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Quaternary structure predictions of transmembrane proteins starting from the monomer: a docking-based approach

Background We introduce a computational protocol for effective predictions of the supramolecular organization of integral transmembrane proteins, starting from the monomer. Despite the demonstrated constitutive and functional importance of supramolecular assemblies of transmembrane subunits or proteins, effective tools for structure predictions of such assemblies are still lacking. Our computational approach consists in rigid-body docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Prediction of the native oligomer is therefore accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis. With this approach, knowledge about the oligomerization status of the protein is required neither for improving sampling nor for the filtering step. Furthermore, there are no size-limitations in the systems under study, which are not limited to the transmembrane domains but include also the water-soluble portions. Results Benchmarks of the approach were done on ten homo-oligomeric membrane proteins with known quaternary structure. For all these systems, predictions led to native-like quaternary structures, i.e. with Cα-RMSDs lower than 2.5 Å from the native oligomer, regardless of the resolution of the structural models. Conclusion Collectively, the results of this study emphasize the effectiveness of the prediction protocol that will be extensively challenged in quaternary structure predictions of other integral membrane proteins.

Background
A number of α-helical transmembrane (TM) proteins organize themselves in supramolecular assemblies, which constitute the functional units (i.e. biological units) of ion/water channels, electron or proton transfer proteins as well as transporters [1]. Also the 7-TM bacteriorhodopsin (BRD) forms trimers, which, in turn, form multimolecular assemblies in the native purple membrane [2,3]. G-pro-tein-coupled receptors (GPCRs), which constitute the largest superfamily of membrane proteins, are not exceptions to this rule. Although they have been classically assumed to exist and function as monomeric entities, the concept that they exist as constitutive dimers/oligomers is now substantiated by ever increasing evidences from in vitro studies (reviewed in refs. [4][5][6][7]). Predictions of the likely interfaces in GPCR dimers done so far essentially relied on sequence-based methods [8][9][10][11][12][13] (reviewed also in Ref [14]).
Despite the demonstrated constitutive and functional importance of supramolecular assemblies of TM subunits or proteins, effective tools other than sequence-based methods for structure predictions of such assemblies are still lacking. Indeed, almost all the docking algorithms and approaches to quaternary structure predictions developed so far work with water-soluble proteins and most of them employ geometrical constraints, including symmetry information [15][16][17][18][19][20]. The very few approaches intended for quaternary structure predictions of TM-helical proteins include a method based on Monte Carlo sampling of single TM helices [21]. The method is limited to very simple homo-oligomers and requires knowledge about the oligomerization state of the protein, since symmetry information is used to filter the most realistic solutions. The information from cryo-electron microscopy and evolutionary data is employed by an alternative automated method for orienting TM helices [22]. The method has been probed on a number of membrane proteins, including the multimeric TM portion of the acetylcholine receptor [22]. Another docking method, i.e. GRAMM [23], can deal with predictions of helix-helix packing interactions involving either water-soluble or TM-systems. In fact, GRAMM can run in a "helix docking mode" that discards configurations with large displacements along the helix axes and angles between helices larger than indicated cutoffs [24]. This is instrumental in saving computational time and facilitating the analysis of the docking results.
Herein, we present an approach based upon rigid body docking simulations, membrane topology-based filtering, and cluster analysis to predict the quaternary structure of homo-oligomeric integral membrane proteins. This approach does not employ symmetry constraints either for improving sampling or in the filtering step. Furthermore, there are no size-limitations in the systems under study, which are not limited to the TM domains but include also the water-soluble portions.
Benchmarks of the approach were first carried out on the tetrameric KcsA potassium channel H + gated (384 amino acids, PDB code: 1BL8; Figure 2) [25], pentameric MscL and eptameric MscS mechanosensitive channels (540 amino acids, PDB code: 1MSL, Figure 3 [26], and 1771 amino acids, PDB code: 1MXM, Figure 4) [27], respectively), and trimeric BRD (698 amino acids, PDB code: 1BRR; Figure 5) [2]. Selection of these supramolecular systems followed an accurate inspection of the database of Flowchart of the docking-based stepwise oligomerization approach Figure 1 Flowchart of the docking-based stepwise oligomerization approach. The quaternary structure prediction approach consists in a number of dense docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Thus, prediction of the native oligomer is accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis. For each stepwise quaternary structure prediction, the docking runs that succeeded the first one were carried out by using the original monomer as a probe and the intermediate oligomer as a target. The selected proteins, indeed, fulfilled at best the following requirements: (a) the biological unit is a homo-oligomer; (b) the asymmetric crystallographic unit contains the biological unit; (c) the monomers in the biological unit are α-helical TM proteins; and (d) significant structural diversity exists among the selected oligomers concerning oligomeric order, architecture and extension of Native and native-like structures of tetrameric KcsA Figure 2 Native and native-like structures of tetrameric KcsA. (a) View of the crystal structure seen from the extracellular side; the monomers are differently colored. (b) and (c) The superimposition between native (green color) and the best native-like (violet color) structures is shown. The native-like structure shown in this figure, i.e. the A-Bs1-Cs500-Ds2 tetramer, has been achieved through a dipole moment-based reorientation approach (see Table 1 and Figure 6). In panel (b) the superimposed structures are seen from the extracellular side, whereas in panel (c) the structures are seen in a direction parallel to the membrane surface. Drawings were done by means of the software PYMOL 0.98 [39].

Docking-based stepwise oligomerization
Native and best native-like structures of pentameric MscL Figure 3 Native and best native-like structures of pentameric MscL. The best predicted native-like pentamer (violet color) is the one encoded as A-Bs8-Cs1-Ds1-Es6 (see Table 1 and Figure 7). The description of this figure is like that of Figure 2.
The structure of the biological unit from the PDB, obtained by symmetry operations, was used as a native complex for each of these proteins.
For all the systems under study, the approach led to native-like quaternary structures, i.e. with a Root Mean Native and best native-like structures of eptameric MscS Figure 4 Native and best native-like structures of eptameric MscS. The best predicted native-like eptamer (violet color) is the one encoded as A-Bs3-Cs1-Ds1-Es6-Fs1-Gs1 (see Table 1 and Figure 8). The description of this figure is like that of Figure 2.
Native and native-like structures of trimeric BRD Figure 5 Native and native-like structures of trimeric BRD. The predicted native-like eptamer (violet color) is the one encoded as A-Bs162-Cs14 (see Table 1 and Figure 9). The description of this figure is like that of Figure 2.
Square Deviation of the C α -atoms (Cα-RMSD) lower than 2.5 Å from the native oligomer, regardless of the resolution of the structural models.
The effectiveness of the approach makes it suitable for predictions of the supramolecular architecture of other integral membrane proteins.

Results
The computational approach developed in this study consists of a number of dense docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Thus, prediction of the native oligomer is accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis (schematized in Figure 1)).
The only requirement with this approach is the structural model of the monomer and the knowledge of a set of C αatoms, which lie at the two lipid/water interfaces, defining two parallel planes. In the target monomer, these two planes must be parallel to the xy plane and, hence, perpendicular to the z-axis. If these planes are parallel to the xy plane and, hence, perpendicular to the z-axis, the orientation of the monomer is considered good and no reorientation is needed. In contrast, if such planes are not parallel to the xy plane, the monomer needs a reorientation. This reorientation is done by computing the rotations necessary to bring the two planes parallel to the xy plane and then using an average between the two rotations to reorient the whole monomer. The correct membrane topology of the target monomer is, indeed, necessary for the membrane topology filter to work properly (see Methods).
The following paragraphs summarize the results of benchmarks done on ten selected oligomers.

Quaternary structure predictions of oligomeric channels: KcsA, MscL and MscS
The A subunit of KcsA is constituted by 96 amino acids (i.e. 23-119 sequence) organized in two TM α-helices connected by the 30 amino acid pore region [25]. The A subunit extracted from the crystal structure was used both as a target and a probe in the first step, and as a probe in the following docking simulations. Prior to the first docking run, a reorientation of the KcsA A subunit was needed to put it in the right orientation with respect to the putative membrane. Two different ways for reorienting the subunit were probed, i.e. a dipole moment-based approach and a membrane topology-based approach. The first consisted in rotating the native oligomer so as to put the dipole moment of the tetramer parallel to the z-axis and then extracting the A subunit. The second consisted in the following steps: (a) prediction of two sets of C α -atoms of the monomer, which lie at the two membrane-water interfaces; (b) determination of the planes that fit at best these sets of atoms; and (c) orientation of these planes parallel to the xy plane. The first reorientation approach, applied to the native oligomer, was instrumental in designing and benchmarking the prediction protocol, whereas the second approach, applied to the monomer, is instrumental in blind predictions.
Quaternary structure predictions of KcsA consisted of two alternative two-step growing paths differing for the fact that one employed a trimer (Figure 6a), whereas the other employed a dimer (Figure 6b and 6c) as a target at step 2.
The two different paths shared in common step 1, i.e. A vs A docking. A vs A docking gave 231 realistic solutions, i.e. those solutions, which passed the membrane topology filter. These solutions essentially grouped into two clusters, which also showed similar indices of membrane topology goodness (i.e. MemTop indices of 0.495 and 0.494; see the Methods section for the MemTop definition). The best scored solutions from each of these two clusters, s2 and s1 (the letter "s" followed by a number indicates the solution number, or rank number, in the ZDOCK output list), were selected for the following growing step. These solutions contributed to the growth of a cyclic oligomer, likely to represent subunits D and B, respectively, i.e. labeled in a clockwise manner as seen from the extracellular side (Figure 6a). As for the path passing through an intermediate trimer, A vs A-Bs1-Ds2 (the upper case letter indicates the subunit) docking led to 41 realistic solutions, which essentially grouped in four clusters. The best scored solution from the third cluster (characterized by the best MemTop score: 0.375), s500, was selected to produce the A-Bs1-Cs500-Ds2 tetramer. Such tetramer was characterized by a C α -RMSD of 1.79 Å from the native structure (Figures 2b, 2c and 6a, Table 1).
The alternative path passing through an intermediate dimer consisted of two parallel ways, depending on whether the A-Bs1 or the A-Ds2 dimer was employed as a target (Figure 6b and 6c, respectively). In the first case, 242 realistic solutions were filtered, which essentially grouped in two clusters. The best solutions from each of these two clusters, s2 and s1, were, respectively, characterized by contacts with the A and Bs1 monomers in the target dimer, leading to the A-Bs1-Cs1-Ds2 tetramer, which showed a C α -RMSD of 2.45 Å from the native structure ( Figure 6b, Table 1). The parallel pathway, which used the A-Ds2 dimer as a target at step 2, led to 199 realistic solutions, which essentially grouped in two clusters characterized also by similar MemTop indices (i.e. 0.567 and 0.490). The best solutions from each of these two clusters, s2 and s1, led to the A-Bs2-Cs1-Ds2 tetramer, showing a C α -RMSD of 2.27 Å from the native structure ( Figure 6c, Table 1).
In summary, the three different growing paths shared in common the first step, i.e. A vs A docking, and all led to native-like tetramers ( Figure 6). Among these tetramers, the one obtained by employing a trimer as a target at step 2 showed the lowest C α -RMSD from the native complex (i.e. 1.79 Å, Table 1 and Figures 2b, 2c and 2a). The intermediate and final oligomers, with only one exception, were made of solutions falling amongst the top four out of 4000 in the respective output list ( Figure 6). The results presented above were achieved by employing a dipole moment-based criterion for the initial reorientation of the target A subunit. However, employing a membrane topology-based approach for subunit reorientation equally led to native-like solutions. In fact, the path employing a trimer as a target at step 2 led to the A-Bs2-Cs12-Ds4 tetramer, showing a C α -RMSD of 0.94 Å from the native structure, whereas one of the two paths employing a dimer as a target at step 2 led to the A-Bs1-Cs52-Ds4 Prediction paths for KcsA Figure 6 Prediction paths for KcsA. Each of the three different growing paths ((a), (b) and (c)) is characterized by selection, at each growing step, of the best scored solution within the most populated cluster/s, characterized also by similar and significantly low MemTop index. The number of solutions filtered at each step is reported under the arrow. The circle on the arrow indicates the probe, whereas the circles that precede the arrow are the targets. The monomers that constitute these targets are indicated by gray circles except for the last added monomer/s, which are indicated by white circle/s and by the solution number in the ZDOCK output list. The final oligomer is indicated by a string of letters and characters in a way that each subunit is associated with the docking solution. In detail, the upper case letter indicates the subunit, whereas the letter "s" followed by a number indicates the solution number in the ZDOCK output list. Finally, the C α -RMSD (Å) between native and predicted quaternary structures is also reported. All the amino acid residues have been included in C α -RMSD calculations. The same general approaches were employed for quaternary structure predictions of the the pentameric MscL and eptameric MscS mechanosensitive channels, but neither one of the two systems required an initial reorientation of the monomer. In detail, the A subunit of the MscL is constituted by 108 amino acids (i.e. 10-118 sequence), organized in two TM α-helices and a third cytoplasmic helix ( Figure 3) [26]. The A subunit of MscS is constituted by 253 amino acids (i.e. 27-280 sequence), organized in three TM α-helices and a huge cytosolic domain (i.e. 113-280 sequence, Figure 4) [27].
For MscL, two-step and three-step growing paths were probed, differing for the fact that the first path utilized a trimer (Figure 7a), whereas the second one utilized a dimer (Figure 7b and 7c) a Abbreviated name of the membrane proteins subjected to the benchmarks. Asterisks indicate the systems, for which the asymmetric crystallographic unit contains the biological unit. b PDB code of the membrane proteins subjected to the benchmarks. c Atomic resolution (Å) of the crystallographic structures. d Oligomeric order of the biological units. e Probed growing paths (see the text and Figures 6-9 for a detailed explanation). For each protein system, the path that produced the best oligomer, in terms of C α -RMSD from the native assembly, is highlighted in bold. f Predicted oligomers: the upper case letter indicates the subunit, whereas the letter "s" followed by a number indicates the solution number in the ZDOCK output list. The best oligomer, in terms of C α -RMSD from the native assembly, is highlighted in bold. g The predicted BtuCD dimer was achieved following a dipole-moment-based approach for A subunit reorientation. The same path, but using a membrane topology-based approach for A subunit reorientation, led to the A-Bs1584 dimer characterized by a C α -RMSD of 0.48 Å from the native dimer. h The best predicted KcsA tetramer was achieved following a dipole-moment-based approach for A subunit reorientation. The same path, but using a membrane topology-based approach for A subunit reorientation, led to the A-Bs2-Cs12-Ds4 tetramer characterized by a C α -RMSD of 0.94 Å from the native oligomer. i C α -RMSD (Å) between the native and the predicted oligomer from each growing path. For each system, the lowest C α -RMSD is highlighted in bold.
ure 7a, bold labels). A vs A-Bs5-Es6 docking led to 121 realistic solutions, which essentially grouped in the first two clusters. The best scored solutions from each of these clusters, s1 and s4, were characterized by contacts with the Bs5 and Es6 monomers, respectively, closing the oligomeric cycle. The A-Bs5-Cs1-Ds4-Es6 pentamer showed a C α -RMSD of 3.45 Å from the native structure ( Figure 7a, Table 1).
As for the three-step paths, the best scored solutions from the two most populated clusters, s5 and s6, were used to produce the A-Bs5 and A-Es6 dimers. Each of these two dimers was used in turn as a target in two parallel sets of docking simulations, which finally led to the A-Bs5-Cs3-Ds2-Es1 and A-Bs8-Cs1-Ds1-Es6 pentamers, characterized by almost comparable C α -RMSDs, i.e. 2.83 Å and 2.47 Å, respectively, from the native oligomer (Figure 7b and 7c, Table 1). Thus, employing a dimer as a target at step 2 was better than using a trimer (Figure 7). Also, all the predicted pentamers were made of solutions falling amongst the top ten out of 4000 in the respective output list ( Figure  7, Table 1).
For quaternary structure predictions of MscS, three-step and four-step growing paths were probed, differing for the fact that the first utilized a trimer (Figure 8a), whereas the second utilized a dimer (Figure 8b and 8c) as a target at step 2. Also in this case, the two different growing paths shared in common the first step, i.e. A vs A docking. Thus, A vs A docking gave 111 realistic solutions, essentially grouped in two clusters showing also significantly low MemTop indices (i.e. 0.258 and 0.312).
As for the three-step growing path, the first run was instrumental in obtaining the A-Bs3-Gs1 trimer by using the best scored solutions from the first two most populated clusters, s3 and s1 (Figure 8a). A vs A-Bs3-Gs1 docking led to 79 realistic solutions, which essentially grouped in the first two clusters. The best scored solutions from these clusters, s1 and s3, were, respectively, characterized by contacts with the Gs1 and Bs3 monomers, likely to represent the F and C subunits. these clusters, s1 and s3, were, respectively, characterized by contacts with the Fs1 and Cs3 monomers and completed the oligomeric cycle, likely to be the E and D subunits, respectively. The thereof obtained A-Bs3-Cs3-Ds3-Es1-Fs1-Gs1 eptamer showed a C α -RMSD of 2.21 Å from the native oligomer ( Figure 8a, Table 1).
As for the four-step paths, the best scored solutions from the first two most populated clusters, s3 and s1, were used to produce the A-Bs3 and A-Gs1 dimers. These dimers were used in turn as a target in two parallel sets of docking simulations, which finally led to the A-Bs3-Cs3-Ds1-Es165-Fs3-Gs1 and A-Bs3-Cs1-Ds1-Es6-Fs1-Gs1 eptamers, characterized by C α -RMSD of 11.30 Å and 2.06 Å, respectively, from the native oligomer (Figure 8b and 8c, Table 1). In summary, the solutions constituting the three alternative eptamers, which were obtained following different paths, fell amongst the top ten out of 4000 in the respective output list (Table 1 and Figure 8). Also, one of the two four-step growing paths produced the eptamer with the lowest C α -RMSD from the native oligomer (i.e. 2.06 Å, Table 1 and 4b, 4c and 8c), whereas the other fourstep growing path gave the worst eptamer in terms of C α -RMSD (i.e. 11.30 Å, Figure 8b). Since the two predicted quaternary structures are significantly different from each other (i.e. the C α -RMSD between them was 10.88 Å), and contain large water-soluble domains, they were subjected to a quality check by means of the 3D-Profile program [33], to help selection of the best one. Interestingly, for the native-like A-Bs3-Cs1-Ds1-Es6-Fs1-Gs1 eptamer, the 3D-Profile score was higher than that of the alternative eptamer (i.e. 438.0 vs 392.4, respectively), quite indicative of a more suitable packing of the seven subunits.

Quaternary structure predictions of BRD
For BRD, A vs A docking, followed by the application of the membrane topology filter, led to 77 solutions ( Figure  9). These solutions essentially grouped in two clusters. One of these two clusters showed a better MemTop index than the other cluster (i.e. 0.537 vs 0.732, respectively) and was, thus, used to select the best scored solution, leading to the A-Bs162 dimer. A vs A-Bs162 docking led to 45 solutions, grouped in seven clusters. The solution selected based upon the criteria described above, s14, led to the A-Bs162-Cs14 trimer, characterized by a C α -RMSD of 1.04 Å from the native trimer (Table 1 and Figures 5b, 5c and 9 top). This is a striking result, considering also that the lipid molecules, which are present at the interfaces between the monomers in the crystal structure of the trimer, were completely neglected in docking simulations. Furthermore, the predicted trimer is made of identical monomers, whereas, in the crystal structure of the oligomer, slight differences exist between the monomers, concerning their internal coordinates and the main-chain length.
We probed also a different crystal structure of BRD, 1KME [34]. This structure differs from the one extracted from the native oligomer both in the backbone (i.e. 1.0 Å C α -RMSD in the 5-231 sequence, and 1.5 Å C α -RMSD in the intrac- Figure 8 Prediction paths for MscS. See Figure 6 for the description of this figure. Prediction path for BRD (top) and for AmtB and AcrB (bottom). The description of this figure is the same as that in Figure 6.

Prediction paths for MscS
For BRD, only one growing path has been pursued. In contrast, for quaternary structure predictions of AmtB and AcrB, the growing paths (a), (b) and (c) were probed. Black bold labels refer to AmtB, whereas gray bold labels refer to AcrB predictions.

(b)
Quaternary structure predictions of AmtB and AcrB A-Bs1-Cs1: RMSD = 0.75 Å A-Bs1-Cs1: RMSD = 1.64 Å ellular and extracellular domains) and side-chain conformations. Following exactly the same path and approach as those used for the monomer extracted from the native trimer, a trimer was achieved characterized by a C α -RMSD of 4.0 Å from the native oligomer. It is worth noting that such C α -RMSD value comprises also the structural differences between the monomers in the native and predicted trimers.

Quaternary structure predictions of the set of membrane proteins for which the asymmetric unit did not correspond to the biological unit
The subset of membrane proteins, KcsA, MscL and MscS and BRD, for which the asymmetric units corresponded to the biological unit, were employed for setting the prediction protocol. Benchmarks were then extended to a number of homo-oligomers, for which the asymmetric unit did not contain the biologic unit that was, hence, obtained by symmetry operations. These oligomers included: the tetrameric AQP1, GlpF and KirBac1, the trimeric AmtB and AcrB, as well as the dimeric BtuCD.
Differently from BRD, for AmtB and AcrB, the MemTop indices of the two most populated clusters from the first docking step were similar. This made it possible to achieve trimers following either path (a) or (b) or (c) (Figure 9 bottom). In detail, for AmtB, A vs A docking led to 38 solutions essentially grouped in two equi-populated clusters showing also good and similar MemTop indices (i.e. 0.317 and 0.319). The best scored solutions from these two clusters, s1 and s7, were likely to form the C and B subunits, respectively. The A-Bs7-Cs1 trimer was characterized by a C α -RMSD of 1.17 Å from the native trimer. A vs A-Bs7 and A vs A-Cs1 docking, led to 53 and 62 reliable solutions, respectively, which essentially grouped in one cluster. The best solution from this cluster, s1, in both cases, led to the A-Bs7-Cs1 and A-Bs1-Cs1 native-like trimers characterized, respectively, by C α -RMSDs of 0.83 Å and 0.75 Å from the native trimer (Table 1 and Figures 9 and  10d). For AcrB, A vs A docking led to 123 solutions, essentially grouped in two clusters showing low and similar MemTop indices (i.e. 0.220 and 0.135). The best scored solutions from these two clusters led to the A-Bs4-Cs1 trimer showing a C α -RMSD equal to 2.78 Å from the native trimer. A vs A-Bs4 and A vs A-Cs1 docking, led to 48 and 26 reliable solutions, respectively, which essentially grouped in one cluster. The best solution from this cluster, s1 in both cases, led to the A-Bs4-Cs1 and A-Bs1-Cs1 native-like trimers characterized, respectively, by C α -RMSDs of 1.56 Å and 1.64 Å from the native trimer (Table  1 and Figures 9 and 10e).
For BtuCD, whose membrane-spanning domain is a dimer, A vs A docking led to 47 solutions distributed in 11 small clusters. Since the cluster population in this case is meaningless, we considered only the cluster characterized by the best MemTop index (i.e.0.384). The lowest scored solution from this cluster, s178, led to a dimer characterized by a C α -RMSD of 0.63 Å from the native complex (Table 1 and Figure 10f). These results were achieved following a dipole moment-based approach for subunit reorientation (see the Experimental Procedure section). The membrane topology-based reorientation approach, led to 48 reliable solutions, 30 of which were divided into 7 clusters, whereas the remaining 18 couldn't be clusterized. The 25 unique solutions contained the native-like dimer, characterized by one of the best MemTop index and a C α -RMSD of 0.48 Å from the native complex. However, in this case, retrieving the native-like solution couldn't be unequivocally done, as it didn't hold either the best ZDOCK score or the best MemTop index.

Discussion
Following benchmarks, we have defined an effective computational protocol to predict the supramolecular structure of integral α-helical TM proteins, starting from the monomer. The approach was first tested on the trimeric BRD, tetrameric KcsA, pentameric MscL and eptameric MscS. These systems were selected following an accurate database search for high resolution structures of oligomeric membrane proteins, significantly different from each other and whose asymmetric crystallographic unit contains the biological unit. Docking samplings on these proteins were instrumental in setting the structure prediction protocol. Benchmarks were then extended to a number of homo-oligomers, for which the asymmetric unit did not contain the biologic unit that was, hence, obtained by symmetry operations. These oligomers included: the dimeric BtuCD, the trimeric AmtB and AcrB, and the tetrameric AQP1, GlpF and KirBac1.
For all the considered systems, native-like quaternary structures were achieved regardless of the resolution of the structural models and the extensions of the TM and waterexposed domains (Figures 2, 3, 4, 5, 6, 7, 8, 9, 10 and Table 1), thus, proving the effectiveness of the prediction approach. The latter consists in a number of dense docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Thus, prediction of the native oligomer is accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis (Figure 1).
The only requirement with this approach is the structural model of the monomer that must hold the proper membrane topology. The correct membrane topology of the monomer, which in the first docking run of each growing path is employed in two identical copies (one for the target and the other for the probe), is, indeed, necessary for the membrane topology filter to work properly. This filter generally discards more than 94% of the total solutions provided by each docking run (i.e. 4000 solutions), thus allowing for an easy individuation of the clusters holding the native-like solutions. These clusters are, in fact, the one or two most populated, and hold also the best membrane topology indices, i.e a MemTop index close to zero. For almost all the docking runs, the best hit from the selected clusters fall within the first 10 out of the 4000 output solutions provided by the docking algorithm.
A significant number of trials on the four highly different systems BRD, KcsA, MscL and MscS allowed us to define the best criteria for selecting the proper docking solutions at each growing step and the best growing path. These criteria proved validity in quaternary structure predictions of the second set of membrane proteins.
Selection of the proper solution/s at each growing step should be based on the docking score as well as on the population and membrane topology of the solution clusters. In detail, if the realistic solutions group essentially in two clusters characterized also by similar and significantly low MemTop indices, the best scored solutions from both clusters should be selected to grow the oligomer. In contrast, if the two most populated clusters have significantly different MemTop indices, only the one showing the lowest index must be chosen for extracting the best scored solution. Finally, if one cluster is significantly most populated than the others, only that cluster must be considered for extracting the best scored solution.
As for the growing paths, in general, those should be pursued, which employ a dimeric target in the second step (Figures 6,7,8,9). For cyclic oligomers, our approach is able to predict the oligomeric order of the functional unit, since completion of the cycle determines the end of the growth. Once established the oligomeric order, for systems like KcsA, AQP1 and GlpF, which are characterized by an even number of monomers, a growing path, which employs a trimer as a target at step 2, is worth attempting. In fact, in our benchmarks, this path led to the best tetramer, in terms of C α -RMSD from the native structure (Table 1, path (a)). Based on these results, we infer that, for even order oligomers, growing paths characterized by the employment of a trimeric target at step 2 may be preferred, whereas, for odd order oligomers, the preferential growing paths are those employing a dimeric target at step 2. Interestingly, both these paths share the characteristic that, in the final step, only one monomer is needed to complete the oligomer, i.e. to close the cycle. For odd order oligomers, this would imply pursuing two parallel growing paths, finally leading to two predicted quaternary structures. When the two predicted oligomers differ significantly from each other, quality checks are needed to select the final structure. In our study, this was the case of the MscS oligomer (Figure 8b and 8c). In fact, the two parallel growing paths passing through a dimeric target led to two significantly different oligomers (i.e. the C α -RMSD between them was 10.88 Å). In this case, given the significant extension of the water-exposed domains compared to the TM ones, the 3D-Profile score proved effectiveness in individuating the native-like oligomer.
For non-cyclic oligomers, the growth of the supramolecular assembly must stop when no more realistic solutions can be found. Low resolution information from Atomic Force Microscopy (AFM) or cryo-electron microscopy, if available, should be combined with the results of docking analysis. We couldn't do benchmarks on non-cyclic oligomers characterized by oligomeric orders higher than two or three due to the lack of high resolution structures for such systems. As an example, for BRD, we stopped the growth at the trimer, since the native structure, required for benchmarks, is available only for the trimer. However, we know from AFM images that BRD trimers organize in higher order oligomers [3]. Preliminary results of trimer vs trimer docking, reconstructed a BRD esamer similar to that inferred from AFM images (results not shown) [3]. We observed that the number of filtered solutions decreases significantly ongoing from monomer vs monomer to trimer vs trimer docking (i.e. from 77 to 15, respectively). This is in line with the fact that intra-trimer contacts should be stronger than the inter-trimer ones, which are mediated also by lipid molecules.
The effectiveness of predictions is independent of the content of water-exposed domains in the monomeric units, suggestive of the fundamental role of the TM domains in driving the supramolecular organization.
An important result is that predictions were excellent also for those systems, for which the asymmetric unit consists of the monomer and not of the biologic unit. These may be considered as unbound-unbound docking cases. Moreover, quaternary structure predictions can not be considered as bound-bound docking cases neither for those cases, in which the crystallographic asymmetric unit contains the biologic unit, and, hence, the monomer used for simulations was extracted from the X-ray structure of the complex. In fact, in none of the docking steps the situation is such that probe and target are the same components of the native complex. Moreover, for BRD, the predicted trimer is made of identical monomers, whereas, in the crystal structure of the oligomer, slight differences exist between the monomers, concerning their internal coordinates and the main-chain length. Moreover, the lipid molecules, which are present at the interfaces between the monomers in the crystal structure of the trimer, were completely neglected in our docking simulations. The employment of an "unbound" BRD structure (i.e. PDB code 1KME) [34], differing both in the backbone and side-conformations from the monomer extracted from the native trimer did not prevent the approach from leading to a correct quaternary structure.

Conclusion
In conclusion, the benchmarks carried out in this study on ten homo-oligomeric membrane proteins with known quaternary structure validate the proposed structure prediction protocol that, for all the tested cases, led to native-like quaternary structures regardless of the resolution of the structural models.
Quaternary structure predictions will be, hence, extended to other integral membrane proteins, including GPCRs. An attempt in this respect has been already reported, though based on an early and different version of the computational protocol, proving usefulness in aiding the interpretation of biophysical data and the design of novel in vitro experiments [14,35]

Overview of the computational approach
Quaternary structure predictions were carried out through a stepwise approach consisting of a combination of rigidbody docking, membrane topology filtering, cluster analysis, and solution selection for the growth of the oligomer. Solution selection was essentially based on the docking score and the quality of the membrane topology ( Figure  1).
Rigid body docking was carried out by means of the program ZDOCK 2.1, which utilizes the Pairwise Shape Complementarity (PSC) scoring function, neglecting desolvation [36].
For each protein system, the first docking run consisted of docking two identical copies of the crystal structure of monomer A, keeping one monomer fixed (i.e. target) and allowing the other copy of the monomer to move around the target (i.e. probe). For the membrane topology filter to work properly, the two identical copies of the starting monomer must have the appropriate orientation with respect to the putative membrane. This is due to the fact that ZDOCK expresses its docking solutions in terms of a x,y,z-translation and a RzRxRz-rotation of the probe. If both target and probe are properly oriented in a membrane parallel to the XY plane, the translation along the zaxis can be considered as an offset out of the membrane and the Rx component of the rotation as a deviation from the original orientation in the membrane. The membrane topology filter, indeed, discards all the solutions characterized by a deviation angle from the original z-axis, i.e. tilt angle, and a displacement of the geometrical center along the z-axis, i.e. z-offset, above defined threshold values. For the tilt angle and the z-offset, thresholds of 0.4 radians and 6.0 Å were, respectively, employed. The check of the correct orientation and the eventual reorientation of the monomer are automatically carried out in the following way. Membrane topology predictions allow for the definition of two sets of C α -atoms, which putatively lie at the two lipid/water interfaces of the membrane. The planes that fit at best these C α -atoms are found by means of a multiple linear regression approach. If these planes are parallel to the xy plane and, hence, perpendicular to the z-axis, the orientation of the monomer is considered good and no reorientation is needed. In contrast, if the monomer needs a reorientation, the rotations necessary to bring the two planes parallel to the xy plane are computed and then an average between the two rotations is employed to reorient the whole monomer. A number of membrane topology predictors were probed [37]. Overall, the PRODIV-TMHMM_0.91 predictor allowed for the most effective subunit reorientation. A reorientation was needed only for KcsA, BtuCD and the two different monomers of BRD (i.e. the one extracted from the trimer, PDB code 1BRR, and the monomeric structure encoded as 1KME). For KcsA and BtuCD we probed also an approach consisting in orienting the native oligomeric structure so that the dipole moment of the oligomer was parallel to the z-axis.
The codes for monomer reorientation and membrane topology filtering have been included in our FIPD software for the analyses of ZDOCK outputs [see Additional file 1][40].
For each set of docking simulations aimed at reaching the final oligomerization state of the protein, the runs that succeeded the first one were carried out by using the original monomer A as a probe and the intermediate oligomer as a target. For each of these runs, neither the probe monomer nor the target oligomer require a reorientation, since the former molecule is the same as that used in the first step, whereas the oligomeric target inherits the orientation of the original target.
Docking samplings were carried out by using a 128 × 128 × 128 point grid with a spacing of 1.2 Å and a rotational sampling interval of 6°, i.e. dense sampling. From each docking run, the first best 4000 solutions, according to the ZDOCK score, were retained. These solutions were subjected to the "membrane topology" filter described above. The filtered solutions from each run were merged with the target protein, leading to an equivalent number of oligomers that were subjected to cluster analysis. Clustering was carried out by using an algorithm from Dr. M. Schaefer (Michael Schaefer, Syngenta Crop Protection AG, unpublished work). The algorithm first calculates the Cα-RMSD for each superimposed pair of dimers/oligomers and then it computes the number of neighbors for each dimer/oligomer, using a threshold Cα-RMSD. The dimer/ oligomer with the highest number of neighbors is considered as the center of the first cluster. All the neighbors of this configuration are removed from the ensemble of configurations to be counted only once. The center of the second cluster is then determined in the same way as for the first cluster, and this procedure is repeated until each structure is assigned to a cluster. The Cα-RMSD threshold value for each superimposed pair of dimers/oligomers was set equal to 3.0 Å. All the amino acid residues in the dimer/oligomer were included in C α -RMSD calculations.
To identify the cluster of solutions with the best membrane topology, i.e. with the lowest values of both tilt angle and z-offset, we defined the MemTop index, , were <tilt nor > and <Z offnor > are, respectively, the normalized tilt angle and the z-offset averaged over all the members of a given cluster. Normalization of each tilt angle and z-offset value was carried out by dividing each value for the respective cutoff value, i.e. 0.4 radians, for the tilt angle, and 6.0 Å, for the z-offset. The optimal value for such index is zero.
The quality of some of the predicted oligomeric structures of MscS was estimated by means of the 3D-Profile program [33] within the Quanta 2000 package.
As indicative values, a monomer vs monomer and monomer vs esamer docking for MscS required 87 hours and 147 hours, respectively, of CPU time on a 2.2 GHz Opteron with 2 GB RAM.


extfieldsofstudy: Biology Medicine

text:
Production and Characterization of Monoclonal Antibodies Against the Nucleocapsid Protein of SARS-COV

DNA encoding the N protein was amplified from cDNA prepared from the Urbani isolate (CDC) and cloned into the pBAD-TOPO protein expression vector (Invitrogen). The N protein was expressed in E. coli as a 6-His tagged fusion protein and purified using Ni-NTA columns (Qiagen). BALB/c mice were immunized with purified recombinant N protein at 2-week intervals for 8 weeks. Splenocytes from immunized mice were fused with NS-1 myeloma cells and cultured on 24-well plates. Cell culture supernatants from wells containing hybridoma colonies were initially screened by ELISA using recombinant N protein. Hybridoma cells from positive wells were subcloned, expanded, and retested. For Western blot analysis, Vero cells (ATCC) were mock-infected or infected with SARS-CoV (Urbani strain). The cell lysate was separated on a 15% polyacrylamide gel, transferred to PVDF membranes, and then incubated with an anti-N MAb. After extensive washing, the membranes were incubated with a HRP conjugated goat antimouse IgG (Jackson Laboratories). After 60 minutes, the blot was washed extensively

INTRODUCTION
pathogenesis of the disease and in aid of developing diagnostic tests for SARS-CoV, we produced a panel of monoclonal antibodies (MAbs) against SARS-CoV N protein. We further tested their application to the detection of SARS-CoV infection using the methods of Western blotting, immunofluorescent staining (IFA), and ELISA.

MATERIALS AND METHODS
DNA encoding the N protein was amplified from cDNA prepared from the Urbani isolate (CDC) and cloned into the pBAD-TOPO protein expression vector (Invitrogen). The N protein was expressed in E. coli as a 6-His tagged fusion protein and purified using Ni-NTA columns (Qiagen). BALB/c mice were immunized with purified recombinant N protein at 2-week intervals for 8 weeks. Splenocytes from immunized mice were fused with NS-1 myeloma cells and cultured on 24-well plates. Cell culture supernatants from wells containing hybridoma colonies were initially screened by ELISA using recombinant N protein. Hybridoma cells from positive wells were subcloned, expanded, and retested.
For Western blot analysis, Vero cells (ATCC) were mock-infected or infected with SARS-CoV (Urbani strain). The cell lysate was separated on a 15% polyacrylamide gel, transferred to PVDF membranes, and then incubated with an anti-N MAb. After extensive washing, the membranes were incubated with a HRP conjugated goat antimouse IgG (Jackson Laboratories). After 60 minutes, the blot was washed extensively For IFA, infected Vero cells were fixed with 2% paraformaldehyde at 24 h after infection and then permeabilized with PBS containing 3% normal goat sera and 0.2% saponin (blocking buffer) for 15 minutes. MAbs were diluted 1:500 in blocking buffer and incubated with cells for 1 h at room temperature. After extensive wash, the cells were incubated with a goat anti-mouse IgG antibody conjugated to AlexaFluor 594 (Molecular Probes) or FITC (Jackson Laboratories) and the nuclear stain ToPro-3 (Molecular Probes). After incubation, the cells were washed and mounted onto microscope slides. Samples were analyzed on a Zeiss LSM 510 confocal microscope.
For ELISA, detergent extracted, gamma-irradiated SARS-CoV or recombinant SARS N protein were coated onto 96-well plates (Dynex) in bicarbonate buffer (pH 9.6) overnight at 4 0 C. The plates were blocked with 2% BSA for 1 h at 37 0 C. After washing, 100 µl of serially diluted MAbs were added and incubated for 1 h at 37 0 C, and then HRPgoat anti-mouse IgG/IgM was added and incubated for 45 minutes. Plates were developed using ABTS, and OD values were determined using an ELISA plate reader.

RESULTS AND DISCUSSION
Initial hybridoma screening by ELISA using recombinant N protein yielded three candidate MAbs, SA 87-A1, SA 46-4, and SA 41-48. The isotypes of these MAbs were determined using an IsoStrip Kit (Serotech) and results showed that SA 87-A1 was an IgG1, SA 46-4 was an IgG2a, and SA 41-48 was an IgM.
We further tested the reactivity of these MAbs by IFA on Vero cells expressing recombinant EGFP-N protein. The SARS-CoV (Ubani strain) N gene was cloned into pEGFP-C2 vector (Clontech), and Vero cells were transfected with plasmid pEGFP-C2-N. MAb SA 46-4 showed the highest sensitivity and specificity in recognizing the EGFP-N protein in Vero cells, but the other two MAbs showed weak fluorescence and high background (data not show). To further confirm the specificity of MAb SA 46-4, it was tested for cross-reactivity with other coronaviruses and arteriviruses. It did not cross-react with porcine transmissible gastroenteritis virus (TGEV), mouse hepatitis virus (MHV), or porcine reproductive and respiratory syndrome virus (PRRSV).
We further explored the sensitivity and specificity of these MAbs for detection of SARS-CoV infection using Western blotting, IFA, and ELISA. Protein specificity of MAbs SA 87-A1 and SA 46-4 was first studied by Western blot analysis of Vero cell lysates infected with SARS-CoV. The results in Fig. 1 showed that MAbs SA87-A1 and SA46-4 reacted with N from infected cells and not with proteins from mock-infected cells. These MAbs reacted with a viral protein, which migrated as a doublet close to 50 kDa, the predicted size for the N protein. The presence of N in two forms was also shown by Western blotting using in vitro expressed N protein (data not shown). The presence of N in two forms may reflect a post-translational modification. This result is consistent with the results reported by Leung et al. 1 In their experiments, Western blots prepared using lysates from SARS-CoV infected cells probed with sera from patients showed several N protein-specific bands. They indicated that the N protein cleavage site is at the C-terminal, and this type of cleavage is specifically found in every preparation of the crude extracts. Post-translational cleavage of N would be a unique property of SARS-CoV, which is a subject of further study in our laboratory. To further define MAb specificity, IFA was performed on Vero cells infected with SARS-CoV Urbani strain. The cells were fixed and incubated with MAbs SA87-A1 and SA46-4, and then double stained with AlexaFluor 594 (Molecular Probes) conjugated goat anti-mouse IgG and a nuclear stain ToPro-3 (Molecular Probes). As shown in Fig. 2, both MAbs SA46-4 and SA87-A1 specifically recognized the viral N protein, which is accumulated in the cytoplasm, in contrast with the ToPro-3 nuclear staining.
A commonly used method for the diagnosis of SARS-CoV infection is ELISA. To determine if these MAbs have any value for future development of serological tests, we tested MAbs SA46-4, SA87-A1, and SA41-48 in indirect ELISA using either in vitro expressed recombinant N protein or viral lysate as antigen. Antibodies were applied as sensitivity (Fig. 3).
Currently, although SARS-CoV appears to be under control, its future reemergence is still possible. Accurate and timely diagnosis of SARS infection is a critical step in preventing another global outbreak. Diagnostic reagents for SARS that are highly sensitive, specific, and convenient need to be developed. In this study, we produced a panel of anti-N MAbs, and MAb SA 46-4 consistently showed good sensitivity and specificity in all the tests conducted. Preliminary epitope mapping determined that MAb SA 46-4 recognizes an epitope located in the region between amino acids 50 to 200.  Immunon-2 plates. Serially titrated anti-N MAbs were reacted for 1 h at room temperature and the bound MAbs were detected by peroxidase labeled goat anti-mouse IgG/IgM followed by color development with ABTS.
Other anti-N MAbs have been reported. 2,3 Some of them have been mapped to epitope sites on the N protein, and they recognize different epitopes than reported here. 2 Previous studies showed that none of the antigenic epitopes reacted with all test sera from SARS patients. 4 Therefore, a "MAb cocktail" combining currently available anti-N MAbs recognizing different antigenic epitopes could improve detection. Especially, because previous studies demonstrated that N protein detection exhibited a high positive rate between day 3 and day 5 after the onset of symptoms, these anti-N MAbs will be suitable reagents for early detection of SARS-CoV infection. 5 Furthermore, the N protein has been determined to be the most antigenic structural protein, expressed in high abundance in infected cells, and N protein sequences are relatively conserved among different strains of SARS-CoV. 1,4,6 These MAbs may have a substantial impact on the development of diagnostic tests for SARS.


extfieldsofstudy: Chemistry Medicine

text:
Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution

Background The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution. Methods We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20). Results We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2. Conclusion These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.

Background
During pregnancy, the uterus enlarges, which in rats is mainly caused by an increase in the amount of collagen and hypertrophy of the uterine smooth muscle cells. After parturition, the uterus undergoes involution during which it returns to its prepregnancy state. Matrix metalloproteinases (MMPs) are a group of structurally related endopeptidases that catalyze the degradation of various macromolecular components of the extracellular matrix and basement membrane [1,2], and induce various forms of tissue remodeling, including wound healing [3,4], trophoblast invasion [5,6], organ morphogenesis [7,8], and uterine [9][10][11], mammary gland [12,13], and prostate gland [14,15] involution. We previously reported that an increase in the expression levels of both membrane type 1-MMP (MT1-MMP) and MMP-2 plays a key role in tissue remodeling during corpus luteum structural involution both in rats and humans [16][17][18].
To obtain additional information on the activity of MMPs during uterine involution, we have initiated studies using a rat model to examine MMP expression and function in the uterus during pregnancy and after parturition. Although MT1-MMP is abundant in the uterus [19,20], little is known about its activity or that of MMP-2 during uterine involution. To the reason for this, we investigated the expression patterns of MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 and the activation of MMP-2 in the rat uterus during postpartum involution.

Rat uterus
Pregnant Sprague-Dawley rats were obtained from Hokudo Co., (Sapporo, Japan) on day 17 of gestation, after which they were kept in our laboratory and maintained on a 12-hour light and 12-hour dark regimen (light 7:00-19:00) with free access to water and a standard diet. Uterine tissue for postpartum involution analysis was obtained from five rats per group on gestation day 20 and then 18 h, 36 h and 5 days after parturition. The Animal Care and Use Committee of the Sapporo Medical University School of Medicine approved all procedures of this study, which are in accordance with the standards described in the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Each uterine tissue sample was divided into two pieces. One piece was fixed in 4% paraformaldehyde/PBS and embedded in paraffin for immunohistological analysis. The other was used for biochemical studies (zymography and northern blotting); all tissue samples were frozen on dry ice and then stored at -80°C until use.

Northern blotting
Total RNA was extracted from uterine tissue samples using an Ultraspec RNA isolation system, after which the extracted RNA (20 μg/lane) was electrophoresed on 1% agarose/formaldehyde gels (100 V; 2 h), transferred overnight onto nylon membranes in 20 × SSC (3 M sodium chloride, 0.3 M trisodium citrate) and fixed using a UV linker. Filters were prehybridized for 4 h and then hybridized overnight at 42°C with a 32P-labeled cDNA probe. The probes used for Northern blotting were a 1.2-kb Hind-III-and EcoR-digested fragment of MT1-MMP cDNA, a 1.5-kb EcoRI-and Bam-HI-digested fragment of MMP-2 cDNA, a 0.6-kb Cla-I-and Bam-HI-digested fragment of TIMP-1 cDNA, and a 0.7-kb Eco-RI-and Bg-II-digested fragment of TIMP-2 cDNA [21]. A 450-bp cDNA fragment encoding the ribosomal protein L38 was used as an internal control [22].
The probes were radiolabeled with 32P-dCTP using Prime-It II random primer labeling kits, after which the labeled probes were purified on a Nick column before hybridization. After hybridization, the filters were washed four times: twice for 15 min each with 2 × SSC containing 0.1% SDS at room temperature, and twice for 15 min each with 0.2 × SSC containing 0.1% SDS at 65°C. The filters were then exposed to a Fuji RX X-ray film at -70°C for 1-2 days.

Gelatin zymography
Gelatin zymography was carried out as previously described [16]. Briefly, uterine samples were homogenized (100 mg wet weight/ml of PBS containing 0.2% Triton X-100) using a Teflon glass tissue grinder on ice (15 strokes). The resulting homogenates were centrifuged at 12,000 × g for 20 min at 4°C, after which the supernatants were collected as the uterine extract. After assaying the protein concentration, aliquots of the extract (40 μg of protein) were electrophoresed in 10% polyacrylamide gels containing 1 mg/ml gelatin.

Gelatin zymography using crude membrane fraction from rat uterus
The crude membrane fraction from the rat uterus was isolated as previously reported [17,23]. Briefly, uterine tissue samples were homogenized in 2 ml of Tris buffer containing 0.25 M sucrose using a Teflon glass tissue grinder at 45 × g on ice. The resulting homogenates were filtered through a nylon mesh (42 μm) and centrifuged at 120 × g for 10 min. The supernatant was then collected and centrifuged at 10,000 × g for 30 min at 4°C, and the pellet was collected as the crude membrane fraction and stored at -80°C until use. To examine the expression level of membrane-bound pro-MMP-2, aliquots of the crude uterine membrane fraction (20 μg) were mixed with 1 μl of FCS as a source of procollagenase and incubated for 2 h at 37°C. After terminating the procollagenase activity by adding of the sample buffer, the expression level of membrane-bound pro-MMP-2 as estimated from the liberated MMP-2 activity as determined by gelatin zymography.

Immunohistochemistry
The uterine tissues embedded in paraffin were cut into 5μm-thick sections and mounted on APS-coated glass slides. The sections were then deparaffinized with xylene and the slides were placed on a hot plate at 90°C, covered with a STUF solution for 10 min, and then rinsed several times with PBS. Endogenous peroxidase activity was blocked by incubating the slides in 0.6% H 2 O 2 in methanol for 30 min at room temperature, after which Block Ace was applied for 30 min at room temperature to minimize nonspecific antibody binding. Primary antibodies were then applied to the sections for 60 min at room temperature, after which the sections were incubated with a biotinylated secondary antibody for 30 min. The sections were then stained using the ABC method with DAB as a substrate; hematoxylin and eosin stain was used as the counterstain. As a negative control, the slides were processed without incubation with primary antibodies. The sections were also stained with hematoxylin and eosin.

Densitometry
Bands showing gelatinase activity were analyzed using NIH Image software (Version 1.61). Bands on Northern blots were analyzed using a BAS 2000 Bio-Imaging Analyzer (FUJI, Tokyo, Japan). Radioactivity was normalized to that in the L38 RNA band.

Statistical analysis
Data are presented as mean ± SEM. The differences between groups were evaluated using one-way ANOVA with post hoc Schaffer's F-test and unpaired Student's ttest. Values of P < 0.05 were considered significant.

Time-dependent localization of MMP-2 and MT1-MMP proteins in rat uterus
Immunohistochemical staining revealed that both MT1-MMP and MMP-2 proteins mainly localized in the cytoplasm of uterine interstitial cells 18 h (not shown) and 36 h after parturition, although uterine smooth muscle cells also showed weak MMP-2 protein staining (Fig. 1). Weak MMP-2 protein staining was also generally observed in the uterine tissue on day 5 after parturition, and a weaker MMP-2 protein staining was observed in the uterine tissue from 20-day pregnant rats.

Expression levels of MMP-2, MT1-MMP, TIMP-1 and TIMP-2 mRNAs in rat uterus
As shown in Fig. 2, the uterine expression levels of both MMP-2 and MT1-MMP mRNAs time-dependently increased for at least 36 h after parturition, with larger increases observed in the MT1-MMP expression level. The expression levels then decreased markedly 5 days after parturition, although they remained above the levels expressed in the uterine tissue from pregnant rats. The expression level of TIMP-1 mRNA reached a peak within 18 h, the earliest time point measured. In contrast, the expression level of TIMP-2 mRNA increased only slightly 18 h and 36 h after parturition, but markedly higher expression levels were observed 5 days after parturition.

Gelatinase activity in extracts of rat uterus
Gelatinase activity in uterine tissue extracts was examined by gelatin zymography (Fig. 3). In zymograms generated using uterine samples from pregnant (20 days) rats, most of the gelatinase activity was found in a 68-kDa band, with a lower activity in an approximately 62-kDa band. Treatment with EDTA/orthophenanthroline eliminated the activity, whereas pretreatment with amino phenyl Zymography of homogenate extract from rat uterus mercuric acid, which is known to activate latent collagenase, elicited a marked shift in the activity from the 68-kDa band to the 62-kDa band (data not shown). Although not all MMPs exhibit gelatinase activity, which enables MMPs to be detected by zymography, we suspected that the major MMPs in the uterine tissue extracts from the pregnant rats were MMP-2 and MMP-9, which are present mainly as pro-MMP-2, the 68-kDa precursor of the 62-kDa activated form of MMP-2, and MMP-9, the 92-kDa. After parturition, however, the expression level of activated MMP-2 increased significantly over time corresponding to the expression level of MMP-2 mRNA, whereas the expression level of pro-MMP-2 remained unchanged (Fig. 3). The expression level of MMP-9 increased 18 h and 36 h after delivery.

Expression levels of pro-MMP-2 in uterine plasma membrane fractions
The expression levels of pro-MMP-2 in the plasma membrane fractions of the rat uterus were estimated from MMP-2 activity induced by incubation with FCS as a source of procollagenase and revealed by zymography. As shown in Fig. 4, the levels of expression and gelatinase activity of activated MMP-2 paralleled those of both MT1-MMP mRNA expression and the gelatinase activity of MMP-2 in the uterine tissue extract.

Discussion
Immunohistochemical staining revealed that both MT1-MMP and MMP-2 proteins mainly localized in the cytoplasm of the uterine interstitial cells 18 h (not shown) and 36 h after parturition. Moreover, MMP-2 mRNA and MT1-MMP mRNA expressen levels time-dependently increased for at least 36 h. The level of the 62-kDa activated form of MMP-2 also increased 18 h and 36 h after parturition. The expression levels of gelatinase activity of the uterine tissue extract in the uterine plasma membrane fraction increased 18 h and 36 h after parturition. These results indicate that MMP-2 and MT1-MMP may play key roles in uterine postpartum involution During pregnancy, the uterus is transformed into a large muscular organ sufficient to accommodate the fetus, placenta and amniotic fluid. After parturition, the uterus undergoes involution, a conspicuous feature characterized by the rapid decrease in the amount of collagen resulting from the extracellular degradation of collagen bundles. Activated collagenases cleave the collagen bundles into fragments, which then denature at body temperature into gelatin. Gelatinases then cleave the gelatin in small peptides that are rapidly removed in the blood stream [24]. To better understand the functions of MMPs in various uterine processes, we have initiated studies using a rat model for studying the activities of various MMPs in the uterus during pregnancy and after parturi-tion. MT1-MMP, for example, has various functions that include the degradation of several types of collagen, the activations of other MMPs and activities related to apoptosis. This study showed that the expression levels of MT1-MMP and MMP-2 were significantly increased in the cytoplasm of uterine interstitial cells in the first five days after parturition, which suggests that these two enzymes play 9 are involved also in the cervical ripening process [25]. Lyons et al. reported that MMP-9 may be involved in preterm labor, cervical maturity, and postpartum uteruine involution [26]. Roh et al. reported that the proMMP-9 in the uterine tissue maybe related to the remodelling of human myometrium during labor [27]. We also consider that MMP-9 is related to postpartum uterine involution from the results of our gelatine zymograpy experiment ( Fig.  3(a)).
Evidence suggests that MT1-MMP causes the degradation of collagen types I, IV and V and elastin [28,29], and activates membrane-bound pro-MMP-2 [30][31][32][33]. Although it is thought that the main function of the activated MMP-2 is the degradation of type IV collagen in the basement membrane, it also cleaves type I collagen [34], which is consistent with the finding that during postpartum uterine involution, MT1-MMP and MMP-2 act together to degrade the abundant type I collagen.
We found that the time courses of the expressions of MMP-2, TIMP-1 and TIMP-2 differed from those described in the reports mentioned above. This difference is reflected in part by the transiently high expression level of TIMP-1 observed 18 h after parturition. Because TIMP-1 is known to bind stoichiometrically to almost all MMP active sites, thereby irreversibly inhibiting the enzyme's activity [33,35], its upregulation would be expected to mitigate the effect of the abrupt concurrent increase in MMP expression level. On the other hand, the suppression of TIMP-1 transcription 36 h after parturition would likely contribute to the maintenance of a high MMP activity.
In contrast to TIMP-1, TIMP-2 transcription was enhanced only slightly 5 days after parturition. Although TIMP-2 exerts a strong inhibitory effect on MMPs similar to TIMP-1 [36], it also forms MT1-MMP/TIMP-2 and MT1-MMP/ TIMP-2/pro-MMP-2 complexes, which are involved in the activation of pro-MMP-2 [37][38][39]. This suggests that at 18 h and 36 h postpartum the slight increase in TIMP-2 expression level induced proMMP-2 activation. In contrast, the much larger increase in TIMP-2 expression level observed 5 days after parturition, which coincides with the decrease in the activities of MT1-MMP and MMP-2, mediated pro-MMP-2 suppression. Taken together, these findings strongly suggest that MT1-MMP, MMP-2 and MMP-9 are time-dependently regulated and play important roles in tissue remodeling during postpartum uterine involution.


s2fieldsofstudy: Biology Medicine

text:
mRNA Expression and RNA Editing (2451 C-to-U) of IL-12 Receptor β2 in Adult Atopic Patients

Interleukin (IL)-12 activates T helper (Th) 1 cells to produce interferon (IFN)-γ which inhibits atopic inflammation. IL-12 acts through interaction with its receptor, especially β2 subunit. In several studies, the low production of IFN-γ in peripheral mononuclear cells of atopic patients on response to IL-12 stimulation has been reported. Therefore we investigated the IL-12 receptor β2 (IL-12Rβ2) mRNA expression and RNA editing, nucleotide 2451 C-to-U conversion, to find the cause of low responsiveness to IL-12 in atopy. Quantitative real time PCR for mRNA expression and sequence analysis for RNA editing were performed in 80 atopic patients and 54 healthy controls. The expression of IL-12Rβ2 mRNA was significantly lower in atopic patients than healthy controls (p<0.05). In sequence analysis, RNA editing on nucleotide 2451 was not found from either atopic patients or healthy controls. In additional evaluation, there was no relationship between expression of IL-12Rβ2 mRNA and serum total IgE or blood eosinophil count. Reduced IL-12Rβ2 mRNA expression in atopic patients indicate the reduced capacity to respond to IL-12 which induce IFN-γ production and this may contribute to Th2-skewed immune response in atopy.

INTRODUCTION
Atopy is characterized by enhanced immunoglobulin E (IgE) response to common environmental antigens and leads to clinical disorders such as asthma, eczema and rhinitis (1). Allergic inflammation and IgE synthesis are known to be a T helper (Th) 2 phenomenon promoted by a distinctive allergen-specific Th2 cell subset (2).
Th cells can be divided into two subpopulations, Th1 and Th2, based on the cytokines they produce. Th2 cells produce interleukin (IL)-4, IL-5 and IL-13, which cause eosinophilia and promote immunoglobulin class switching to IgE (3). Th1 cells produce interferon (IFN)-, which inhibit Th cells to differentiate to Th2 cells and down-regulates the production of IgE.
IL-12 is the single most powerful factor inducing the synthesis of IFN-and acts by binding to its specific receptor. The receptor of IL-12 (IL-12R) is composed of two subunits, 1 and 2, that assemble to form a high affinity IL-12 receptor complex (4). While the 1 subunit is relatively constantly expressed, the 2 subunit expression is restricted to Th1 cells and highly regulated by several cytokines.
Several investigators have shown that peripheral blood mononuclear cells (PBMCs) from atopic patients produced less IFN-than those of healthy individuals when they were stimulated with IL-12 (5)(6)(7)(8)(9). The underlying mechanisms of the low responsiveness to IL-12 in atopic patients have not been known. In one study, reduced expression of IL-12R 2 mRNA was found to have relation with low production of IFN-in atopic patients (13). They explained that the decreased capacity of up-regulation of IL-12R 2 might decrease the responsiveness of Th1 cells to IL-12. In another study, RNA editing, especially, nucleotide 2451 C-to-U conversion of IL-12R 2, was reported in atopic patients (1). They suggested that above RNA editing might give rise to a conformational change in IL-12R 2 and cause impairment of Th1 cells to respond to IL-12.
The aim of this study was to investigate mRNA expression of IL-12R 2 as well as the RNA editing, nucleotide 2451 C-to-U conversion, of IL-12R 2 as a possible cause of low responsiveness to IL-12 in atopic patients. In additional evaluation, the correlation of the IL-12R 2 mRNA expression with serum total IgE concentration and blood eosinophil count were evaluated in atopic patients.

Subjects
Eighty atopic patients (52 male, 28 female, 34.2±18.9 yr) with major allergic diseases such as bronchial asthma (10 male, 4 female, 30.7±22.1 yr), atopic dermatitis (26 male, 15 female, 36.5±18.5 yr) or allergic rhinitis (17 male, 8 female, 31.6±18.1 yr) were studied. The diagnosis of allergic diseases was established by specific allergic symptoms, positive results of allergen specific IgE test and/or increased serum total IgE concentration. Fifty four healthy controls (23 male, 31 female, 47.1±14.3 yr) who had no history of atopic disease and had normal serum total IgE concentration were studied.

Methods
Venous blood was drawn from atopic patients and healthy controls. PBMCs were isolated by gradient centrifugation method using Ficoll-Paque TM plus (Amersham Biosciences, Uppsala, Sweden) and were suspended in the RPMI 1640 (Invitrogen, Grand Island, NY, U.S.A.) culture medium in 1×10 6 density. Then the cells were cultured in the presence of 10 g/mL PHA for 24 hr at 37℃ in a humidified atmosphere containing 5% CO2 (Forma CO2 incubator model 3039, Formal Scientific Inc, Ohio, U.S.A.).
Total cellular RNA was extracted from cultured PBMCs using High Pure RNA isolation kit (Roche, Penzberg, Germany) and 5 g of RNA was converted to complementary DNA (cDNA) by reverse transcription (RT) PCR in a total reaction mixture volume of 20 L. The real time PCR reactions for quantification of IL-12R 2 cDNA and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were performed, respectively. GAPDH was analyzed as an internal control. The master PCR reaction mixure was contained 2 L of Light-Cycler � FastStart DNA master SYBR green I (Roche Molecular Biochemicals, Mannheim, Germany), 2.5 L of MgCl2 in final concentration of 4 mM, each 10 pM of sense and antisense primers, 2 L of cDNA and distilled water in final volume of 20 L. The LightCycler � FastStart DNA master SYBR green I was composed of Taq DNA polymerase, SYBR green I dye, 10 mM MgCl2 and dNTPs (dATP, dCTP, dGTP, dUTP) in the reaction buffer. The PCR thermal conditions were as follows: 10 min at 95℃ and 40 cycles of 10 sec at 95℃, 5 sec at 57℃ and 4 sec at 72℃ for IL-12R 2 and 10 min at 95℃ and 40 cycles of 10 sec at 95℃, 5 sec at 55℃ and 15 sec at 72℃ for GAPDH. The primer sequences were summarized in Table 1. The quantitative real time PCR was performed using LightCycler � system (Roche Molecular Biochemical, Mannheim, Germany). During each cycle on the real time of reaction and after completion of primer extension at 72℃, fluorescence at 530 nm (F1 channel) was recorded. The amount of IL-12R 2 mRNA was calculated relative to the amount of GAPDH present in each sample and described as IL-12R 2/GAPDH ratio. All the analysis was performed in duplicate. Melting curve analysis was used to detect nonspecific amplification which could be occurred during PCR procedures using SYBR green I (Fig. 1).
Fragment of exon 13 including nucleotide 2451 of IL-12R 2 was amplified from mRNA and genomic DNA, respectively. The PCR thermal conditions were as follows: 5 min at 94℃ and 40 cycles of 20 sec at 94℃, 20 sec at 65℃ and 45 sec at 72℃ for mRNA and 10 min at 94℃ and 40 cycles of 1 min at 94℃, 1 min at 55℃ and 1 min at 72℃ for genomic DNA. The primer sequences were summarized in Table 1. The amplified products were purified and sequenced in automatic sequencer ABI prism � 3730xI (Applied Bisosystems, Foster City, CA, U.S.A.). Serum total IgE concentration was measured using RIDA AllergyScreen test kit (R-biopharm AG, Darmstadt, Germany). Peripheral blood eosinophil count was measured using ADVIA120 (Bayer, Dublin, Ireland).

Statistical analysis
The comparison of IL-12R 2 mRNA expression between atopic patients including 3 disease groups and healthy controls was analyzed using independent t test. The comparison of IL-12R 2 mRNA expression among 3 disease groups was analyzed using one way ANOVA. The relationship between IL-12R 2 mRNA expression and serum total IgE were analyzed using one way ANOVA. The relationship between IL-12R 2 mRNA expression and blood eosinophil count was analyzed using Spearman's correlation test. All the statistical analysis was performed with SPSS program (version 12.0, SPSS Inc, Chicago, IL, U.S.A.) and the p value of <0.05 was considered as significant.

RESULTS
The PHA stimulated PBMCs from atopic patients had significantly lower expression of IL-12R 2 mRNA than the cells from healthy controls (p<0.05) (Fig. 2). The average IL-12R 2/ GAPDH ratio was 0.035±0.02 in healthy controls and 0.018 ±0.047 in atopic patients. When patients were divided into 3 disease groups (asthma, atopic dermatitis, allergic rhinitis), each disease group showed lower IL-12R 2 mRNA expression than controls (p<0.05, respectively), also. But there was no significant difference of IL-12R 2 mRNA expression between 3 groups (p>0.05) ( Table 2).
In the results of sequencing analysis, all the nucleotide 2451 was cytidine (C) in cDNA as well as genomic DNA from atopic patients and healthy controls (Fig. 3). Therefore, the RNA editing phenomenon, C-to-U conversion at nucleotide 2451 of IL-12R 2 was not found in present study.

DISCUSSION
IL-12 is the single most powerful factor to induce Th1 immune response. It is produced by antigen present cells (APCs), including monocytes and dendritic cells. IL-12 is a heterodimeric cytokine composed of 35 and 40 kDa peptides and the genes are located on chromosome 3 and 5, respectively (11). By binding to its specific receptor complex, 1 and 2, IL-12 promotes the development of Th1 responses and induces production of IFN-. The 2 subunit is the signal transducing component of the receptor and highly regulated in contrast to the 1 subunit. IFN-inhibits Th cells to differentiate to Th2 cells and down-regulates the production of IgE in B lymphocytes. IFN-is also able to augment IL-12. This IL-12/IFN-loop helps the predominant Th1 cell differentiation and inhibits Th2 cell differentiation (12).
Since it was reported that PBMCs from atopic patients produced less IFN-than those of healthy individuals when they were stimulated with IL-12 (5-9), several investigations to find genetic mechanisms from interaction between IL-12 and IL-12R have been reported. Several IL-12 polymorphism or IL-12R 2 mutations such as truncated (2496 del 91) or missense (1577 A to G and 2799 A to G) mutation has been reported (13). Although these mutations were thought to lead to change in the conformational structure of 2 receptor, these are controversial, yet. In a recent study, the relationship between RNA editing of IL-12R 2 and atopy was reported by Kondo et al. (1). RNA editing is defined when there is any specific alteration in primary sequence of a RNA molecule. Such changes have marked effects on gene expression. They found that the patients who had nucleotide 2451 C-to-U conversion showed lower IFN-production and faint tyrosine phosphorylation of STAT-4 than those who did not have editing. This C-to-U conversion cause substitution of amino acid alanine to valine. Therefore, they proposed that the RNA editing might cause impairment of the IL-12 signal cascade and the subsequent reduction in IFN-production by disturbing conformational binding of IL-12 to IL-12R. Unfortunately, we could not find the C-to-U conversion at nucleotide 2451 from either atopic patients or healthy controls.
In other studies, the expression of IL-12R 2 mRNA was studied in atopy (10,14). PBMCs from children with atopic airway symptoms showed lower mRNA expression of IL-12R 2 than non-atopic children and the production of IFNafter stimulation with IL-12 was also lower in atopic children. In present study, we also found that PBMCs from atopic patients expressed lower IL-12R 2 mRNA than those of healthy controls. This low expression findings of IL-12R 2 mRNA was found in all 3 atopic disease groups, but significant differences was not found between groups. These results correspond with previous studies and support their hypothesis that reduced capacity of up-regulation of IL-12R 2 may contribute to the Th2-skewed immune response in atopic patients.
The underlying mechanism of low mRNA expression of IL-12R 2 in atopic patients cannot be known yet. But there was a suggestion to introduce association of CD2 defects and low responsiveness to IL-12. In previous and present studies, the PBMCs were stimulated with phytohaemagglutinin, which activates lymphocytes via CD2. CD2 is an accessory molecule of T cell and has been thought to synergize the IL-12 action by up-regulation of the 2 chain (15,16). Besides, the lower proportion of CD2+ lymphocytes was detected in atopic children (17). But further studies will be required whether there is connection between CD2 and atopy.
In additional evaluation, we also analyzed the relationship between IL-12R 2 mRNA expression and serum total IgE concentration or eosinophil count. There were not any significant associations between them. This result indicates that Blood eosinophil count (/ L) Fig. 5. Relationship between IL-12R 2/GAPDH ratio and blood eosinophil count in atopic patients. Significant relationship between IL-12R 2 mRNA expression and blood eosinophil count is not found (Spearman's correlation test, p>0.05).
the IL-12R 2 mRNA expression would not affect production of IgE or eosinophils, directly. However, we thought that this analysis would be more reliable if we analyzed serum IgE concentration with quantitative method. In one study, a negative correlation between IL-2-induced IL-12R 2 mRNA expression and serum IgE concentration had been reported (10).
In conclusion, RNA editing which might cause the conformational changes in IL-12R 2 was not found, but less IL-12R 2 mRNA on response to PHA stimulation was found in atopic patients. This low expression of IL-12R 2 did not have relationship with atopic disease category, serum IgE concentration or blood eosinophil count. We guess low expression of IL-12R 2 may contribute to low responsiveness to IL-12, low production of IFN-and Th2-skewed immunity in atopic patients, subsequently.