigotica Fort. (IsatidisFolium ), Commelinacommunis L. and Isatisindigotica Fort. (Isatidis Radix), was developed by Dr. Yan De-xin, a famous Chinese medicine (CM) expert. The prescription has been employed in clinical use since 1960s for the treatment of exogenous fever and is well-received by patients in China and other Asian countries. Based on the rich practical experience in the treatment of epidemic diseases, the prescription had exerted signifi cant adjunctive effect for pneumonia induced by causing severe acute respiratory syndrome (SARS) in China in 2004. These results provided indirect evidence for QZD's application to anti-influenza infection. However, treatment and prevention of H1N1 infection with QZD has not been investigated. The major components of QZD have been studied for their biological effects as following. N. incisumis reported to exert inhibitory effects on coronavirus, herpetic and hepatitis B virus; (7)(8)(9) Isatidis Folium has been shown to have activity against herpes simplex virus type Ⅰ (HSV-Ⅰ); (10) and Commelinacommunis L. and Isatidis Radix have activity against influenza-virus in vitro. (11,12) However, these four Chinese herbs in combination has not been studied for anti-H1N1 virus activity. The innate immune system within the airway and lungs can respond with rapid identification and elimination of invading virus by local and systemic inflammation. (13) Therefore, modulation of the host immune response has a potential advantage of exerting less-selective pressure on viral populations. In order to understand the protective mechanism of QZD, we hypothesized that QZD would induce distinct innate immune responses, and modulate the balance between suppression and activation of inflammatory cytokines to alleviate the lung injury. Therefore, we investigated antiviral effect of QDZ in lethal infl uenza A infection model and its immunodulatory potential in vivo and in vitro.

Virus Strains
A mice-adapted strain A/FM/1/47 (H1N1) of influenza A virus, was obtained from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Beijing, China). Infl uenza virus A3/ Beijing/30/95 (H3N2) was kindly provided by Shanghai Municipal Center for Disease Control and Prevention (Shanghai, China). The virus strains were stored at -70 ℃. Viruses were thawed and freshly prepared for each experiment. (14)

Cell Cultures
Madin Darby Canine Kidney (MDCK) cells, Ana-1 cells (a murine macrophage cell line) and airway epithelial carcinoma A549 cells were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium (Gibco, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, USA), 100 U/mL of penicillin, 100 μg/mL streptomycin, 2 mmol/L L-glutamine and non-essential amino acids. The cells were maintained in a humidifi ed atmosphere containing 5% CO 2 at 37 ℃.

Laboratory Animals
Inbred ICR mice, 100 male and female, weighing 16-18 g were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China with Certificate No. SCXX(SH)2003-0003, and maintained at the Animal Center of Fudan University. The animals had free accesses to food pellets and tap water. All animals were used in accordance with the policies and guidelines of Ethic Committee for Animal Use in Fudan University, China.

Protective Effect of QZD in H1N1 Pneumonia Mice
Mice were randomly divided into the control and QZD treatment groups (12 mice per group, 6 males and 6 females). After the mice were anesthetized with ether, all mice were infected with H1N1 virus intranasally at 10 median lethal dose (LD 50 ) dosage in a volume of 30 μL per mouse. The initial drug treatment was at 2 h post virus challenge; QZD at dosages of 960, 320 or 107 mg/(kg • d) and Tamiflu at dose of 24 mg/(kg • d) were gavaged twice daily for 7 days. Mice in the normal control group were given saline alone under the same conditions. The survival of mice was monitored until day 14 post virus infection.

Pathological Damages of Lung Tissue in H1N1 Infected Mice
For the acute tissue damage, mice were randomly divided into the control and the QZD treatment groups (8 mice per group, 4 males and 4 females). Mice, anesthetized with ether, were infected with H1N1 virus intranasally at 10 LD 50 dosage (30 μL per mouse). QZD and Tamiflu were intragastrically administered to the mice twice daily for 4 days. The mice from each group were euthanized at day 4, and lungs were collected. Lung tissues were fi xed in formaldehyde for 24 h, embedded in paraffin, sliced into 5-μm sections, and stained with haematoxylin and eosin (HE). Histopathologic changes of the lungs from each mouse were examined by two blinded observers to determine the number of focal lesions and lung consolidation microscopically. Consolidation was scored using the following assigned values: 0 = no consolidation; 1+ = mild consolidation encompassing <25% of the lung; 2+ = moderate consolidation (25%-50% of lung); 3+ = extensive and dense consolidation (>50% of lung) as previous report. (10)

Assay for Pulmonary Viral Titer
Viral load was presented as hemagglutination (HA) titer, similarly to a previous report. (7) Briefl y, each lung was homogenized to a 10% (w/v) suspension with phosphate buffered saline (PBS), and the homogenates were serially diluted 2-fold. Fifty microliters of two-fold serial dilutions of the homogenate was added into each well of V-bottom microplate, and 50 μL of 1% chicken red blood cells suspension in PBS was added to each well. The suspensions were mixed and incubated at room temperature for 30 min. The final dilution of homogenates that agglutinates red blood cells was considered the end point of the titration and the HA titer was calculated.

Antiviral Effect of QZD In Vitro
The antiviral effect of QZD on infl uenza virus was measured by cytopathic effect (CPE) assay. MDCK cells were seeded into 96-well culture plates at a density of 1×10 4 cells per well and incubated for 24 h for until 90% confl uency. Monolayer MDCK cells were washed twice with serum-free 1640 culture medium, and then infected with influenza virus H3N2 at 100 tissue culture infectious dose 50% (TCID 50 ), and dissolved in cooled serum-free 1640 culture medium containing 0.025% trypsin. After incubation at 37 ℃ in 5% CO 2 for 1.5 h, uninfected virus was removed by pipetting. Samples of QZD (below the limit of toxic concentration) were added into the 96-well plates for further 72 h incubation. Cell viability was observed by a modifi ed MTT assay.

Effect of QZD on Chemokine
A549 cells were seeded into 6-well culture plates at a density of 1×10 5 cells per well and incubated for 24 h for until 90% confluency. Then the A549 cells were infected with influenza virus H3N2 at TCID 50 . After incubation for 1.5 h with the virus, QZD dilutions were added into the 6-well plates for 24 h. Culture supernatants were collected for the RANTES ELISA assay.

Effect on Cell Proliferation and Cytokine Production
Ana-1 cells were seeded into 6-well culture plates at a density of 1×10 6 cells per well and incubated for 24 h until 90% confl uency. Then the cells were infected with infl uenza virus H3N2 at 10 TCID 50 . After 24 h of infection, cell viability was assayed and the inhibitory effect of QZD on cell proliferation after infection was evaluated. On the other plate, the Ana-1 cells were infected with infl uenza virus H3N2 at TCID 50 . After incubation for 1.5 h, the uninfected virus was removed and QZD dilutions were added for further incubation. Culture supernatants were collected at 6 and 12 h after the infection. The levels of TNF-α were determined by an ELISA method.

Statistics
Data was analyzed with Stat View ver. 5.01 SAS Institute Inc and SPSS 11.5 software package (SPSS Inc, Chicago, IL). All experimental values obtained from above assays were expressed as mean ± standard deviation, and examined by the two-sample test or one-way analysis of variance (Dunnett's t test). A probability of less than 0.05 was considered to be statistically signifi cant.

Protective Effects of QZD in vivo
Mice infected intranasally with infl uenza virus showed signs of piloerection, lethargy, and reduced food intake 3 days after infection. The infection was lethal in several animals by day 4. After day 11, no additional animals died, and the survival rate (survival/total) remained stable in each group. No mice survived in the control group, or in the QZD 107 mg/(kg • d) group. In contrast, after treatment with QZD 320 and 960 mg/(kg • d), the survival rate of mice on day 14 was 41.7% (5/12, P<0.05) and 66.7% (8/12, P<0.01), respectively ( Figure 1). The mean survival days (MSD) of the control mice was 5.39±0.98 days. Administration of QZD at 107, 320, and 960 mg/(kg • d) increased the MSD of mice to 6.50±1.93 days, 9.91±3.96 days (P<0.01) and 12.4±2.57 days (P<0.01), respectively. The MSD increased by 20.6%, 83.8% and 130%, respectively, compared with the control group. Additionally, there was a clear correlation between treatment with QZD and subsequent survival in mice (r=0.93, P<0.01).

QZD Protected the Pulmonary Damages
Viral pneumonia was the main pathological damage in the mice infected with influenza virus H1N1, which was characterized by interstitial pneumonia. The pathological damages were observed on the day 4 after virus inoculation. Most infected mice showed severe infiltration of monocytes and lymphocytes, thickened alveolar walls, and exudation of infl ammatory cells into the alveolar space, whereas treatment with QZD ameliorated the pathological injury in a dose-dependent manner ( Figure 2). QZD at the dose of 960 mg/(kg • d) significantly decreased the number of influenza-related focal lesions and lung consolidation (P<0.05, Figure 3). In addition, the infiltration of monocytes and lymphocytes were signifi cantly reduced.

QZD Inhibited HA Titers
The pulmonary viral HA titer in mice treated with QZD of 960, 320, 107 mg/(kg • d) was reduced to 68.75%, 33.3% and 25%, respectively, as compared with the infected control ( Figure 4). The HA titer in the lungs in QZD treated mice [960 mg/(kg • d)] were significantly lower than that in the control mice (P<0.05).

QZD Suppressed Cytokine/Chemokine Production
Influenza virus infection induces a robust inflammatory reaction, hallmarked by the production of the antiviral cytokines typeⅠand typeⅡIFN. (15)(16)(17) To determine the immune regulatory effect of QZD, serum and lungs were collected at day 4 post-Virus control Tamifl u Pathological index score infection and cytokines were measured. Treatment with QZD [960mg/(kg • d)] showed a significant inhibition of IFN-γ, TNF-α and IL-6, as well as ICAM-1 and RANTETS production compared with saline controls from influenza virus-infected mice ( Figure 5). Importantly, QZD-dependent downregulation of the production of cytokine/chemokine/ ICAM-1 by innate immune cells, including epithelial cells and macrophages, did not increase viral burden. This demonstrates that QZD enhanced protection without altering the host's ability to control infection. Quantitative analysis showed that QZD treatment reduced cytokines in a concentration-dependent manner.

CPE Induced by H3N2 Infl uenza Virus Was Not Inhibited by QZD
Microscopic examination showed that the virus-infected control MDCK cells were nearly destroyed by infl uenza virus 3 days after incubation. Treatment with QZD did not exert significant antiviral activity in infected MDCK cells (data not shown).

QZD Increased the Cell Viabilty under Virus Infection
Microscopic examination showed that the proliferation of macrophage Ana-1 cells was signifi cantly The results revealed that the forty-five percent of cells were killed after H3N2 infection with a 10TCID 50 virus titer. However, QZD at 30 and 15 μg/mL significantly increased cell viability to 95% and 82% of the control cells, respectively ( Figure 6).

Figure 6. Effect of QZD on Cell Viability of Ana-1 Cell Line by MTT at 24 h after H3N2 Infection
Notes: P<0.05, P<0.01, compared with the cells infected with H3N2

DISCUSSION
In our preliminary study, the toxicity of QZD was evaluated in ICR mice. According to the commendatory dose [120 mg/(kg • d)] of QZD for adults, three-fold and eight-fold doses were tested. The results showed that administration of 960 mg/(kg • d) had no toxic effects on mice (data not shown). Therefore, we chose three lower doses for the current experiments.
H1N1-infected mice are a common experimental model for acute viral pneumonia. In such model, mice with viral infection showed various symptoms, from morbidity to death at different times. Out results showed that mice treated with QZD 960 mg/(kg • d) had a significant improvement in the overall survival rate, reduction of adverse pathological changes and inhibition of virus titer in pulmonary homogenates. Tamiflu administration was found to exert a much better inhibitory effect on the virus titer in the lungs than QZD. However, QZD could significantly reduce the cytokine/chemokine/ICAM-1 production in the infected mice.
In order to provide further evidence for the suppression of infl ammatory cytokines, we used various cell models for evaluation the immune modulation with QZD treatment. While QZD had no antiviral activity in MDCK cells, it reduced mouse macrophage apoptosis, and suppressed the release of TNF-α and RANTES from macrophage and endothelial cells after infl uenza virus infection. Combined with the data in vitro, our In the early lethal influenza virus infection, the prognosis of viral pneumonia depends on the innate immune response, in which cytokines and other inflammatory mediators are released from immune cells. Mice infected with influenza virus produce cytokine and chemokine responses, including IL-1, IL-1α, IL-6, IFN-γ, TNF-α, IL-8, macrophage inflammatory protein (MIP)-1α, RANTES, and ICAM-1 at days 3-5 post-infection. (9,18,19) The interplay of IL-1β, TNF-α, IFN-α/β, IL-18, IFN-γ, and chemokines forms a complex positive feedback network leading to inflammatory response and the development of influenza-specific Th1 response. (20) TNF-α and IL-6 possess multifunctional activities, and are associated with morbidity during influenza virus infection. Chemokines, such as RANTES, induce the recruitment of innate immune cells into the lung, which can release more cytokines exacerbating cytokine storm and further damage the lung. (21) This study showed that the influenza virus transiently altered endothelial function expressed as ICAM-1. Endothelial dysfunction may be explained by the expression on the endothelial surface of adhesion molecules, including ICAM-1, and promote the adherence of leukocytes with the consequent initiation of vascular damage. (19) RANTES, a member of the C-C chemokine family, is a chemoattractant for monocytes, T lymphocytes, basophils, and eosinophils, and expressed by endothelial cells and T lymphocytes. It has been found in nasal secretions of patients suffering from upper respiratory tract infection with infl uenza virus, parainfl uenza virus and adenovirus. (22) RANTES may contribute to the accumulation of these inflammatory cells into the epithelium during viral infection.
We found that QZD exerted no antiviral activity in vitro, but robustly inhibited the expression of viral protein HA in the lung of mice. The signifi cant antiviral effi ciency in vivo may due to suppression of cytokine/ chemokine/ICAM. Immune response is a doubleedged sword. The immune response presents a powerful barrier against virus and can target virusinfected cells. However, the uncontrolled immune response may cause more damage to host cells than could be attributed to the replication of the virus alone. (23,24) Excessive infl ammatory cytokine released from immune cells leads to lung injury and dysfunction of respiratory system. Administration of QZD signifi cantly dampened interferon, cytokine, chemokine and ICAM-1 release into the bronchoalveolar lavage, and resulted in a signifi cant decrease in infi ltration of monocytes, macrophages, neutrophils in the lung. In addition, we found that QZD had the ability to inhibit RANTES release by H3N2 infected A549 cells. As a result of no obvious cytopathic change in A549 cells 48 h after virus inoculation, the suppression of RANTES by QZD might not contribute to inhibition of virus replication. Influenza virus-infected monocytes and macrophages responded with a cell-specific response involving cytokines (TNF-α, IL-1, IL-6, and IFN-α/β). (25) We found that with the higher virus titer, QZD at the concentration of 30 μg/mL inhibited cell apoptosis, whereas with the lower virus titer, QZD reduced TNF-α in Ana-1 cells.
As a result, pathological changes in the lung were reduced through inhibition of this cytokine storm. It has been confirmed that N. forbesii and I. indigotica have anti-infl ammatory activity to reduce cell damage caused by virus, lipopolysaccharide, or radiation. (25)(26)(27) QZD, which contains these herb components, may exert multiple anti-inflammation activities for indirect modulation of virus replication.
In conclusion, the results presented here clearly demonstrate that QZD has a promising anti-viral effect against infl uenza virus infection, leading to improved survival rate, attenuation of lung lesions, reduction of virus HA titers in the lung of infected mice, and attenuation of the cytokine network after viral infection. However, further understanding to cellular signaling pathways and cytokine storm would provide insight into infl uenza pathogenesis, and provide the potential application for CM in infectious disease.