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Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment.

INTRODUCTION
Ionotropic glutamate receptors (iGluRs) are glutamate-gated cation channels that mediate the majority of fast excitatory synaptic transmission in the central nervous system (1) and are key players in synaptic plasticity, a process that underlies learning and memory (2). iGluRs have also been implicated in various diseases and therefore are important drug targets (3)(4)(5)(6).
iGluRs assemble into homo-or heterotetramers, with a twofold symmetric extracellular region (ECR) and an~4 -fold symmetric TMD. The ECR accounts for~80% of an iGluR and has a unique organization, with the distal NTDs forming two pairs of stable dimers that associate into tetramers via a small interface. Subunits cross over and swap dimeric partners at the level of the LBD with regard to the NTD ( Fig. 1 A) (16)(17)(18). This results in a loosely organized but at the same time interwoven ECR assembly, which will have key consequences on allosteric communication and ultimately on gating. Recent structural data show that both ECR layers can drastically reorganize in response to channel activation and desensitization (19,20), confirming earlier findings (21). This notion is supported by a recent analysis of the collective motions that are accessible to intact AMPARs and NMDARs (22).
The iGluR NTD and LBD, both of which belong to the periplasmic binding protein (PBP) superfamily, have bilobate structures that evolved to bind ligands in the interlobe cleft (see Fig. 2 B) (23). The NMDAR NTD is believed to function like a classic PBP allosteric module that is stabilized in a closed-cleft conformation upon binding of the cleft ligand zinc, which triggers a reduced channel open probability (10,24,25). Related dimeric, allosterically active type I PBPs are found in natriuretic peptide receptors (NPRs) (26)(27)(28) and type C G-protein-coupled receptors (GPCRs), including metabotropic glutamate receptors (mGluRs) and the GABA B receptor (29,30).
To date, a signaling role has not yet been described for the NTD in non-NMDARs (AMPARs and KARs), where this domain forms tight dimers and is mostly involved in subtype-selective subunit assembly (31,32). In addition to tighter dimeric packing, reduced allosteric activity in non-NMDARs is also suggested by the overall ECR architecture, which is loosely organized. In the more tightly packed NMDAR (16,17), extensive interactions between NTD and LBD permit allosteric coupling and hence changes in the channel open probability in response to NTD ligands (6). Nevertheless, the flexible ECR assembly in non-NMDARs permits dynamic rearrangements, which appears to facilitate interactions with other synaptic components, including auxiliary subunits in cis (9) and perhaps other proteins in trans, i.e., via presynaptic interactions (33)(34)(35), as has been described for GluDs (36,37). These reconfigurations could drastically alter the allosteric landscape of non-NMDARs and their anchorage/diffusion at synapses.
To provide a better understanding of the iGluR ECR, a strategic drug target, we revisit NTD dynamics at the level of the monomer, dimer, and tetramer, as well as whole-receptor dynamics. We review recent data on NTD structure/dynamics and further elaborate on NTD dynamics at the level of the monomer, the dimer, and the intact receptor tetramer using coarse-grained and all-atom molecular-dynamics (MD) simulations. Motions are benchmarked against the fold-related mGluR and GABA B ligand-binding cores (LBCs), which we also investigate in this study.

MATERIALS AND METHODS
The anisotropic network model (ANM) treats each residue as a node at the Ca position, with interactions within a cutoff distance of 15 Å modeled as springs using harmonic potentials with uniform spring constants for all interacting residues (38). The ANM yields a unique spectrum of modes for each examined structure, ranging from low-frequency (or soft) modes to high-frequency (or stiff) modes. Each mode k (3N-6 of them for a structure of N residues) is characterized by a shape (3N-dimensional eigenvector u k ) and frequency (eigenvalue l k ) determined by eigenvalue decomposition of the 3N Â 3N Hessian matrix H. Mode 1 is the softest mode (with the smallest eigenvalue, or effective force constant), mode 2 is the next softest, and so on. Soft modes are usually highly distributive, as illustrated in Movies S3, S4, and S5. The modes at the other end of the spectrum are highly localized. To generate a conformation along a given mode k, we add the quantity [5 al k À1/2 u k ] to the 3N-dimensional position vector R composed of the original Ca coordinates, where the arbitrary coefficient a is varied to generate the movies as described earlier (39). We chose these multiplication factors so that we could clearly illustrate each motion. We used the ANM web server (40) and PyMOL (41) when creating the movies and the panels containing structures shown in Figs. 2, 3, and 5 and Fig. S2 in the Supporting Material. The amplitude of motion for a given mode is taken as the square displacements along that mode that scale with l k À1 . As such, we were able to compare the relative mode amplitudes by comparing the eigenvalues. All comparisons in this work (Figs. 2, C and D, and 3 C) were made relative FIGURE  (64). The dashed red circles in GluA3 and GluN1 highlight the lack of LL packing. A more detailed analysis of NTD dimer interfaces can be found in our recent studies (32,56,57). To see this figure in color, go online.
Biophysical Journal 109(6) 1136-1148 iGluR NTD Structure and Dynamics to the interlobe twisting motions of an open mGlu1 clamshell (PDB ID: 1EWV) (30). The normalized amplitude of each ANM mode (in arbitrary units) was taken as a fraction of the normalized amplitude of mGlu1 mode 1 by dividing mGlu1 eigenvector 1 by the eigenvector of the mode with which it was compared. Long N-and C-terminal extensions were cleaned when necessary. The structures used are listed in Table S1 along with their eigenvalues and relative amplitudes.
To compare the ANM modes with experimentally observed structural differences (Figs. 2, E-G, 3 B, and S2 B), we first calculated the vector corresponding to the structural differences (displacements of corresponding Ca atoms) and then calculated the correlation cosine (or overlap) between this vector and each ANM mode vector. Cumulative overlaps are defined as the square root of the sum of correlation cosines squared. In a similar way, we compared ANM modes between smaller and larger systems (Fig. 4). We used subsets of the mode vectors for the large systems that corresponded to only those residues found in the relevant small systems. We ran all calculations in Python using the ProDy package (42).
We carried out all-atom MD simulations using GROMACS 4.6 (43) and the CHARMM27 force field (44). Bonds that contained hydrogen atoms were constrained with LINCS (45), allowing 2 fs time steps. The starting structure was an isolated NTD dimer (PDB ID: 3H5V) (46), which was imbedded in a rectangular box extending at least 9 Å away from the protein in any direction. This was filled with TIP3P water molecules (47), which were then randomly replaced with sodium and chloride ions to neutralize the system and reach a concentration of 70 mM (a total of 53 sodium and 49 chloride ions). Two rounds of steepestdescent energy minimization (5000 steps), NVT equilibration (1 ns), and NPT equilibration (1 ns) were performed before production MD. The first round included restraints on the protein heavy atoms to equilibrate the solvent around the protein. The temperature was set at 300 K by means of a stochastic velocity rescaling thermostat (48) throughout. The pressure was set at 1 bar using the Berendsen barostat (49) during equilibration and the Parrinello-Rahman barostat (50) during production MD. Short-range noncovalent interactions were cut off at 12 Å using the particle-mesh Ewald method for long-range electrostatics (51), and van der Waals interactions were smoothly switched off between 10 Å and 12 Å . This protocol was carried out for two independent replicas, each lasting 100 ns.
The homology model of TARP g-2 shown in Fig. 5 was generated using MODELLER (52). Template selection and alignment were carried out using the HHPred server (53,54).
iGluR NTDs have a PBP-like clamshell structure with a cleft separating the upper lobe (UL) and lower lobe (LL) (Figs. 1 B and 2 B), which are connected via three peptide hinges (a characteristic of type I PBPs) (69). NTDs show an intermediate cleft angle compared with other, allosterically active PBPs (38), which is similar across all AMPARs (56) and KARs (59,60). Current structures of NMDAR NTDs appear to be in a closed-clamshell conformation, in both the absence and presence of the GluN2 cleft ligand Zn 2þ (62,63); hence, cleft motions have only been inferred from simulations (56,70) and have not been observed crystallographically.
A number of differences among the AMPAR, KAR, and allosterically active NMDAR NTDs are evident. At the level of the monomers, the following features stand out (Fig. 1 B).
1) The NMDAR NTD lobes are uniquely twisted (62,63,71), creating a coordination site for zinc. The GluK5 NTD also exhibits an interlobe twist relative to the other AMPARs and KARs (59), although to a much lesser extent. 2) A wing-like helical extension of hinge 2 is seen in AMPAR and KAR NTDs. The AMPAR NTD wing is highly dynamic in all-atom MD simulations (56) and thus may be of functional relevance. This segment is absent in NMDARs, which likely permits increased intraprotomer dynamics. 3) A loop facing the dimer interface varies in length, and this side loop is shortest in the AMPARs. The side loop projects into the dimer interface in KARs and has been suggested to affect receptor assembly (60). 4) Loop variation is also apparent at the top of the protomer, with this top loop, or flap, being longest in AMPARs, also suggested to play a role in subfamily-selective assembly (46,55,60,72). Collectively, these features likely impact intra-and interprotomer dynamics, which are described below.
Similarly to other receptor families that harbor NTD-like modules (26,27,29,30), iGluR NTD dimers may be the functional unit, i.e., ligands binding to monomers will result in dimer rearrangements that in turn trigger allosteric signal transmission. NTDs assemble into dimers with their clefts facing in opposite directions, and, as noted above, dimeric packing differs between iGluR subfamilies. AMPAR and KAR NTDs exhibit more extensive associations involving both the UL and LL, whereas NMDAR dimers are mostly held together via their ULs, which will facilitate intradimer motions ( Fig. 1 C). AMPAR NTD dimers generally show tighter packing between the ULs, as evidenced by their higher local atomic contact density (LD) (24) (quantified in Fig. 1 C and previous studies (32,56,57)). This feature contrasts with KAR NTD dimers, which exhibit more uniform LDs between the ULs and LLs (32). Within the AMPAR subfamily, differences are mostly seen across the variable LL dimer interface, with GluA2 and GluA3 at the extremes showing extensive and minimal LL packing, respectively ( Fig. 1 C) (32,56), in line with dimer affinities measured by analytical ultracentrifugation (61,64,72,73).

iGluR NTD monomers can undergo classical PBP cleft motions
To elucidate the dynamic spectrum of iGluR NTDs, we utilized all-atom MD simulations and elastic network model calculations. In particular, we used the ANM, an elastic network model in which each residue is represented as a node centered at its Ca position with springs connecting interacting residues (74,75). The resulting harmonic potential can be solved analytically to determine all possible motions, which are decomposed into a series of normal modes. The first, energetically favorable (low-frequency) modes describe correlated, global motions, whereas high-frequency modes (denoted by higher mode numbers) represent localized motions. Previous ANM analyses provided first insights into the dynamics of the AMPAR and NMDAR NTDs (56,57,70). Here, we extend those analyses by comparing these NTDs with the other non-NMDAR subfamily, KAR, as well as the related LBCs of mGluRs and GABA B receptors.
The two most accessible global modes at the level of NTD monomers exhibit interlobe twisting (Fig. 2, A and C) and opening/closing motions of the clamshell cleft ( Fig. 2, B and D) (56,57). These NTD cleft motions are observed to various degrees, presumably depending on the specific interactions that constrain the hinges connecting the two lobes. They are also apparent in the related mGluRs (30) and the iGluR LBDs (type II PBPs) (76). Although no cleft ligand has been identified yet for non-NMDAR NTD clefts, electron density has been observed in GluA2 (55,57) and GluK2 (60). Interlobe twisting has been described for NMDAR NTDs (70,71) and also in the GluA2 LBD, where it has been linked to partial agonist binding (77).
In line with our previous ANM analysis of AMPAR and NMDAR NTDs (56), we find that both modes of motion are conserved across the iGluRs, but the extent varies (Fig. 2, C and D). For example, both the low-affinity KARs (GluK2 and K3) and the high-affinity KAR (GluK5) are very similar to AMPARs (red and blue blocks in Fig. S1, A and B). There are, however, subtle differences due to an extension of the wing toward the LL, especially in the low-affinity KARs, causing the twisting mode to exhibit a cleft-closure component. To benchmark motions in iGluR NTDs and to aid comparison, we analyzed the dynamics of the related modules from mGluRs (mGlu1 subunit in open and closed conformations) and GABA B receptors (GB1 and GB2 subunits), which are allosterically active receptor PBPs that have been crystallized in various ligand-bound states (29,30,78,79).
Despite its large wing element, the twisting motion is largest for the open mGlu1 monomer, and the remaining structures are shown relative to this value (Fig. 2 C; Table  S1). Most iGluR NTDs (including GluN2B) cluster together on the extent scale, with the exception of GluN1, which aligns more closely with the GABA B modules (yellow cluster). GABA B receptors may indeed use a twist motion for activation. We find that a rotation of the GB2 LL toward the dimer interface is apparent when the apo and GABAbound structures are superposed (PDB ID: 4MQE and 4MS3) (29). The same two conformations are also observed in crystal structures of GB2 extracellular domain alone (78). We next assessed how well this twisting of GB2 corresponds to its ANM modes. For this purpose, we first calculated the vector of differences for the Ca position of each corresponding GB2 residue between the two heterodimeric structures. Next, we calculated the overlap between this vector and each ANM mode vector as a correlation cosine (blue bars in Fig. 2 E). This analysis indeed showed that ANM mode 1 of GB2 from the apo structure (asterisk in Fig. 2 E) could account for the difference between these two crystallographically observed GB2 conformations. Interestingly, GluN2B, where twisting has been observed structurally (63), and which has been linked to function (71), clusters with the AMPAR and KAR NTDs (Fig. 2 C, blue cluster). Hence, AMPAR and KAR NTD protomers also have capacity to twist and untwist to an extent similar to that observed for GluN2B. Accordingly, we find that AMPAR and KAR NTDs have the intrinsic ability to twist toward the conformation seen in GluN2B via ANM mode 1 (shown for GluA3 in Fig. 2 F).
Classic PBP cleft motions (30) are also observed for all NTD-like protomers analyzed, with the degree of motion again illustrated relative to mGlu1 twisting (Fig. 2 D; Table  S1). We can see that the extent and overall order of cleft motions are generally comparable to the twisting. However, we observe no clear segregation between the known allosteric modules (i.e., NMDA and GABA B ) and non-NMDA iGluRs at this level. Moreover, GB2 (which is not believed to bind ligands) has the ability to undergo cleft motions similar to those observed for the ligand-coordinating GB1 subunit, which closes upon GABA binding (asterisks in Fig. 2 G) (29). This may be linked to transmission of the signal of agonist binding from the GB1 cleft to the G-protein-binding GB2 subunit, which is required for receptor activation (80,81).
In summary, our analysis reveals that NTD (and NTDlike) monomers of these receptors share two major types of intraprotomer motions, twisting and cleft opening/ closure, consistent with the intrinsic flexibility of the type I PBP fold that they share. The analysis further shows that these modular structures do not exhibit discrete states (or switches between these states) but rather a continuum of conformations along the mode coordinates. The extent of motion is defined by the specific sequence (or side-chain Biophysical Journal 109(6) 1136-1148 iGluR NTD Structure and Dynamics interactions) of the particular receptor or by the ligands (e.g., Zn 2þ ) that stabilize particular conformers, whereas the global hinge mechanism is robustly retained. These intraprotomer motions are coupled to dimeric rearrangements as described in the next section.

iGluR NTD dimers undergo rearrangements similar to those observed in mGluRs
Dimeric rearrangements of NTD-like modules in response to ligand are believed to trigger allosteric signal transduction in type C GPCRs. This has been documented in mGluRs, where ligand-free NTD dimers appear to exist in equilibrium between displaced and parallel dimer conformations, both of which were captured by crystallization (Fig. 3 A) (30) and were apparent in previous ANM simulations (57). The more parallel conformation is favored in the agonist-bound, closed-cleft state (the active (A) state) and the displaced conformation (the resting (R) state) (Fig. 3 A) (30). These states are characterized by an intradimer rotation mediated by the UL dimer interface. Negatively charged residues at the LL dimer interface couple the cleft conformation to the dimeric state: the A state is unstable with both clefts open and can only be obtained with at least one cleft closed (30). Cations can bind and stabilize this LL dimer interface, enabling the closure of both clefts (82) and maximal receptor activity (83). This cation modulation has been observed in both heterologously expressed mGluRs and neurons (84). As noted previously (57), a single ANM mode (the first and thus lowest-energy mode, M1), which features an interprotomer counterrotation, accounts for most of the transition between the R and A states (Fig. 3 B). These findings  Table S1. (E) The ANM modes of the GB2 NTD-like module are compared against the conformational transition induced upon activation of the dimer by GABA binding to the GB1 subunit. Correlation cosines (or overlaps) are shown as blue bars. The red curve shows the cumulative overlap (root mean square of the correlation cosines). A twisting motion stands out (the asterisk marks mode 1). (F) The twisting motion (the asterisk marks mode 1) also stands out when we assess the overlaps between the ANM modes of the GluA3 NTD monomer and the difference between the structures of GluA3 and GluN2B NTD monomers. (G) A comparison of the ANM modes of the GB1 module against its conformational change upon GABA binding reveals a cleft closure motion (the asterisk marks mode 2).
Analogous dimer rotations are evident in ANM simulations of GABA B extracellular domain dimers (mode 1; Fig. S2 A). However, the crystal structures of this domain (both apo and GABA bound) contain parallel dimers (29); the major difference between the two states appears to be a closure of the LL dimer interface. A comparison of the GABA-induced conformation change with ANM modes accessible to the apo form (Fig. S2 B) shows that activation involves both dimer rotation (albeit small) and LL closure (mode 2), as well as intraprotomer conformational changes (twisting and cleft closure), which are seen to various extents in higher-frequency modes 3-8. This analysis highlights parallels and differences in the dynamic spectra of these closely related structures.
Dynamics related to mGluR1 can be inferred from GluN1/2B NTD heterodimeric structures (64). The NMDAR NTD dimer can access a similar mode of interprotomer counterrotation to reach a parallel, A-like state (Fig. S2 C) (56). In this case, the A state could be stabilized by polyamines (e.g., spermine), positive allosteric modulators that bind to the LL dimer interface to alleviate likecharge repulsion (24). Contrary to the case in mGluRs, it has been suggested that in NMDARs the A state is coupled to an open cleft and that cleft closure drives it to the R state (resulting in negative modulation). This prediction is confirmed by our ANM analysis (Fig. S2 C; Movie S1), which shows that cleft motions are apparent in GluN1 but reduced in GluN2B. This is readily explained by the formation of an interface between the GluN2B LL and the GluN1 UL (restricting GluN2B cleft motions). The  Table S1. (D) Right: GluA3 was captured in a conformation resembling the mGluR A state. Left: ANM mode 1 enables the GluA3 NTD dimer to reach a conformation resembling the R state. (E) NTD dimers also exhibit cleft motions in the ANM (56) and in all-atom MD simulations. Variations in the Ca distance between A47 and D193 in simulations of a GluA2 NTD dimer (PDB ID: 3H5V) (46) reveal an interlobe twist similar to that of monomer ANM Mode 1. The first 25 ns portion of one simulation is shown for this distance in one subunit. The horizontal stippled line denotes the 17.5 Å Ca distance seen in the crystal structure. The GluA2 NTD structure on the right shows the position of the marker residues (A47, blue; D193, red) and the distance between their Ca residues (stippled line between dark gray spheres).
Biophysical Journal 109(6) 1136-1148 iGluR NTD Structure and Dynamics heterodimer is trapped in an R-like, displaced state by the NMDAR-negative allosteric modulator ifenprodil, which binds to the UL dimer interface (64). Ifenprodil derivatives hold promise for the development of subtype-selective NMDAR-negative modulators.
Related dimer rotations are also accessible to AMPAR (57) and KAR NTDs, despite their more extensive dimer associations. This is most evident for GluA3, which stands out from other AMPARs and KARs in a map of relative mobilities (Fig. 3, C and D; see also Table S1) due to its looser LL packing (Fig. 1 D) (56,57). The dynamics of GluA3 NTDs can be explained by their substantially weaker dimer associations (72) that uniquely give rise to a variety of dimeric arrangements in GluA3 NTD crystal structures (57) (I.H.G., J. Garcia, and B. Herguedas, unpublished data). The loosely packed GluA3 conformation resembles the mGluR A state (57), with a root mean-squared deviation in the positions of the corresponding Ca atoms of~5 Å (when comparing the 3O21 dimer CD with 1ISR and 1EWK). This arrangement is likely to be unstable due to like-charge repulsion of the LLs in the absence of a stabilizing anion, analogous to the situation for mGluRs and cations (57). The functional consequence of this unique behavior in GluA3, relative to the other AMPAR NTD paralogs, remains to be determined.
We observe that dimer rotations are coupled to concerted intraprotomer twisting and cleft motions in mode 1 of all AMPARs and KARs, as well as the agonist-bound GABA B module, which features tighter LL associations (compared with the apo form). In particular, the LLs twist upward (closing the cleft) and toward the dimer interface as the protomers become displaced (R-like) (shown for GluA2 in Movie S2). Thus, the AMPAR and KAR NTD dimers approach an shows the overlap between modes of monomer motions (ordinate, label on far right and shared by AMPARs and NMDARs) and dimer motions (abscissa). A zoom into the first few elements illustrates that many dimer modes show a significant overlap with both prominent modes of monomer motion. The matrix above (panel 2) compares the same dimer modes of motion (shared abscissa) with the modes of motion of the NTD tetramer (ordinate, label on far right). In this case, there are many darker blocks, indicating a higher correlation. The top-left matrix shows the overlap between the motions of the NTD tetramer and those of the whole receptor. The starting structure for the AMPAR is a GluA2 homotetramer (18); the NMDAR is a GluN1/2B heteromer (PDB ID: 4PE5) (16). Lines connect some modes of motion that show good correlations all the way from monomers to whole receptors. Dimeric motions are more conserved through the levels in the NMDAR, as illustrated for dimer mode 1, where dark dots illustrate good correlations along the way and the endpoints are the circled modes 6-8 of the whole NMDAR. The monomer shown is GluN1, where cleft motions are also well retained. In the AMPAR monomer, cleft motions are again retained but dimer rearrangements are dampened in the whole receptor. This results in poor correlations transitioning from the NTD tetramer to the whole receptor (white dots and stippled trapezium). Also evident is a higher dominance of tetramer motions in lower-frequency modes of the NMDAR, where the tighter ECR packing restricts rearrangements in which the NTD moves as a rigid body.
Biophysical Journal 109(6) 1136-1148 NMDAR-like structure in which the LL of one subunit contacts the UL of the other. In addition to showing the same motion as the AMPAR NTD dimers in this mode, the dimeric KAR NTD behavior is very similar to that of the AMPARs up to~15 ANM modes (Fig. S1 C). The GluK5 KAR NTD homodimer has an intermediate structure along this mode, showing greater intraprotomer twisting and interprotomer displacement relative to the other AMPARs and KARs. The retention of intraprotomer modes of motion after dimerization was also apparent in our previous analysis of AMPAR NTD dynamics (56). This coupling likely results from the packing between LLs, and cleft motions are reduced as LL associations loosen up (for a given extent of rotation). As in previous all-atom MD simulations of AMPAR NTDs (56), we observed cleft twisting in our allatom MD simulations of a GluA2 dimer (PDB ID: 3H5V), especially during the first 10 ns, during which time the LL of one chain twisted toward the dimer interface, thereby decreasing the distance between D193 and A47 to~14 Å (compared with 17.5 Å in the crystal structure; Fig. 3 E).
In addition to enabling dimer rotations and clamshell motions (56,57), the looser packing in the GluA3 NTD homodimer also permits a splaying apart of the LLs, which has been observed in all-atom MD simulations (56). This motion resembles that observed in NPRs, where NTD-like dimers also exhibit an opening between LLs, and closure is triggered via binding of a cyclic peptide (natriuretic peptide) to the LL dimer interface (26). Protomer counterrotations have also been observed in NPR crystal structures (27,28), and our ANM analysis shows that both LL interface closure and protomer counterrotations are important for peptide-induced conformational changes in both NPR-A and NPR-C (data not shown). LL dimer interface opening and closure are also apparent in an ANM analysis of GluA3 (mode 4), the A state mGluR1 (mode 3), and the GABA B apo form (mode 2 discussed above).
Overall, a common theme for receptors harboring type I PBP dimeric modules emerges in which intraprotomer cleft motions are coupled to displacement and LL closure of NTD-like dimers. This set of motions appears to form a principal pathway of signal propagation for these diverse receptor families. Next, we examine how dimer dynamics are transmitted within the tetrameric iGluR ECR.
Monomer and dimer motions are coupled to larger rearrangements in NTD tetramers and intact iGluRs NTD dimers associate via a small interface (~400 Å ) in AMPARs and KARs. These tetramer contacts are mediated by the LLs and are apparent in some isolated NTD structures (46,55,(59)(60)(61) as well as in intact receptors (18)(19)(20)(66)(67)(68). Helices engaged in this interface (aF and aG in GluA2) show structural heterogeneity (57) and are highly dynamic in solution when analyzed at the single-molecule level (85) as well as in all-atom MD simulations (56). Together with the global motions described above, these local fluctuations could impact the formation, stability, and dynamics of the NTD layer. NMDAR NTD dimers appear to come together via an interface involving related segments in the GluN2B LL, although the overall arrangement of the NTD layer diverges (16,17).
We recently studied the global dynamics of whole AMPARs and NMDARs, and found unexpected similarities between the two iGluRs (22). We observed some conservation of NTD tetramer and dimer dynamics in intact iGluRs (22), which, together with the finding that NTD monomer dynamics are conserved in the dimer (28), suggests that these motions persist in whole receptors. Here, to unravel allosteric communication in iGluR ECRs, we focused on this hierarchy of motions, i.e., how monomer and dimer dynamics relate to motions in NTD tetramers and whole receptors. For this purpose, we performed a stepwise comparison of smaller and larger systems, i.e., we compared NTD monomers with dimers, NTD dimers with tetramers, and the isolated NTD tetramer layer with whole receptors. These comparisons are depicted in correlation matrices arranged end to end for the level that comprises a large system in one and a small system in the next (gray arrows, Fig. 4). Each matrix element represents the overlap between the directions of motion of the selected small system in isolation and in the context of the larger system that contains it, for a given ANM mode of each system. We did not perform this analysis on KARs because no sufficiently complete wholereceptor structure is available and KAR NTD tetramers behave the same as AMPAR NTD tetramers (Fig. S1 D).
First, we investigated the interplay of ECR dynamics in NMDAR, which is of functional consequence (6). In this receptor, the two ECR layers interdigitate, which presumably restricts their dynamics (22) but at the same time forms an allosteric unit (16,17). Localized motions in the NTD layer, such as cleft opening/twisting in the protomers and dimeric rotations, are indeed still apparent and are affected by the packing within and between ECR layers. As outlined in Fig. 4 A (and detailed in the legend), we observe significant conservation of the two dominant monomer motions (Fig. 2,  A and B) in the first global modes of the dimers (Fig. 4 A, panel 1 and zoom) as indicated by red and blue elements, which represent high correlations (positive and negative overlaps, respectively; this sign is not relevant, as ANM modes are harmonic fluctuations with arbitrary starting directions). At the next level, dimer motions are retained in isolated tetramers (Fig. 4 A, panel 2), and the tetramer modes of motion that contain these also show good correlations in the whole receptor (Fig. 4 A, panel 3).
Whole-receptor modes 6-8 ( Fig. 4 A, panel 3, M6-M8) are of particular interest because they feature NMDAR clamshell dynamics. As shown above, these are more pronounced in GluN1 and less so in GluN2B. GluN2B is known to bind Zn 2þ ions, which trigger an allosteric Biophysical Journal 109(6) 1136-1148 iGluR NTD Structure and Dynamics cascade, ultimately resulting in a decreased channel open probability (15,86). Zn 2þ has been suggested to stabilize the closed-cleft conformation to initiate this negative modulation (6,10,24,63,86). Focusing on mode 6, we observe that GluN1 cleft closure triggers a displaced R-like state, whereas GluN1 cleft opening is coupled to the dimer adopting a more parallel, A-like conformation (Movie S3; Fig. S2  C). This state could be stabilized by polyamine binding between the LLs, which would potentiate the channel (24). Dimer rotations are associated with substantial reorientations of the NTD layer and the entire ECR. Approximation of the dimer A state is accompanied by a hinging at the tetramer interface that results in a more upright NTD layer that makes less contact with LBD, as well as a stretching of the LBD-TMD linkers. Related to this, our recent study (22) showed that modes related to mode 6 bring the NMDAR toward an AMPAR-like structure, highlighting the close similarities in the dynamic spectra of the two subfamilies. Conversely, the transition toward the R state results in a flipping toward a horizontal NTD layer, with the GluN2B subunits pushing down on the LBD (Movie S3), which could explain the finding that zinc binding to the NTD destabilizes the LBD dimers, resulting in a desensitized-like state (86).
For the GluA2 AMPAR, NTD clamshell motions are also evident in isolated NTD tetramers and the whole receptor (Fig. 4 B). Like NMDAR, AMPAR shows a clear conservation of monomer dynamics in the dimers and dimer motions in the NTD tetramer (compare correlations in Fig. 4, A and B, panels 1 and 2). However, in the whole AMPAR (panel 3), these are found in higher ANM modes, which are characterized by more localized (high-frequency) motions. This may result from the looser packing of the AMPAR ECR, which enables a greater variety of global rearrangements (see below) (22). Another difference is that in AMPAR, NTD dimer rotations can be discerned in the isolated NTD tetramer but are reduced in the intact receptor. This likely results from the tighter LL packing of AMPAR (GluA2) NTD dimers. This difference is illustrated in Fig. 4 B (panels 2 and  3), where higher tetramer modes that correlate well with the dimer (dark dots in panel 2) exhibit lower overlaps in the whole receptor (top trapezium in panel 3), whereas tetramer modes that correlate poorly with those of the dimer (white dots in panel 2) show higher overlaps in the whole receptor (bottom trapezium in panel 3).
Surprisingly, two modes of NTD tetrameric motion dominate the dynamics of the intact AMPAR (each correlating well with multiple whole AMPAR modes): 1) NTD dimer sliding (tread-climb mode, e.g., mode 11; Movie S4) and 2) rotation of the NTD dimers toward each other (e.g., mode 12; Movie S5). These motions resemble those seen in NMDAR modes 6-8 and are likewise accompanied by an approximation of the NTD and LBD at one extreme of the motion. Unlike the case with NMDAR, in AMPAR the NTD dimers are approximately symmetric, with both clefts equally able to undergo clamshell motions. The NTDs that form the tetramer interface (i.e., the inner or proximal chains, equivalent to GluN2) are coupled to hinging of the NTD tetramer interface, resulting in increased cleft motions in these chains. A clear cleft opening/closure component is seen in mode 11, whereas a twisting motion dominates in mode 12. Whether this capacity for intrinsic motions in the AMPAR ECR is related to allosteric communication remains to be elucidated.

Large-scale AMPAR NTD rearrangements, global bending motions, and interactions with synaptic components
In addition to internal rearrangements, including those described above, the AMPAR tetramer interface can also rupture to enable rearrangements associated with receptor desensitization (19)(20)(21). In KARs, which have an ECR organization closely resembling that of AMPAR, the NTD tetramers do not seem to dissociate (20,67), which may be due to the greater stability of the KAR tetrameric interface (K d 1 5 mM) (61) compared with AMPAR NTD tetramers (at least 10-fold weaker) (46,55,72,73). The relative mobilities in the first few ANM modes of GluK2 and GluA2 NTD tetramers are consistent with the relative stability of the GluK2 versus GluA2 ECRs (data not shown).
At excitatory synapses, iGluRs are in close proximity to a multitude of pre-and postsynaptic components, which may vary in a synapse-specific fashion. Large-scale receptor motions could impact interactions with synaptic proteins. Non-NMDARs are also associated with auxiliary subunits, both secreted and membrane bound, many of which have been identified for AMPARs (87)(88)(89)(90) and modulate their trafficking and gating (91). We recently showed that transmembrane AMPAR regulatory proteins (TARPs) can interact with the NTD in vitro (9). A cross talk between the membrane-distal NTD with the membrane-proximal TARP would require substantial movements of the ECR, resulting in receptor reconfiguration, which could impact receptor function, diffusion, and ultimately synapse organization. Bending motions, which would facilitate such interactions, are indeed apparent in AMPAR and NMDAR ANM. In fact, the first and hence highest-amplitude mode of the intact AMPAR features this motion (Fig. 5). We note that the energy/frequency of such large bending events cannot be accurately predicted from the ANM as a result of the use of uniform spring constants. Conformational changes within the NTD layer and global rearrangements of the ECR may also have an impact on interactions with N-cadherin (34), secreted pentraxins (33,35), and other, yet to be identified components. This cross talk may even be related to the reported synaptogenic role of the GluA2 NTD (92)(93)(94), a function that is well established for the related GluD1 and GluD2 NTDs (36,37).

CONCLUSION
iGluRs have a unique architecture among other ligand-gated channels, which is characterized by a large, modular ECR (accounting for 80% of the receptor mass). The ECR is made up of eight PBP-like clamshell domains that are connected via flexible linkers between the ECR layers and are associated via interfaces, featuring a range of affinities, within the layers (72,73,95). This architecture offers a multitude of motions and sites for drug action, such as the clamshell cleft, dimer and tetramer interfaces, and interdomain regions connected by linkers. Here, we analyzed the NTD modules in a hierarchical manner by examining their monomeric, dimeric, tetrameric, and whole-receptor dynamics using existing crystal structures in combination with the ANM (75) and all-atom MD simulations. In line with earlier studies (22), we found differences and similarities between the NMDAR and AMPAR ECR dynamic spectra at all hierarchical levels.
At the monomer level, interlobe twisting and opening/ closure motions are seen across the spectrum of NTD-like modules (Fig. 2). Surprisingly, twisting, which was first observed in GluN2B, is widely seen and therefore may not be unique to NMDAR NTDs. In fact, the ability to undergo this motion is equally accessible to AMPAR and KAR NTDs, and is prominent in mGluRs despite their large wing element. The wing is mostly packed against the UL but exhibits contacts (predominantly water mediated) with the LL in AMPARs, a structural element that is missing in NMDAR NTDs. No clashes are apparent upon visual examination of these AMPAR ANM modes, suggesting that the wing does not restrict these motions. At the level of the dimer, dominant motions include interprotomer rotations and opening of the LL dimer interface. These two themes are seen to various extents and levels across the examined structures, and are likely a core mechanism of signal transmission in receptors containing NTD-like signaling modules. For example, in the GABA B receptor, the LL dimer opening/closing motion dominates with a smaller rotational component. Not surprisingly, we generally observe that large-scale motions in isolated systems (i.e., a PBP-like monomer) are restricted in larger systems to various extents. Nevertheless, motions of individual clamshells, including opening/closing and lobe twisting, are still apparent in intact receptors, where they are expected to trigger complex allosteric pathways that ultimately control channel gating.
Several key questions remain to be answered. Are the signs of non-NMDAR NTD dynamics functionally relevant or is the NTD in these iGluR subfamilies an orphan PBP that lost its signaling function (a scenario that is somewhat related to inactive enzymes (96,97))? In this case, the sole role of the NTD would be restricted to subunit assembly (72,98) and synapse organization/synaptogenesis (33,92). How is allosteric communication via the NMDAR NTDs transmitted? Specifically, does Zn 2þ binding trigger GluN2B NTD cleft closure followed by (largescale) dimeric rearrangements (as shown in Figs. S2 C and Fig. 3) or are localized changes surrounding the Zn 2þ FIGURE 5 ANM of the whole GluA2 AMPAR shows global bending motions that could bring the NTD into proximity with auxiliary subunits such as TARPs. An AMPAR crystal structure (PDB ID: 3KG2) (18) is shown on the left, with the two nonequivalent chain pairs colored in red (distal from the NTD tetramer interface) and gray (proximal and interface forming). A homology model of TARP g-2 based on the related claudins is shown in cyan.
The structure on the right shows a conformation along ANM mode 1 in which the NTD bends down and can contact the TARP. To see this figure in color, go online.
Biophysical Journal 109(6) 1136-1148 iGluR NTD Structure and Dynamics coordination site (His-127 and Glu-284 in GluN2B) propagated to the dimer interface and the GluN1 NTD? We find that the GluN1 NTD exhibits large cleft motions, consistent with GluN1 allosteric activity (71). Moreover, the GluN2 NTD-LBD linkers, which have been shown to mediate NTD signaling, are packed between the NTD and LBD layers, forming an interface as revealed in current crystal structures (16,17). How this interface and the NTD-LBD linkers, particularly of GluN2B (10,15), mediate NTD signaling remains to be elucidated. The rapid progress that is being made in electron cryo-microscopy (20,99) is expected to increase the suite of intact iGluR structures and reveal conformational intermediates. These will serve as substrates for further simulations to ultimately reveal the complex allosteric routes through these large signaling machines.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Macrophage‐mediated cholesterol handling in atherosclerosis

Abstract Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

Introduction
Macrophages are key players in all stages of atherosclerosis. Initially, monocytes attracted by pro-inflammatory signals coming from the inflamed endothelium attach to the problematic arterial sites and infiltrate the intima [1]. To be attractive for monocytes and other immune cells, the problematic vascular sites should be injured or atheroprone because of the abnormal haemodynamic forces and/or accumulation of oxidized lipids in the arterial wall [1]. In the subendothelial layer, monocytes differentiate to macrophages that in turn polarize to pro-inflammatory/anti-inflammatory phenotype depending on the local stimuli and transform to foam cells [2].
Formation of foam cells that occurs in the initial stages of atherogenesis is a hallmark of atherosclerotic disease [3,4]. Increased uptake of oxidized low-density lipoprotein (oxLDL) and/ or reduced cholesterol efflux leads to the deposition of esterified cholesterol in the cytoplasm of macrophages and generation of foam cells [5]. In macrophages, oxLDL is taken up with help of scavenger receptors (SR) such as CD36 (also known as fatty acid translocase), SR-A1 and lectin-like oxLDL receptor-1 (LOX-1) (Fig. 1) [6]. Acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) and neutral cholesteryl ester hydrolase (nCEH) are involved in the formation of cholesterol esters [7]. ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI contribute to the reverse transport of cholesterol from macrophages [8].
The purpose of this review is to characterize key mechanisms of cholesterol efflux, specifically in macrophages. In this work we briefly consider all constituents of the system responsible for cholesterol metabolism in macrophages.

Cholesterol uptake
Cholesterol enters the macrophage cytoplasm through mechanisms of SR-dependent phagocytosis and pinocytosis [9]. CD36 and SR-A are principal contributors to cholesterol uptake accounting up to 90% of oxLDL loading in macrophages [10].

Scavenger receptor CD36
CD36 is a 88 kD glycoprotein that is a member of SR class B family [11]. The human CD36 gene is located on chromosome 7q11. 2 [11]. This receptor includes two extracellular and two transmembrane domains that flank the extracellular domain [12]. CD36 binds oxLDL with high affinity. A major oxLDL-binding site is located between amino acids (a.a.) 127 and 279, with an additional site located between a.a. 28-93 [13]. Interaction of oxLDL with CD36 causes endocytosis of CD36-oxLDL complex via lipid raft-dependent mechanism [13].
The pro-atherogenic role of oxLDL depends on the abundance of CD36. Handberg et al. [14] found a correlation between the plasma levels of soluble CD36 (sCD36) and indices of insulin resistance, carotid atherosclerosis and fatty liver. Furthermore, increased levels sCD36 were associated with insulin resistance and higher risk of type 2 diabetes [15,16]. Elevated sCD36 levels were detected in the monocytes of patients with coronary artery disease [17] and acute coronary syndrome [18]. Statin therapy reduces sCD36 concentrations and suppresses oxLDL uptake by monocytes and macrophages [19]. Similarly, low molecular CD36 inhibitors were shown to reduce lipid deposits in the arterial wall and improve insulin sensitivity and glucose tolerance [20]. However, patients deficient in CD36, had including CD36, SR-A1, and lectin-like oxLDL receptor-1 (LOX-1). In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and scavenger receptor SR-BI. The main acceptors of free cholesterol from ABCG1 and SR-BI are high density lipoprotein (HDL) and from ABCA1 is apolipoprotein A1 (ApoA1). In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells. advanced atherosclerosis [21]. Indeed, the role of CD36 in atherosclerosis is complicated and should be further studied in detail.
In macrophages, expression of CD36 is regulated by many factors. An antioxidant curcumin up-regulates the CD36 expression through stimulating nuclear erythroid-related factor 2 (Nrf2) [22]. Astaxanthin, an oxidized carotenoid, increases CD36 expression by the activation of the peroxisome proliferator-activated receptor-c (PPAR-c) [23]. Palmitic acid stimulates monocyte production of CD36 by the induction of de novo ceramide synthesis [24] as ceramides reduce CD36 expression and decrease oxLDL uptake by monocytes [25]. Similarly, lipopolysaccharide from the bacterium Porphyromonas gingivalis, a causative agent of gingivitis, up-regulates CD36 in macrophages by stimulating c-Jun/activator protein-1 (AP-1) pathway [26]. Interestingly, a 12-week dietary intake of champignon, Agaricus blazei, by Apolipoprotein E (ApoE)-deficient mice resulted in elevated levels of CD36 and higher lesion instability suggesting for pro-atherogenic properties of this mushroom [27].
Some compounds were shown to down-regulate CD36 expression. For example, tanshinone IIA, a diterpene from a sage herb Salvia miltiorrhiza Bunge, reduces CD36 expression by antagonizing PPAR-c [28]. Squalene (a component of olive oil) and endomorphin-1 (an opioid peptide) decrease CD36 and disrupt lipid overload in macrophages [29,30]. Quercitrin, a plant pigment glycoside, also suppresses CD36 expression in macrophages by altering protein kinase C (PKC)/PPAR-c signalling pathway [31]. Exposure of the macrophages to another flavonoid and antioxidant (kaempferol) prevents nuclear translocation of transcription factor AP-1, thereby inhibiting expression of CD36 [32]. Finally, walnut that is rich in c-3 polyunsaturated fatty acids and antioxidants was shown to display an anti-atherosclerotic activity in ApoE-deficient mice by decreasing the CD36 expression [33]. Indeed, CD36 expression could be nutritionally modulated such that it could represent a valuable tool for the prevention of atherosclerosis.

Scavenger receptor A1
Scavenger receptor-A1 (also known as macrophage SR MSR1 or CD204) belongs to the class A SR family [34]. The human SCARA1 gene encoding this receptor is situated on chromosome 8p22 [35]. Three alternate transcripts are produced from this gene. Isoforms 1 and 2 are functional and able to mediate endocytosis of modified LDL. The isoform 3 cannot internalize oxLDL because of altered processing that leads to the localization of this isoform in the endoplasmic reticulum (ER) making it unable to perform endocytosis. This isoform suppresses the activity of the isoforms 1 and 2 when co-expressed, thereby acting as a negative regulator of SR-A1 expression in macrophages [36]. SR-A1 functions as a homotrimer consisting of three 77-kD glycosylated subunits.
In macrophages, SR-A1 is involved in the uptake of modified LDL. In ApoE mice, SR-A1 knock-down reduces the generation of foam cells and atherosclerosis progression [37]. In LDL receptor-deficient mice, inhibition of either CD36 or SR-A1 alone had atheroprotective effect. However, suppression of both SRs showed no positive effect on atherosclerosis suggesting that compensatory activation of these receptors is sufficient for the intake of modified LDL [38]. Similarly, in hyperlipidemic ApoE-deficient mice, deletion of either CD36 or SR-A1 significantly reduces lipid accumulation in macrophages, but does not diminish atherosclerosis [39]. These data suggest for the existence of alternative mechanisms of lipid uptake by macrophages that are independent of CD36 or SR-A1.
Like CD36, expression of SR-A1 could be influenced by a variety of modulators. Pro-inflammatory cytokines such as tumour necrosis factor a (TNF-a) and interleukin-6 (IL-6) promote SR-A1 expression by activation of transcription nuclear factor (NF)-jB [40]. Indeed, pharmacological inhibition of both cytokines decreases oxLDL accumulation and foam cell formation [41]. Berberine, a plant alkaloid from Berberis sp., was shown to have pro-atherogenic effects on culture mouse monocytes by up-regulating SR-A1 and increasing the cholesterol uptake by suppressing negative cell cycle regulator phosphatase and tensin homologue (PTEN) and thereby preserving protein kinase Akt from PTEN-dependent dephosphorylation [42].
Yang et al. [43] showed that voltage-gated potassium channel Kv1.3 is involved in the modulation of activity of SR-A1. Kv1.3 activation led to increased activity of SR-A1 and enhanced uptake of oxLDL. Indeed, antibody-dependent Kv1.3 blockade resulted in reduced oxLDL entrance, decreased cholesterol esterification and enhanced apoA-I-mediated cholesterol efflux [44]. This finding may have important pharmaceutical consequences as it allows specific targeting and modulation of lipid accumulation in macrophages.
Modulators such as curcumin, polyphenolic extracts of mulberry leaves and hydrogen sulphide (H 2 S) were shown to decrease SR-A1 levels [45]. In ApoE-deficient mice, curcumin induced SR-A1 ubiquitination and calpain-mediated proteolysis in macrophages [45]. Mulberry-derived polyphenols down-regulate SR-A1 through the suppression of PPAR-c [46]. In the vascular system, H 2 S is mainly produced by cystathionine c-lyase (CSE). However, in apoE-deficient mice, this pathway is impaired resulting in reduced H 2 S production. Deregulation of CSE-dependent generation of H 2 S contributes to oxLDL-mediated inflammation in macrophages [47]. H 2 S possesses anti-atherosclerotic properties by decreasing plaque size and production of intercellular adhesion molecule-1 [48]. H 2 S also inhibits foam cell formation by suppressing SR-A1 via K ATP /Erk 1/2 mechanism [49].

Lectin-like oxidized low-density lipoprotein receptor-1
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a membrane glycoprotein that contains a short N-terminal cytoplasmic domain, a transmembrane domain, a neck region that controls receptor oligomerization and an extracellular C-type lectin-like extracellular domain, involved in oxLDL binding [50,51]. Human LOX-1 is encoded by the OLR1 gene located on chromosome 12p13.2-p12. 3 [52]. The gene encodes several LOX-1 isoforms of which the longest isoform contains a 273-a.a. polypeptide [53]. Another variant lacks an exon that results in a frameshift and early stop codon. Indeed, the isoform 2 is shorter than isoform 1 and contains a distinct C-terminus [54].  Compared with CD36, LOX-1 is able to bind moderately modified, but not fully oxidized LDL suggesting for crucial role of this receptor in initial stages of atherosclerosis [55]. LOX-1 is a major oxLDL-binding receptor in endothelial cells, but it could be up-regulated in macrophages in atherosclerosis [56]. LOX-1 could not be found in monocytes, but may be induced in differentiated macrophages [54].
Multiple regulators are involved in the up-regulation of this receptor especially in inflammation. Pro-inflammatory cytokines such as IL-1, interferon (IFN)-c and TNF-a stimulate LOX-1 expression in macrophages [57]. Modified lipids such as oxLDL and products of its hydrolysis (lysophosphatidylcholine) were shown to activate LOX-1 expression in macrophages [51]. Hypertension-related stimuli including angiotensin II and endothelin-1 were found to induce the expression of LOX-1 in macrophages [58]. In diabetic patients, hyperglycaemia and advanced glycation end-products (AGEs) play a key role in the up-regulation of LOX-1 expression in macrophages [59, 60].
Macrophage-specific deletion of LOX-1 did not reveal significant changes in oxLDL uptake compared with wild-type cells [61]. Therefore, in normal conditions, impact of LOX-1 to intake and catabolism of oxLDL by macrophages is small probably because of the high contribution of other SRs. LOX-1 knock-down does not greatly influence oxLDL uptake by non-stimulated macrophages as this receptor contributes for only 5-10% of a total oxLDL intake by macrophages, whereas in pro-inflammatory-stimulated macrophages, the impact of LOX-1 could increase up to 40% [61].
Monocytes were shown to differentiate to dendritic cells (DCs) [62], a common event in atherosclerosis [63]. High levels of LOX-1 expression were found in mature DCs suggesting for a significant contribution to oxLDL uptake because an antibody against LOX-1 decreases oxLDL influx by 48% [64].
A markedly increased expression of LOX-1 was detected in atherosclerotic lesions compared to the normal vascular tissue [55]. In LDL-deficient mice with genetic deletion of LOX-1, a less pronounced development of atherosclerotic plaques was observed in comparison with LDL-deficient mice fed on high-cholesterol diet. In mice, LOX-1 knockout was accompanied by the decreased expression of the pro-inflammatory transcription factor NF-jB and variable pro-inflammatory markers [65]. In contrast, ApoE-deficient mice transgenic for LOX-1 showed advanced atherosclerosis [66]. In rabbits with experimental hypercholesterolaemia, LOX-1 was found to be predominantly expressed in unstable lesions [67]. Intraplaque LOX1 levels correlated with the expression of tissue factor and apoptosis suggesting a possible involvement of this SR to plaque instability [67]. All these observations suggest the pro-atherogenic role of LOX-1.

Formation of cholesterol esters
The rate of cholesterol esterification could determine the fate of macrophages to be transformed to foam cells or not. After engulfment, lipoproteins are transferred to late endosomes/lysosomes where LAL hydrolyses cholesterol esters with the generation of free cholesterol. Free cholesterol in turn is de novo esterified by ACAT1 and stored as lipids in the ER. In case of overload with cholesterol esters, macrophages could be transformed to foam cells (Fig. 2). Indeed, cholesterol excess should be removed from the cell. The nCEH is responsible for the secondary hydrolysis of cholesterol esters [68]. Free cholesterol is then transferred out with cholesterol transporters residing on the plasma membrane. Therefore, balance between hydrolysis and esterification of cholesterol plays a key role in maintaining cholesterol homoeostasis and prevention of generation of foam cells. Two enzymes, nCEH and ACAT1, are crucially involved in this mechanism.
Acyl coenzyme A: cholesterol acyltransferase-1 In macrophages, ACAT1 [or sterol O-acyltransferase 1 (SOAT1)] (EC 2.3.1.26) is mainly located in the tubular rough ER [69]. The human SOAT1 gene resides on chromosome 1q25 and encodes a 550 a.a. polypeptide. The enzyme contains two transmembrane domains [70]. In murine peritoneal macrophages, lack of ACAT1 was reported to elevate free cholesterol levels and increase cholesterol efflux [71]. In opposite, ACAT1 overproduction resulted in an enhanced accumulation of cholesterol esters followed by the formation of foam cells [72]. However, other investigations showed that the role of ACAT1 in atherogenesis is not ordinary. For example, in ApoE-deficient mice, ACAT1 suppression by a small molecular inhibitor F-1394 had an atheroprotective effect [73]. In contrast, macrophage-specific depletion of ACAT1 in mice caused advanced atherosclerosis [74,75]. The discrepancy in these results could be explained by the non-specific action of F-1394 that inhibits both ACAT isoforms (ACAT1 and ACAT2) and therefore has a systemic effect [74]. Human ACAT2 is mainly expressed by intestinal cells and hepatocytes where it is responsible for lipoprotein assembly [76]. In addition, free cholesterol produced by ACAT1-catalysed reaction could be seriously cytotoxic for macrophages and other vascular cells. In macrophages, free cholesterol overload could lead to the formation of cholesterol crystals (Fig. 3) that are highly cytotoxic and may not only impair intracellular cholesterol metabolism but also enhance release of proinflammatory cytokines IL-1b and IL-18 [77]. These processes might progress leading to cell death, accompanied by the release and accumulation of 'free' cholesterol crystals in the extracellular matrix of atherosclerotic lesions (Fig. 4).
The ACAT1 expression and activity are extensively modulated by different signalling messengers. Leptin, a hormone produced by adipose tissue, stimulates ACAT1 expression through Janus-activated kinase 2 (Jak2)/phosphatidylinositide 3-kinase (PI3K) mechanism [78]. Insulin also up-regulates ACAT1 expression in macrophages via extracellular signal-regulated kinase (Erk)/ p38MAP kinase/Jnk-dependent activation of CCAAT/enhancer binding protein a, a transcriptional regulator [79,80]. Except regulation of SR-A1 activity, voltage-gated potassium channel Kv1.3 is involved in the up-regulation of ACAT1 that leads to enhanced uptake of oxLDL and accumulation of cholesterol ethers in macrophages [44].
Neutral cholesteryl ester hydrolase nCEH also known as hormone-sensitive lipase (EC 3.1.1.79) is responsible for the hydrolysis of cholesterol esters to generate free cholesterol for further release from the cell. The human enzyme is encoded by the LIPE gene located on chromosome 19q13.2 [86]. Two transcripts (long and short nCEH isoforms) are produced from this gene. The long isoform is expressed in testis and other steroidogenic tissues where it converts cholesteryl esters to free cholesterol for steroid hormone production. The short isoform is present in adipocytes, macrophages and other cells [87]. nCEH is a 1076-a.a. membrane polypeptide that contains three domains (lipid-binding, catalytic and N-terminal). The N-terminal domain serves as an anchor to recruit nCEH to the ER membrane where the enzyme exposes its catalytic domain to the lumen. nCEH is a glycoprotein, with three N-glycosylated sites at N270, N367 and N389. All these sites are glycosylated, and glycosylation at N270 is essential for catalytic activity [88].
Inhibition of nCEH greatly accelerates formation of foam cells [89]. Overexpression of nCEH results in the increased hydrolysis of cholesterol esters in lipid-laden macrophages [90]. However, nCEH overexpression alone without decreasing the expression of ACAT1 and stimulation of cholesterol efflux was not enough to protect   against atherosclerosis [90]. Transgenic mice overexpressing nCEH and cholesterol acceptor such as ApoA4 do have reduced atherosclerosis [91]. Macrophage-specific overexpression of nCEH also diminished atherosclerosis and decreased plaque necrotic core size in LDL receptor-deficient mice [92] suggesting a critical role of macrophages in lipid handling in atherosclerosis.
Recently, a new neutral cholesterol ester hydrolase (NCEH1; also known as arylacetamide deacetylase-like 1, AADACL1) was found [93]. Human enzyme is encoded by the NCEH1 gene located on chromosome 3 [94]. Like nCEH, NCEH1 resides in the ER membrane and contributes to the hydrolysis of cholesterol esters in macrophages. Knockout of NCEH1 in ApoE-deficient mice promoted the development of atherosclerosis without altering the serum lipid profile [95] suggesting the atheroprotective role of this enzyme. Indeed, both nCEH and NCEH1 are involved in the generation of free cholesterol from cholesterol esters, thereby preventing formation of foam cells from macrophages.
Insulin was found to be involved in the regulation of nCEH expression in macrophages. In the initial stages of type 2 diabetes associated with the hyperfunction of insulin-producing b-cells, increased levels of insulin could down-regulate the nCEH expression in macrophages, thereby contributing to atherogenesis [96]. Interleukin 33, a member of the IL-1 cytokine family, suppresses nCEH in macrophages through the stimulation of ST-2/NF-jB signalling. Furthermore in ApoE-deficient mice, IL-33 significantly reduced accumulation of macrophages in atherosclerotic plaques and generation of foam cells [97].

Cholesterol efflux
Free cholesterol could be removed from macrophages through active transfer mediated by cholesterol transporters or by passive transmembrane diffusion. High density lipoprotein (HDL) or ApoA1 then capture the released cholesterol. Cholesterol transporters such as ABCA1, ABCG1 and SR-Bi play the major role in active free cholesterol efflux.

ATP-binding cassette transporter, ABCA1
The ABCA1, also known as cholesterol efflux regulatory protein, regulates cholesterol efflux and phospholipid homoeostasis [98]. The human ABCA1 gene is located on chromosome 9q31.1 [99]. The 200-kDa transporter is ubiquitously expressed throughout the body. Interestingly, mice deficient for ABCA1 and SR-BI had severe hypocholesterolaemia mainly as a result of HDL loss, but showed no atherosclerosis because of the absence of pro-atherogenic lipids [100]. ApoA1, the major protein component of HDL, serves as an acceptor of cholesterol released by ABCA1 [101]. Unexpectedly, in LDL receptor-deficient mice, hepatic ABCA1 overexpression led to the deposition of pro-atherogenic lipids and advanced atherosclerosis because of the enhanced transfer of HDL cholesterol to LDL and delayed catabolism of cholesterol-enriched LDL [102].
As a result of the key role in cholesterol reverse transport, ABCA1 expression is controlled by various regulators and bioactive molecules. In macrophages, the expression of ABCA1 is regulated by liver X receptor a (LXRa), a nuclear transcription factor [103]. Quercetin was observed to stimulate LXRa through the activation of PPARc that finally results in the up-regulation of ABCA1 transcription and increased cholesterol efflux from macrophages [104]. Apelin-13, a vasoactive peptide, enhances cholesterol out-flow from foam cells through the stimulation of PKCa and inhibition of calpain, a protease that is involved in ubiquitination-mediated ABCA1 degradation [105,106]. Similarly, various proteasome inhibitors and ApoA1, an acceptor of cholesterol transferred by ABCA1, restore cholesterol efflux in macrophages by suppression of ABCA1 degradation [107,108]. S-allylcysteine, a major garlic extract constituent, possesses the atheroprotective activity by increasing ABCA1 expression through unknown mechanism [109]. However, lipid-lowering properties of S-allylcysteine appear to play a secondary role in a summary of its anti-atherogenic effects as this cysteine derivative only moderately inhibited lipid accumulation in macrophages [110]. The main beneficial effects of Sallylcysteine on macrophage function in atherosclerosis are inhibiting inducible nitric oxide synthase and suppressing the production of hydroxyl radical, thereby suggesting for the primary role of the antioxidant activity [111].
Unsaturated free fatty acids such as palmitic acid or linoleic acid down-regulate ABCA1 expression in macrophages by epigenetic repression of LXR genes and in LXR-independent post-translational level involving ABCA1 phosphorylation and destabilization by protein kinase Cd that induces degradation of the transporter [112,113]. Pro-inflammatory cytokines IL-12 and IL-18 suppress ABCA1 expression through IL-18 receptor/NF-jB-mediated induction of zinc finger protein 202 (ZNF202), a transcriptional repressor [114]. MicroRNA (miR)-26 that is critically involved in the regulation of vascular smooth muscle cell differentiation [115] was shown to target LXRa and therefore down-regulate ABCA1 expression [116]. MiR-144 directly decreases ABCA1 mRNA levels in macrophages and liver as two miR-144-binding sites were found in the 3 0 untranslated region of the ABCA1 mRNA [117]. Indeed, development of miR-26 and miR-144 inhibitors has a therapeutic potential to suppress the formation of foam cells in atherosclerosis.

ATP-binding cassette transporter ABCG1
This transporter transfers cholesterol to HDL particles, but not to ApoA1. The human ABCG1 gene is mapped to chromosome 21q22.3 [118]. The gene contains alternative start codons that result in the production of multiple transcripts in a tissue-specific manner [119]. In LDL receptor-deficient mice, ABCG1 knock-down inhibition, results in a moderate rise of atherosclerotic plaques [120]. In contrast, a study shows the atheroprotective effect of genetic deletion of ABCG1 in the LDL receptor-deficient mouse [121]. Indeed, as ABCA1 is a primary cholesterol transporter in macrophages and it could compensate lipid efflux.
In fact, the regulation of expression of ABCA1 and ABCG1 is shared between LXRa and LXRb that control tissue lipid intake [8]. Notably, ABCG1 expression could be modulated by dietary components. Cultured macrophages exposed to extra-virgin olive oil developed increased ABCG1 expression and enhanced lipid overflow [122]. Cineole, a monoterpenoid of Eucalyptus sp. was shown to stimulate ABCG1 expression via activation of LXR receptors [123]. Cyanidin-3-O-b-glucoside (Cy-3-G), an anthocyanin derived from blackberry, blueberry, bilberry, cranberry and other herbs is known to have remarkable atheroprotective properties [124]. In the intestine, Cy-3-G could be converted by gut microflora to protocatechuic acid (PCA), a bioactive metabolite that is able to reduce expression of miR-10b, which represses ABCA1 and ABCG1 [125]. Except for inhibiting lipid accumulation in macrophages, PCA exhibits anti-inflammatory properties by decreasing subendothelial monocyte infiltration and recruitment in ApoE-deficient mice [126]. Therefore, regular intake of a proper food could prevent lipid misbalance and cholesterol deposition in macrophages and other cells.

Scavenger receptor BI
Scavenger receptor-BI is responsible for cholesterol transfer to HDL. The human receptor is encoded by the SCARB1 gene located on chromosome 12g24.31 [44]. Two SR-BI isoforms could be translated from the human SCARB1 gene. The longest isoform contains 509 a.a., with a C-terminal distinct from the shortest isoform (506 a.a.) [127]. Both isoforms have two transmembrane domains, two short cytoplasmic tails and a large extracellular loop [128].
In ApoE-deficient mice, macrophage-specific deletion of SR-BI resulted in advanced atherosclerosis, with an 86% increase in average lesion area. Overexpression of SR-BI delayed formation of atherosclerotic plaques in ApoE-deficient mice [129]. However, in LDL receptor-deficient mice, macrophage-specific overproduction of SR-BI protected against advanced atherosclerosis, but contributed to early plaque formation [130]. The extraordinary role of SR-BI in atherosclerosis could be explained by ability of this transporter to move cholesterol bidirectionally [131]. At early atherosclerosis stages, SR-BI in macrophages acts like SR-A1 to conduct cholesterol and phospholipid influx to decrease excessive serum cholesterol levels. Furthermore, SR-B1 was shown to block ABCA1-dependent cholesterol efflux in macrophages suggesting for distinct and competing roles of these transporters in mediating cholesterol flux [132]. Local HDL phospholipid composition could greatly influence the reverse cholesterol transport, with increased phosphatidylcholine enrichment of HDL that stimulates cholesterol efflux [131].
Numerous bioregulators modulate SR-BI expression. Dietary components such as polyphenol resveratrol and 13-hydroxy linoleic acid activate PPARc that in turn up-regulates LXRa and SR-BI [133,134]. Similarly, caffeic acid and ferulic acid, two major phenolic acids of coffee, increase cholesterol efflux from macrophages by stimulation of SR-BI and ABCG1, e.g. carriers that deliver cholesterol to HDL, but not to ApoA1 [135]. In contrast, pappalysin-1 (or pregnancy-associated plasma protein A; PAPPA), a metalloproteinase that cleaves insulin-like growth factor binding proteins could decrease expression of ABCA1, ABCG1, and SR-BI by inhibiting IGF-I-mediated (IGF/PI3-K/ Akt) stimulation of LXRa [136].

Concluding remarks
Macrophages are master regulators of plasma lipid balance being involved in ingestion and storage of excessive lipids that could be then released in a case hypolipidaemia. However, in atherosclerosis, there is a deregulation of cholesterol uptake and reverse transport by macrophages. OxLDL, which is a major source of cholesterol, contributes to the suppression of cholesterol efflux, whereas expression of SRs especially LOX-1 becomes significantly up-regulated. Indeed, it is not surprising that the genetic deletion of either SR-A1 or CD36 has no beneficial effects in ApoE-deficient mice [38, 39] because oxLDL uptake by macrophages could be compensated by SR-BI (in early atherosclerosis) and LOX-1.
It should be stressed that lipid metabolism in macrophages could be modulated by multiple dietary factors that provide a good option to prevent lipid accumulation or improve macrophage function in atherosclerotic patients. For example, several polyphenolic acids derived from blueberries were shown to efficiently suppress generation of foam cells through the down-regulation of CD36 and stimulation of ABCA1 [137].
Targeting components of cholesterol uptake/esterification/efflux should be of great therapeutic value. Statins (lipid-lowering agents) were shown to possess a wide range of anti-inflammatory activities including improvement of lipid handling by macrophages in patients with cardiovascular disease. Simvastatin showed beneficial effects in the treatment of stroke-prone hypertensive rats via decreasing macrophage infiltration and lipid deposition and inhibition of LOX-1 expression [138]. Similarly, pravastatin also reduced LOX-1 expression in intimal macrophages and decreased lipid core size in the atherosclerotic plaques of Watanabe heritable hyperlipidaemic rabbits [139]. In ApoE-deficient mice, K-604 and rimonabant, recently developed ACAT1 inhibitors, suppressed foam cell formation and reduced atherosclerosis [140,141].
Pro-protein convertase subtilisin/kexin type 9 (PCSK9) belongs to the family of PCSKs involved in the activation of peptide hormones and receptors from precursors. Specifically, PCSK9 decreases LDL cholesterol uptake by liver by stimulating degradation of LDL receptors [142]. As a consequence, plasma LDL cholesterol (LDL-C) concentrations become elevated which could increase cardiovascular risk. Increased expression of PCSKs was found in human and mouse atherosclerotic plaques suggesting the involvement of PCSKs in atherogenesis [143]. To date, multiple PCSK9 inhibitors including low-molecular inhibitors and monoclonal antibodies such as alirocumab (Aventis/Regeneron), bococizumab (Pfizer) and evolocumab (Amgen) have been developed [144]. In animal models of atherosclerosis, PCSK9 inhibition resulted in significant lowering of plasma LDL-C levels and diminished atherosclerosis progression [142]. Clinical trials involving anti-PCSK9 monoclonal antibodies showed a notable atheroprotective effect associated with cholesterol decrease, cardiac events and mortality [145].
In cultured human THP-1 macrophages, PCSK9 expression was shown to be up-regulated by oxLDL. Knock-down of PCSK9 expression with PCSK9-specific small interfering RNA resulted in the inhibi- tion of oxLDL-induced degradation of IjB-a, an inhibitor of nuclear transcription factor NF-jB that primes expression of a set of pro-inflammatory genes [146]. Indeed, PCSK9 inhibition prevents pro-inflammatory activation of macrophages.
In addition to SRs, enzymes and transporters characterized in this review, it is necessary to investigate other biomolecules that could contribute to cholesterol handling in macrophages. Further studies are required to evaluate mechanisms that control expression and function of these biomolecules and investigate their involvement in cardiovascular disease. It is important to note here that in atherosclerosis, the disruption of lipid homoeostasis in macrophages, which leads to cholesterol accumulation and formation of foam cells, is only one of the well-known mechanisms responsible for atherosclerotic plaque formation [1][2][3][4][5]. Accumulated evidence indicate that the multi-factorial inflammatory processes are responsible for the progression of atherosclerosis and that different cell types are simultaneously involved in all stages of plaque development [1][2][3][4][5]