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Self Nonself. 2010 Jul-Sep; 1(3): 192–224.
Published online Jun 11, 2010. doi:  10.4161/self.1.3.12794
PMCID: PMC3047783
The SCHOOL of nature
III. From mechanistic understanding to novel therapies
Alexander B Sigalovcorresponding author
SignaBlok, Inc.; Shrewsbury, MA USA
corresponding authorCorresponding author.
Correspondence to: Alexander B. Sigalov; Email: Sigalov/at/signablok.com
Received April 27, 2010; Revised June 10, 2010; Accepted June 11, 2010.
Protein-protein interactions play a central role in biological processes and thus represent an appealing target for innovative drug design and development. They can be targeted by small molecule inhibitors, modulatory peptides and peptidomimetics, which represent a superior alternative to protein therapeutics that carry many disadvantages. Considering that transmembrane signal transduction is an attractive process to therapeutically control multiple diseases, it is fundamentally and clinically important to mechanistically understand how signal transduction occurs. Uncovering specific protein-protein interactions critical for signal transduction, a general platform for receptor-mediated signaling, the signaling chain homooligomerization (SCHOOL) platform, suggests these interactions as universal therapeutic targets. Within the platform, the general principles of signaling are similar for a variety of functionally unrelated receptors. This suggests that global therapeutic strategies targeting key protein-protein interactions involved in receptor triggering and transmembrane signal transduction may be used to treat a diverse set of diseases. This also assumes that clinical knowledge and therapeutic strategies can be transferred between seemingly disparate disorders, such as T cell-mediated skin diseases and platelet disorders or combined to develop novel pharmacological approaches. Intriguingly, human viruses use the SCHOOL-like strategies to modulate and/or escape the host immune response. These viral mechanisms are highly optimized over the millennia, and the lessons learned from viral pathogenesis can be used practically for rational drug design. Proof of the SCHOOL concept in the development of novel therapies for atopic dermatitis, rheumatoid arthritis, cancer, platelet disorders and other multiple indications with unmet needs opens new horizons in therapeutics.
Key words: membrane receptors, single-chain receptors, multichain immune recognition receptors, MIRR, transmembrane signal transduction, cell activation, intrinsically disordered proteins, signaling chain homooligomerization model, SCHOOL model, mechanistic model, protein-protein interactions, therapeutic targets, transmembrane interactions, cytoplasmic homointeractions, immune disorders, platelet disorders, platelet inhibition, viral pathogenesis, human immunodeficiency virus, human cytomegalovirus, severe acute respiratory syndrome coronavirus, inflammatory conditions, thrombosis, stent thrombosis, sepsis, dermatitis, arthritis, inflammatory bowel diseases, cancer, novel therapies, immune response, immunomodulators, receptor inhibitors, peptide inhibitors, peptides, peptidomimetics, small molecules
Specific protein-protein interactions are responsible for the function of numerous processes in the cell and constitute the foundation for the majority of cell recognition, proliferation, growth, differentiation, programmed cell death and signal transduction in health and disease.14 It seems that almost every important pathway includes and is critically influenced by protein-protein interactions. 1 Despite high diversity of protein-protein interactions, all these interactions occur in a highly specific manner determined by structural and physicochemical properties of the interacting proteins. Because of the ubiquitous nature of these interactions and the knowledge that inappropriate protein-protein binding can lead to disease, the specific and controlled inhibition and/or modulation of these interactions provides a promising novel approach for rational drug design, as revealed by recent progress in the design of inhibitory antibodies, peptides and small molecules. A number of recent reviews have addressed this topic.127 Thus, revealing information about specific protein-protein interactions in any particular pathway can provide promising targets for a generation of new drugs.
Cell surface receptors are integral membrane proteins and, as such, consist of three basic domains: extracellular (EC) ligand-binding domains, transmembrane (TM) domains and cytoplasmic (CYTO) signaling (or effector) domains. Transmembrane signal transduction via cell surface receptors is a complex fundamental process by which extracellular information is translated into intracellular signaling sequences and further into physiological cell response. This process plays an important role in health and disease and is central to therapeutic control of multiple diseases.28,29 It is therefore fundamentally and clinically important to mechanistically understand, at the level of protein-protein interactions, how signal transduction occurs. However, until recently, there was no clear mechanistic understanding of TM signaling.
A general platform for receptor-mediated signaling, the signaling chain homooligomerization (SCHOOL) platform,2935 suggests that receptor oligomerization (clustering) induced or tuned upon multivalent ligand binding outside the cell is translated across the membrane into protein oligomerization inside the cell with formation of competent signaling oligomers in CYTO milieu being necessary and sufficient to trigger receptor activation. This uncovers for the first time the major mechanisms coupling recognition and activation functions at the level of protein-protein interactions—biochemical processes that can be influenced and controlled for therapeutic purposes.
Until recently, the lack of our mechanistic understanding how transmembrane signal transduction occurs at the molecular level (Fig. 1A) significantly impeded progress in fundamental studies in biology and life sciences as well as in the development of new therapies. As a result of this, currently, the most widely used strategies of receptor modulation for therapeutic purposes aim to prevent binding of receptors to their ligands. This is usually achieved by using the clinically relevant antibodies or soluble receptor domains (Fig. 1B).3651 These and other recombinant therapeutic proteins are a rapidly growing category of the prescription drug market, with sales projected to reach $50 billion worldwide by 2010. Yet they possess severe disadvantages including a long and costly development process, manufacturing difficulties and a lack of oral bioavailability.51 In addition, their relative inability to cross the cell membrane confines them to extracellular interventions. Still, when compared to harsh interventions, such as chemotherapy, antibodies were initially believed to be rather benign in terms of side effects. However, a recent catastrophic failure of an antibody trial, in which an anticancer antibody against the T cell marker (CD28) administered to four patients in the United Kingdom resulted in severe and life-threatening responses, has shattered this serene sense of confidence. The cause of failure is at present not understood.
Figure 1
Figure 1
Therapeutic inhibition of membrane receptors. Due to the lack of knowledge about the molecular mechanism of ligand-induced receptor triggering (A), a vast majority of current inhibitors block the binding of ligand to receptor (B).
Another option for therapeutic inhibition of membrane receptors is not to prevent binding of receptors to their ligands but interrupt TM signal transduction per se. In this context, protein-protein interactions that are involved in receptor signaling represent an appealing target for innovative drug development. They can be targeted by small molecule inhibitors and by modulatory peptides and peptidomimetics, which represent an alternative to protein therapeutics and avoid many of their disadvantages. However, the lack of a clear molecular understanding of how receptors transduce antigen-binding information across the cell membrane (or, in other words, how they translate this extracellular binding into an intracellular activation signal) significantly impeded the development of novel pharmacological approaches and even more important, the potential transfer of our current and future clinical knowledge, experience and therapeutic strategies between seemingly unrelated diseases.
Recently, an unusual biophysical phenomenon, the ability of intrinsically disordered proteins (IDPs; i.e., proteins that lack a well-defined ordered structure under physiological conditions in vitro) to homooligomerize, has been intriguingly reported for a signaling-related novel class of IDPs.52,53 This key missing piece to the receptor triggering puzzle allowed to accomplish our understanding of TM signal transduction at the level of specific protein-protein interactions and to structure our current multidisciplinary knowledge and views of the mechanisms governing the coupling of recognition to signal transduction and cell response.30,3335,54,55 This also allowed to build a general platform, the SCHOOL platform, for receptor-mediated signaling.30,34,35,55 According to the SCHOOL platform, signaling chain homooligomerization and formation of competent signaling oligomers in CYTO milieu provides the necessary and sufficient event to trigger receptors and induce cell activation.30,31,3335 Thus, receptor oligomerization induced or tuned upon ligand binding outside the cell is translated across the membrane into protein oligomerization in CYTO milieu, providing a general platform for TM signaling.
For single-chain receptors (SRs; i.e., receptors with their binding and signaling domains located on the same protein chain), the SCHOOL platform suggests that multivalent ligand binding results in receptor re-orientation and dimerization (oligomerization) mediated by interreceptor TM interactions5659 with following formation of competent signaling oligomers in the cytoplasm (Fig. 2).30,3335,55,58,6071 In receptor tyrosine kinases (RTKs), for example, this leads to trans-autophosphorylation at defined CYTO tyrosines through intrinsic kinase activity.61 RTKs and some other SRs such as, for example, members of the tumor necrosis factor (TNF) receptor superfamily71,72 can exist as pre-assembled dimers/oligomers on the surface of unstimulated cells. According to the SCHOOL platform,30,3335,55 in this scenario, binding to multivalent ligand results in re-orientation of receptors in these oligomers to adopt an interunit geometry permissive for CYTO homointeractions and therefore for further receptor activation (Fig. 2). Interestingly, not only EC and TM regions of SRs but also CYTO tails of these receptors represent folded and well-ordered domains, thus providing, within the SR signaling platform, the principal functional link between protein order and oligomericity in CYTO milieu (Fig. 2).34,35,55
Figure 2
Figure 2
SCHOOL platform of single-chain receptor (SR) signaling. Ligand-induced SR clustering and reorientation (in pre-existing SR clusters, ligand binding induces receptor reorientation) results in SR oligomerization mediated by transmembrane interactions. (more ...)
In multichain receptors, EC recognition module(s) and intracellular signaling module(s) are found on separate receptor subunits. In this work, I refer to multichain activating receptors, as to multichain immune recognition receptors (MIRRs), the term first introduced by Keegan and Paul in 1992.73 It should be noted, however, that not all members of this family are necessarily immune-related (an example is the major collagen receptor on platelets, glycoprotein VI, GPVI). The MIRR ligand-binding subunits are integral membrane proteins with small intracellular domains that are themselves inert with regard to signaling. Signaling is achieved through the association of the ligand-binding chains with signal-transducing subunits that contain in their CYTO domains one or more copies of the immunoreceptor tyrosine-based activation motifs (ITAMs) with two appropriately spaced tyrosines (YxxL/Ix6–8YxxL/I; where x denotes non-conserved residues)74 or the YxxM motif, found in the DAP10 CYTO domain.75,76 The association of the MIRR subunits in resting cells is driven mostly by the noncovalent TM interactions between recognition and signaling components and plays a key role in receptor assembly, integrity and surface expression.31,7794 Multi- but not monovalent ligand binding and subsequent receptor clustering are required for induction of the MIRR-mediated signaling cascade.29,30,67,95126 The SCHOOL platform for MIRR signaling suggests that multivalent ligand binding-mediated formation of competent signaling homooligomers of MIRR signaling subunits in CYTO milieu is necessary and sufficient to trigger the receptors and induce TM signal transduction and the downstream signaling sequence (Fig. 3).30,31,33,35,54 Similar to SRs, some MIRRs such as T cell receptor (TCR), GPVI and the natural killer group 2D (NKG2D) receptor, can exist as pre-assembled oligomers on the cell surface.114,123,125,127130 Within the SCHOOL platform, in these oligomers, multivalent ligand binding (or antibody stimulation) results in re-orientation of receptors to adopt an interunit geometry permissive for CYTO homointeractions between MIRR signaling subunits and therefore for further receptor activation (Fig. 3).
Figure 3
Figure 3
SCHOOL platform of multichain receptor signaling. Within the platform, formation of competent signaling oligomers in cytoplasmic milieu is necessary and sufficient to generate the activation signal, thus triggering downstream signaling pathways. Ligand-induced (more ...)
Structurally, ITAM-containing CYTO domains of MIRR signaling subunits represent a novel class of IDPs.35,52,55,131 Within the MIRR signaling platform (Fig. 3), this, together with the intriguing ability of these proteins to homooligomerize,52 provides the intriguing principal functional link between protein disorder and oligomericity in CYTO milieu.34,35,55
Introducing the homotypic interactions between the CYTO domains of SRs or MIRR signaling subunits as one of the key functional interactions involved in receptor triggering and TM signaling, the plausible and easily testable SCHOOL platform thus defines TM signal transduction as an outcome of the interplay between three major driving forces: ligand-receptor EC interactions, interreceptor (SR signaling) and intrareceptor (MIRR signaling) TM interactions and interreceptor CYTO homointeractions.3134,132 While, as discussed above, ligand- receptor binding is a commonly accepted and widely used point of intervention for therapeutic receptor inhibition (Fig. 1), the latter two protein-protein interactions (TM and CYTO interactions) represent new intervention points (Fig. 4) that can be used for therapeutic inhibition and/or modulation of cell response in treatment of receptor-mediated disorders.3134,132134
Figure 4
Figure 4
Key protein-protein interactions as new therapeutic targets in receptor signaling. Within the SCHOOL platform, receptor triggering and signaling is an outcome of the ligand-induced interplay between three key protein-protein interactions: (1) ligand-receptor (more ...)
Importantly, in contrast to existing strategies of therapeutic receptor inhibition that target ligand-receptor interactions (Fig. 1),3650,135 the SCHOOL strategy is not to prevent binding of membrane receptors to their ligands but block receptor-mediated signal transduction in TM and CYTO milieu. This “Freedom to Bind not to Signal” strategy allows for effective and selective therapeutic targeting by small molecule inhibitors and modulatory peptides and peptidomimetics. Interestingly, advances in peptide and peptidomimetic pharmaceuticals have already resulted in novel therapies for diabetes, obesity, Crohn's disease, osteoporosis, cancer, cardiovascular disease, immunotherapy, acromegaly, enuresis, pain and antimicrobials.136 The use of D-amino acids, novel amino acids and structure/activity relationships importantly allows us to generate analogs that impart protease resistance and increased bioavailability. This and further improvements in novel formulation and delivery strategies have made it possible to target optimal therapeutic dosing requirements.
Currently, peptides are increasingly making their way into clinical practice. In 2003, Frost & Sullivan estimated that the global therapeutic peptides market is valued at around $1 billion. More than 40 peptides are in the world market. Six are in the registration phase. Approximately 270 peptides are in clinical phase testing and more than 400 are in advanced preclinical phases worldwide.
The SCHOOL model was initially developed in 2004 for MIRR-mediated TM signaling.30 Later, the SCHOOL-based mechanism has been suggested as a general platform for members of both structural families—SRs and MIRRs (Figs. 5 and and66).29,3335,55
Figure 5
Figure 5
Single-chain receptors (SRs). The extracellular portion of the receptors is on top and the cytoplasmic portion is on bottom. The lengths of the receptors as shown are only approximately to scale. The inset shows SR domain organization. Extracellular ligand-binding (more ...)
Figure 6
Figure 6
Multichain immune recognition receptors (MIRRs). Schematic presentation of the MIRRs expressed on many different immune cells including T and B cells, natural killer cells, mast cells, macrophages, basophils, neutrophils, eosinophils, dendritic cells (more ...)
Within the SCHOOL platform, the key interactions involved in receptor triggering, namely interreceptor TM and CYTO interactions in SR signaling as well as intrareceptor TM interactions and interreceptor CYTO homointeractions in MIRR signaling, represent promising novel therapeutic targets.30,31,54,133,137,138 Controlled inhibition/modulation of these protein-protein interactions provides a means to inhibit/modulate receptor-mediated TM signaling and specific downstream signaling pathways, thus inhibiting/modulating the cell response. This can be used in rational drug design and the development of novel strategies for the treatment of a variety of diseases and medical conditions that involve receptor-mediated signaling. In addition, unraveling the molecular basis of receptor triggering and signaling, the SCHOOL platform suggests invaluable and unique powerful tools to dissect fundamental mechanisms of the related cell functional outcomes in response to antigen/ligand and to study many important aspects of viral pathogenesis.3032,54,132,133,137140
Importantly, the platform suggests that within the single- and multichain receptor families (Figs. 5 and and66), the similar architecture of the receptors (the insets, Figs. 5 and and66) dictates similar mechanisms of receptor triggering which in turn provide the similarity of the therapeutic targets (revealed at the level of protein-protein interactions critically involved in receptor signaling).2931,34,35,55,132,141 This builds the structural basis for the development of novel pharmacological approaches as well as the transfer of our current and future clinical knowledge, experience and therapeutic strategies between seemingly unrelated diseases mediated by receptors within SR and MIRR families.
Despite the difference in details of the molecular mechanisms of action, the use of the suggested SR- and MIRR-targeted agents represents a general therapeutic strategy for disorders mediated by members of both receptor families: SRs and MIRRs. Thus, by revealing specific protein-protein interactions critically involved in receptor-mediated signaling, current platform of receptor-mediated TM signal transduction (Figs. 2 and and33) not only provides molecular explanations for many biological phenomena and processes and introduces powerful tools for fundamental and applied research but also suggests novel avenues for drug discovery.3135,132134,138,140 Selected immunomodulatory agents, mechanisms of their action and potential therapeutic applications are summarized in Table 1 and discussed in more detail below. It should be noted, though, that despite most of the immunomodulatory agents discussed are peptides and peptide-based agents, peptidomimetic and small molecule inhibitors of signaling-related protein-protein interactions can also be designed or discovered.127
Table 1
Table 1
Mechanisms of immunomodulatory action and therapeutic potential of selected transmembrane- and cytoplasmic-targeted agents
Concept and evidence for single-chain receptors.
Suggesting critical role of TM interactions that mediate ligand-induced SR dimerization (oligomerization) and CYTO interactions that result in formation of competent signaling homooligomers (Figs. 2, ,44 and and77), the SCHOOL platform of SR signaling reveals these interactions as important control points for modulation of SR-mediated cell activation by using targeted agents.
Figure 7
Figure 7
SCHOOL platform and therapeutic inhibition of single-chain receptors (SRs). (A) Within the SCHOOL platform, specific blockade of interreceptor transmembrane interactions prevents ligand-induced SR oligomerization. Competent signaling oligomers in cytoplasmic (more ...)
Transmembrane interactions. Ligand-induced receptor dimerization/oligomerization is considered to represent a common mechanism of SR triggering and TM signal transduction.12,58,6769, 120,142150 In RTKs, divalent ligand binding is believed to stimulate monomeric receptor dimerization and trans-autophosphorylation at defined tyrosine residues through intrinsic kinase activity.6264 Interestingly, dimerization of SRs is known to be mostly driven by homointeractions between receptor TM doma-ins.58,59,69,120,142,145,147,148,151,152
At present, there is a growing line of experimental evidence indicating that TM-targeted strategy for inhibition/modulation of SR signaling might represent a promising therapeutic approach.58,145,147,151,153158 Within the SCHOOL platform, the TM-targeted peptides/agents block/disrupt/modulate interreceptor TM interactions crucial for ligand-induced receptor oligomerization, thus preventing formation of competent signaling oligomers in CYTO milieu (Fig. 7A). Importantly, peptide drugs possess several advantages over large protein molecules (Fig. 7C). Selected examples of using TM peptides to inhibit SR signaling are described in more detail below.
In line with the SCHOOL platform of RTK signaling, ligand binding-induced association of the TM domains has been proposed to favor productive dimerization of intracellular kinase domains to promote trans-autophosphorylation.151 Studies with the epidermal growth factor (EGF) and ErbB2 receptors have shown that synthetic peptides encompassing the TM domains of these receptors inhibit the autophosphorylation and signaling pathway of their cognate receptor.151,157 These peptides are thought to block/disrupt specific TM interactions, thereby inhibiting receptor dimerization and activation.151,157
Using differential epitope tagging, it has been demonstrated that β2-adrenergic receptors form homodimers and that TM domain VI of the receptor may represent part of an interface for receptor dimerization.153 As shown, a peptide derived from this domain inhibits both dimerization and β-adrenergic agonist-promoted stimulation of adenylyl cyclase activity.153 In contrast, a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) has been found to specifically inhibit D1DR binding and function without affecting receptor oligomerization.154 One possible explanation for this finding is that in addition to ligand-stimulated dimerization of receptors, the correct (permissive) relative orientation in the receptor dimers formed can also play an important role in D1DR signaling. The importance of the relative orientation has been shown for other SRs such as, for example, EGF receptors,159 Epo receptor,68,160162 toll-like receptors (TLRs)163 and the integral membrane receptor LuxPQ.164 Recent studies of vascular endothelial growth factor receptor-2 (VEGFR-2) also demonstrate that SR dimerization is necessary, but not sufficient, for receptor activation and that ligand-mediated receptor activation requires specific orientation of receptor monomers,165 as suggested by the SCHOOL platform of SR signaling (Fig. 2).30,3335,54 Thus, the presence of the TM peptide bound to the D1DR TM domain is likely to prevent ligand-induced formation of receptor dimers with correct intermolecular orientation, thus preventing formation of competent signaling dimers in CYTO milieu and therefore generation of the activation signal.
Another example of TM-targeted inhibitory peptides, the short peptide sequences corresponding to the Neu RTK TM domain, have been also reported to independently fold in membranes, interact with the full-length receptor and inhibit transformation of cells in vitro and in vivo.166
G-protein-coupled receptors (GPCR) are characterized by the presence of seven TM domains and represent a superfamily of proteins that mediate the function of neurotransmitters and peptide hormones and are involved in viral entry and perception of light, smell and taste. Structural analogs of individual TM domains of GPCRs have been reported to serve as potent and specific receptor inhibitors.156 Peptide sequences corresponding to the TM domains of chemokine receptors, CXCR4, also called fusin, an alpha-chemokine receptor specific for stromal-derived-factor-1, and CCR5, the chemokine receptor which HIV uses as a coreceptor to gain entry into macrophages, have been demonstrated to specifically inhibit receptor signaling and the in vitro replication of HIV-1.156 Similarly, peptides mimicking the TM domains of cholecystokinin receptor A, have been found to abolish ligand binding and signaling through the receptor.156
Thus, together, these findings clearly demonstrate the fundamental importance and clinical significance of inhibition/modulation of SRs by using the sequence-based blockade of the interreceptor TM protein interactions (Fig. 7A).
Cytoplasmic interactions. There is a growing line of experimental evidence supporting the SCHOOL platform-driven CYTO-targeted strategy of receptor inhibition/modulation (Fig. 7B). Interestingly, in general, CYTO peptides and peptidomimetics have been already shown to successfully target CYTO hetero- or homointeractions between entire protein molecules or the CYTO domains of TM proteins.167174 This means that once we can identify a new promising therapeutic CYTO target, it is technologically feasible to design, synthesize and use the relevant peptide-based agents, peptidomimetics and small molecules (or screen for the appropriate agents by using high-throughput screening, HTS, assays). Selected examples of CYTO-targeted agents used to inhibit CYTO protein-protein interactions, thus modifying the functional response, are considered in more detail below. These and other findings demonstrate that therapeutic inhibition of SRs using a variety of CYTO-targeted agents and/or mutations (Fig. 7B) is technologically feasible and of both fundamental and clinical importance.
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity.175 Although Fas-associated death domain (FADD) and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain (DD) leads to activation of caspase-8 in the receptor signaling complex.175,176 Recently, ligand-induced formation of surface receptor oligomers has been reported for Fas receptor.70 A cytoplasmic DD of this SR, upon ligand stimulation, binds to the homologous DD of the adaptor protein FADD and homooligomerizes, thus initiating the caspase signaling cascade (Fig. 8A). Interestingly, an autoimmune lymphoproliferative syndrome-linked mutation in Fas CYTO domain (T225K) impairs receptor oligomerization and inhibits Fas-mediated signaling but retains the ability to interact with FADD (Fig. 8A).70 This suggests that homointeractions between signaling CYTO tails themselves play an important role in ligand-induced surface receptor oligomerization and subsequent signaling, providing experimental proof for the SCHOOL platform. This finding also supports the suggested CYTO-targeted strategy (Fig. 7B) and provides a promising direction for future research. One can also hypothesize that similar mutations located in the CYTO domains of other SRs as well as in the CYTO domains of MIRR signaling subunits might occur naturally in receptor-mediated disorders and disturb the homooligomerization interface(s), preventing formation of competent signaling oligomers in CYTO milieu and triggering of the receptor.
Figure 8
Figure 8
Inhibition of cytoplasmic interactions in single-chain receptor signaling. (A) Fas apoptosis signaling by a normal Fas receptor and the receptor with the Fas T225K mutation that naturally occurs in patients with the autoimmune lymphoproliferative syndrome (more ...)
Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that recruits signaling proteins to TLR/IL-1 receptor (IL-1R) superfamily and thus plays a crucial role in the signaling pathways triggered by these receptors in innate host defense.177,178 A critical event in MyD88-triggered signaling pathway is homodimerization of MyD88 mediated by its TLR/IL-1R translation initiation domain (TIR) that is able to heterodimerize with the receptor and homodimerize with another MyD88 molecule (Fig. 8B).171,172,178 Dimerization of MyD88 favors the recruitment of downstream signaling molecules such as two IL-1R-associated kinases (IRAKs): IRAK1 and IRAK4 (Fig. 8B). Recently, eptapeptides that mimic the BB-loop region of the conserved TIR domain of MyD88, have been shown to effectively inhibit homodimerization with either the isolated TIR or full-length MyD88 (Fig. 8B).172 The authors also demonstrated that a cell permeable analog of MyD88 eptapeptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, indicating that the MyD88 homodimerization interface is a good target for specific inhibition of MyD88-mediated signaling in vivo.172
Importantly, a synthetic peptidomimetic compound modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain has been shown very recently to inhibit MyD88 dimerization in coimmunoprecipitation experiments.171 This effect is specific for homodimerization of the TIR domains and does not affect homodimerization of the DDs. The agent causes inhibition of IL-1β-mediated activation of NFκB transcriptional activity.171 After oral administration, the compound results in dose-dependent inhibition of IL-1β-induced production of IL-6 in treated mice.171 In addition, it suppresses B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling.171 These data indicate that the peptidomimetic compound studied blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization. This suggests that inhibition of MyD88 homodimerization in CYTO milieu by peptide-based agents or peptidomimetics may have therapeutic potential in treatment of chronic inflammatory diseases.171
As another example, the processes by which Nef mediates the redistribution of CD80 and CD86 in human monocytic cells can be considered.168 The endocytic mechanism to trigger internalization of CD80 and CD86 is known to involve Nef binding to the CYTO tails of these target proteins.168 In an inhibition assay, synthetic peptides corresponding to the CYTO domains of CD80 or CD86 have been demonstrated to inhibit Nef binding to the same peptides immobilized on polystyrene plates.168 Introduction of these CYTO peptides into Nef-expressing U937 cells using the Chariot reagent at 4°C causes substantial reduction in the loss of CD80 or CD86, respectively, from the cell surface of Nef-expressing cells,168 thus further proving the principal feasibility and the utility of the SCHOOL platform-driven CYTO-targeted strategy.
Interestingly, unlike wild-type Nef, the Nef D123G mutant has been shown to lose its ability to mediate efficient internalization of cell-surface CD80 or CD86 or bind to the CYTO peptides of CD80 or CD86.168 On the other hand, mutation of a conserved D123 residue is known to affect the ability of Nef to form dimers and results in impairment of other Nef biological functions such as major histocompatibility complex (MHC) class I downmodulation and enhancement of viral infectivity, indicating that the oligomerization of Nef may be critical for its multiple functions.179 In this regard, inability of the Nef D123G mutant to form homooligomers has been suggested to explain the impaired function of the mutant with regard to downmodulation of CD80/CD86.32 If true, this means that the rational design of antiviral agents that are able to target CYTO homointeractions in Nef oligomers, may represent an attractive target in the CYTO milieu not only with regard to Nef-mediated modulation of TCR triggering and TM signaling but also with respect to other Nef biological functions.31,32,54
Peptide-based CYTO-targeted strategy has been also successfully applied to modulate outside-in TM signaling mediated by the platelet receptors such as GPIb/IX/V,167 GPIIb,169 and the megakaryocyte- and platelet-specific integrin αIIbβ3.170
The platelet GPIb/IX/V receptor plays a key role in platelet adhesion at sites of vascular damage through its interaction with subendothelial-bound von Willebrand factor (VWF).180,181 However, despite the crucial role that the GPIb/IX/V receptor complex plays in hemostasis, the molecular mechanisms of its signaling are not completely understood. The GPIb/IX/V complex consists of four subunits, namely, GPIba, GPIbβ, GPIX and GPV. An amino acid sequence in the CYTO domain of the GPIbβ subunit between residues R151 and A161 has been shown to be highly conserved across species and play an important physiological role.167 It has been also reported167 that a synthetic CYTO-targeted agent, the cell-permeable palmitylated peptide corresponding to this sequence, completely inhibits low-dose thrombin- and ristocetin-induced aggregation in washed platelets, significantly reduces thromboxane (TXA) production in platelets stimulated by thrombin compared with collagen, substantially decreases activation of the integrin αIIbβ3 in response to thrombin, and significantly reduces the adhesion of washed platelets to VWF under static conditions and the velocity of platelets rolling on VWF. This demonstrates an effective impact of this peptide-based CYTO-targeted agent on platelet function in terms of rolling velocity, adhesion, spreading, signaling to αIIbβ3 and aggregation.
The integrin αIIbβ3 plays an important role in hemostasis mediating platelet adhesion, aggregation and bidirectional signaling.182,183 Little is known about the molecular mechanisms underlying the regulation of αIIb-mediated outside-in signaling. Recently, it has been shown that this signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the β3 CYTO domain, as detected by thromboxane production and granule secretion, and requires ligand cross-linking of αIIbβ3 and platelet aggregation.170 A synthetic CYTO-targeted agent, the cell-permeable palmitylated β3 peptide corresponding to the CYTO sequence R724–R734, has been demonstrated to effectively and specifically inhibit this outside-in signaling,170 thus supporting basic principles and feasibility of the SCHOOL platform-driven CYTO-targeted strategy.
All integrin α subunits are known to contain a highly conserved KXGFFKR motif in their CYTO domains that plays a crucial role in the regulation of integrin affinity for their ligands.169,184186 A synthetic CYTO-targeted agent, the palmitylated peptide corresponding to the K989-R995 sequence of the CYTO domain of the platelet integrin GPIIb (aIIb) subunit, has been shown to specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin.169 The authors conclude that this lipid-modified peptide imitates the CYTO domain of GPIIb and, in a highly specific and effective manner, initiates parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.169
An interesting example of using a synthetic peptide to inhibit protein-protein homointeractions in the intracellular milieu has been recently reported in studies of Ebola virus (EBOV), a filovirus that causes sporadic outbreaks of a fatal hemorrhagic fever in Africa.173,187 Viral protein 30 (VP30), one of seven structural proteins of this enveloped virus,187 is the constituent of the nucleocapsid and represents an EBOV-specific transcription activation factor.188 The essential role of homooligomerization for the function of VP30 and the significance of the self- assembly of VP30 for viral transcription and propagation have been recently reported.173 Interestingly, it has been also shown that the homooligomerization of VP30 can be dose dependently inhibited by a 25-mer peptide derived from the presumed oligomerization interface region.173 Importantly, when this peptide is transfected into EBOV-infected cells, the peptide inhibits viral replication, suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiviral drugs.173 This confirms that, as proposed by the SCHOOL platform for receptor-mediated TM signaling and cell activation,3035,54,55 protein-protein homodimerization/homooligomerization interface(s) can represent an important point of intervention in the CYTO milieu and be targeted by synthetic peptides, their derivatives and peptidomimetics.
Another potential application of the CYTO-targeted strategy involves the use of CYTO-targeted agents to modulate TLR4 signaling. This receptor is activated by monophosphoryl lipid A, derived from the active moiety (lipid A) of bacterial endotoxin (lipopolysaccharide, LPS). As recently demonstrated,189 LPS binds to a secreted glycoprotein MD-2, which in turn binds to TLR4 and induces aggregation and signal transduction. It has been also shown that TLR4 can form homodimers.190 Despite both TLR4 monomers and dimers are able to activate NFκB, this activation is significantly enhanced upon homodimerization.190 However, NFκB activation by TLR4 monomer, but not homodimer, is completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers can activate NF.B through different mechanisms.190 Interestingly, using the protein complementation assay, a novel method to detect protein-protein interactions in vivo,191 the TLR4 homodimerization has been shown to be mediated by the TLR4 CYTO domain.192 Thus, similar to other applications mentioned above, CYTO-targeted agents can be used to modulate TLR4-mediated signaling and cell activation, thus modulating the host immune response to LPS.
Interesting experimental evidence about the importance and utility of the SCHOOL platform has been recently provided in studies of FcγRIIA, the most highly expressed Fcγ receptor and the only receptor for human IgG2, the most common autoantibody isotype.174 This receptor plays an important role in rheumatoid arthritis (RA) and has emerged as a leading target for new drug candidates.174,193,194 Similar to other SRs, within the SCHOOL model of FcγRIIA signaling, formation of competent signaling oligomers in CYTO milieu is necessary and sufficient to trigger FcγRIIA and generate the activation signal, thus triggering downstream signaling pathways (Fig. 9A). Interestingly, dimerization that is known to be a prerequisite for FcγRIIA receptor activation is driven by interactions between not only the TM domains but also between the EC domains of the two monomeric partners.195 Mutagenesis of the EC dimer interface, as identified by crystallographic analyses, affects receptor signaling but not ligand binding.195 Within the SCHOOL model, antibody binding to the FcγRIIA receptor with the altered EC dimer interface results in incorrect relative orientation in ligand-induced receptor dimers/oligomers, preventing formation of competent signaling oligomers in CYTO milieu and blocking triggering of the receptor (Fig. 9B). Intriguingly, the Trojan peptide containing the CYTO tail sequence of FcγRIIA has been demonstrated to result in inhibition of antibody-induced signal transduction and phagolysosome formation.174 Within the model, this Trojan peptide construct specifically blocks (prevents) FcγRIIA CYTO homointeractions, blocking (preventing) formation of competent signaling oligomers and preventing Ig-induced cell activation (Fig. 9C). This finding directly proves the SCHOOL platform-driven CYTO-targeted strategy for therapeutic inhibition of SRs (Fig. 7B).
Figure 9
Figure 9
SCHOOL platform and FcγRIIA inhibition. (A) Within the platform, formation of competent signaling oligomers in cytoplasmic milieu is necessary and sufficient to trigger FcγRIIA and generate the activation signal, thus triggering downstream (more ...)
Thus, together, these data clearly show that inhibition of SRs by using the sequence-based blockade of the interreceptor CYTO interactions (Fig. 7B) is of both fundamental and clinical significance.
Concept and evidence for multichain receptors.
According to the SCHOOL platform, intrareceptor TM interactions and interreceptor CYTO homointeractions represent important points of intervention with targeted agents to inhibit and/or modulate MIRR-mediated TM signaling, thus inhibiting and/or modulating the immune response (Figs. 4 and and1010).
Figure 10
Figure 10
SCHOOL platform and therapeutic inhibition of transmembrane interactions in multichain receptor signaling. Within the SCHOOL model of multichain immune recognition receptor (MIRR) signaling, specific blockade or disruption of transmembrane interactions (more ...)
Transmembrane interactions. Concept. Since it was first published in 2004,30 the SCHOOL model has revealed the intra- MIRR TM interactions as important therapeutic targets as well as control points of great fundamental interest to study the molecular mechanisms underlying the MIRR-mediated cell response in health and disease (Figs. 4 and and1010).3032,34,54,132134,137,138 Importantly, the model has provided a mechanistic explanation at the molecular level for specific processes behind “outside-in” MIRR signaling that were unclear.3032,34,54,132134,137,138 Examples include molecular mechanisms of action of the therapeutically important TCR TM peptides196203 first introduced by Manolios et al. in 1997,204 and the mechanism underlying human immunodeficiency virus type 1 (HIV-1) fusion peptide (FP)-induced inhibition of antigen-dependent T cell activation.205 The relevance of the latter mechanism has since been confirmed experimentally.206
Within the SCHOOL model, upon antigen/ligand stimulation, the intra-MIRR TM interactions balance opposing interactions, the inter-MIRR CYTO homointeractions, and represent one of three major driving forces of MIRR triggering that helps to discriminate ligands/antigens in their functional ability to trigger MIRRs and induce a cellular activation signal (Figs. 3 and and44).30,31,3335,54 The model suggests that specific blockade or disruption of the TM interactions between MIRR recognition and signaling subunits causes a physical and functional disconnection of the subunits (Figs. 4 and and1010).3034,54,132134,137,138 Peptides and their derivatives, small molecule disruptors of protein-protein interactions, site-specific mutations and other similar agents/modifications can be used to affect the MIRR TM interactions.1,422 It should be noted that in this context, a physical disconnection of the subunits means “pre-dissociation” rather than full dissociation. Thus, in the absence of stimulus, the affected subunits can still remain together with the receptor (Fig. 10). Ligand stimulation of these “pre-dissociated” receptors leads to reorientation and clustering of the recognition but not the TM agent-affected signaling subunits (Fig. 10A). As a result, the corresponding signaling oligomers are not formed, ITAM Tyr residues do not become phosphorylated and the signaling cascade is not initiated (Fig. 10A). In contrast, the TM agent-induced “pre-dissociation” does not prevent the formation of competent signaling oligomers when signaling subunits are clustered by specific antibodies that trigger cell activation, e.g., anti-MIRR signaling antibodies (Fig. 10B) such as anti-CD3 (or anti-TCRβ) antibodies for TCR and anti-Igβ antibodies for B cell antigen receptor (BCR).
According to the SCHOOL platform, in MIRRs with more than one signaling subunits, those signaling subunits that are not affected by the TM agents can still form competent signaling oligomers upon antigen/ligand stimulation. Thus, these subunits can still initiate the corresponding cell response. For TCR, this will be illustrated below.
Importantly, our current understanding of the MIRR structure and the nature and specificity of TM interactions between receptor recognition and signaling subunits allows us not only to block or disrupt but also to modulate these protein-protein interactions in a sequence-based approach with using corresponding peptides and/or their derivatives. Strengthening/weakening and/or selective disruption of the association between particular recognition and signaling subunits might allow us not to inhibit, but rather to modulate the ligand-induced cell response. In addition, selective functional disconnection of particular signaling subunits from their recognition partner represents an invaluable tool in studies of MIRR-mediated TM signaling and cell activation. It should be also noted that methods of computational design, synthesis and optimization of TM peptides and peptidomimetics as well as HTS techniques to search for the relevant TM mutations or small molecule disruptors are currently developed and well-established,111,58,202,203,207216 thus making the proposed powerful approach both technologically feasible and of great fundamental and clinical value.
Thus, within the SCHOOL platform, TM interactions between recognition and signaling MIRR subunits represent important points of control in MIRR triggering and cell activation. Since now we can use the SCHOOL model to design the TM-targeted agents effective in inhibition and/or modulation of MIRR-mediated TM signaling (Figs. 4 and and1010) and to have a powerful and well-controlled influence upon MIRR-mediated cell activation, thus controlling the immune response.3035,54,55,132134,137,138 The relevant TM-targeted agents for any particular member of MIRR family can be readily designed using the SCHOOL model and our knowledge about structural organization of this receptor. Examples include the TM peptides of TCR,196,197,199204,208 NK receptors217 and GPVI134,138 tested to inhibit/modulate the receptor-specific response. Importantly, the SCHOOL model unravels the TM-targeted molecular mechanisms underlying ability of different human viruses such as HIV, cytomegalovirus (CMV), severe acute respiratory syndrome coronavirus (SARS) and others, to modulate and/or escape the host immune response.31,32,133,137,139,140 It also demonstrates how the lessons learned from viral pathogenesis can be used practically for rational drug design.32,133,138140 These and other examples that successfully prove the main concept of the SCHOOL model-driven TM strategy are considered in detail below.
Obviously, allowing us to effectively control MIRR signaling and the related immune response, the intrareceptor TM interactions represent an important target of pharmacological intervention as first revealed and suggested by the SCHOOL model in 2004.30 Importantly, it further assumes that a general therapeutic strategy, aiming to disrupt/modulate these interactions, may be used in the existing and future treatments of seemingly unrelated immune diseases. In other words, within the SCHOOL platform, specific therapeutic TM agents that target any particular MIRR involved in pathogenesis of the related immune disorder can be readily designed using primary structural information for the receptor and basic principles of the SCHOOL model.
There is exciting experimental evidence198 of both fundamental and clinical importance of the SCHOOL platform-driven TM approach. This finding is covered in more detail below.
Evidence for T cell receptor. TCR provides an intriguing ability of T cells to discern and differentially respond to MHC-bound peptides that can differ by only a single amino acid. Despite TCR being one of the most studied MIRRs, many of the models of TCR signaling suggested to date are descriptive and often fail in trying to explain most of the known immunological data.
Structurally, TCR is a member of the MIRR family with the α and β antigen-binding subunits that are bound by electrostatic transmembrane interactions with three signaling homo- and heterodimers: ζζ, CD3εδ and CD3εγ (Fig. 11), thus maintaining the receptor integrity in resting T cells.77,78 Within the SCHOOL model of TCR-mediated TM signal transduction, distinct TCR signaling is achieved through ζ and CD3 signaling oligomers (Fig. 11).30,3235,54,55,132 Importantly, the model suggests intrareceptor TM interactions not only as promising therapeutic targets but also as an important point of viral attack (Fig. 12).3133,132,133,139,140
Figure 11
Figure 11
SCHOOL model of T-cell receptor (TCR) signaling. Interaction with multivalent ligand (not shown) clusters the receptors and pushes them to reorientate (I), to bring signaling subunits into a correct (permissive) relative orientation and in sufficient (more ...)
Figure 12
Figure 12
A schematic representation of the SCHOOL-based mechanisms of action of T cell receptor transmembrane inhibitors such as the T cell receptor core peptide (CP) and HIV-1 gp41 fusion peptide (FP). Considering the close similarity in patterns of inhibition (more ...)
Transmembrane peptides capable of inhibiting TCR-mediated cell activation were first reported in 1997.204 These peptides include the TCR core peptide (CP). This synthetic peptide corresponds to the sequence of the TCRα TM domain that is known to interact with the TM domains of CD3δε and ζ.77,78 Interestingly, while TCRα CP inhibits antigen-stimulated interleukin-2 (IL-2) production, T-cell activation via anti-CD3 antibodies is not affected by this peptide.197 As shown, TCRα CP might be a proper treatment for human T cell-mediated dermatoses substituting for corticosteroids (Table 2).218 The peptide might be used also as a treatment for rheumatoid arthritis and other T cell-mediated disorders.198200,202,204 However, despite extensive studies,198,202,203 the mode of action of this clinically relevant peptide has not been elucidated until 2004 when the SCHOOL model of TCR signaling was first introduced (Fig. 11).30
Table 2
Table 2
Effect of TCR core peptide on T cell-mediated dermatoses in man*
Briefly, within the SCHOOL model, TCRα CP competes with the TCRα chain for binding to CD3δε and ζζ, thus resulting in disconnection/pre-dissociation of the affected signaling subunits from the remaining receptor complex (Fig. 12). This leads to inhibition of antigen- but not antibody-mediated TCR triggering and cell activation (Fig. 12). Importantly, as shown recently,219 TCR assembly and cell surface expression is not affected by treatment with TCRα CP. This directly proves the hypothesis about “pre-” rather than full dissociation state of the unstimulated TCR complex in the presence of TCRα CP, whereas upon stimulation, the affected signaling subunits, ζζ and CD3εδ, become physically disconnected from the remaining receptor complex (Figs. 10, ,1212 and and1313). It should be noted that the proposed SCHOOL mechanism is the only mechanism consistent with all experimental and clinical data reported up to date for TM peptides of TCR and other MIRRs as well as for lipid and/or sugar conjugates of these peptides.134,138,196198,201203,218225
Figure 13
Figure 13
A schematic representation of the SCHOOL-based mechanisms of action of different T-cell receptor transmembrane inhibitors. Within the SCHOOL model, upon antigen stimulation of T cells, T-cell receptor α-chain (TCRα) transmembrane peptide (more ...)
The SCHOOL model predicted that the MIRR TM peptides corresponding to the TM regions of not only recognition but also signaling subunits act through the same mechanisms of inhibitory action.30,31,33,34,54,132,133 This was recently confirmed experimentally by showing that the synthetic peptides corresponding not only to the TM sequence of the antigen recognition TCRα subunit (i.e., TCRα CP) but also to the sequences of the TM regions of the signaling CD3 (δ, ε or γ) and ζ subunits are able to inhibit the immune response in vivo (CD3 TM peptides) and NK cell cytolytic activity in vivo (ζ TM peptide).198,217
Interestingly, the model suggests a molecular explanation for the apparent discrepancy in CD3 TM peptide activity between in vitro and in vivo T-cell inhibition (Fig. 13).198 In this study, it has been shown that the CD3δ and CD3γ TM peptides do not impact T-cell function in vitro (the CD3ε TM peptide has not been used in the reported in vitro experiments because of solubility issues). In contrast, all three CD3 TM peptides used (CD3ε, CD3δ and CD3γ) inhibit an immune response in vivo and decrease signs of inflammation in the adjuvant-induced arthritis rat model.198 Within the SCHOOL model, the CD3δ and CD3γ TM peptides functionally disconnect the corresponding signaling subunits (CD3δ and CD3γ, respectively) from the remaining receptor complex (Fig. 13). Thus, upon antigen stimulation, these subunits do not participate in signaling, resulting in the lack of the Bδ and Bγ activation signals (Fig. 11) and the corresponding cell responses. On the other hand, the previously reported in vitro activation studies with T cells lacking CD3γ and/or CD3δ CYTO domains indicate that antigen-stimulated induction of cytokine secretion and T-cell proliferation are intact,226 evidencing that the Bδ and Bγ activation signals provided by CD3δ and CD3γ, respectively, are not important for antigen-induced cytokine production. This explains the lack of inhibitory effect of the CD3δ and CD3γ TM peptides observed in the in vitro activation assays used (IL-2 production).198 However, in vivo deficiency either of CD3δ or CD3γ results in severe immunodeficiency disorders,227 demonstrating the importance the Bδ and Bγ activation signals for the T cell-mediated immune response in vivo. This can explain the inhibitory effect observed in the in vivo studies for all three CD3 TM peptides used, including CD3δ and CD3γ (Fig. 13).198
Another interesting study demonstrated that short, incomplete peptide versions of the TCRβ chain naturally occur in the thymus and are sorted preferentially to the mitochondrion.228 As a consequence of the mitochondrial localization, apoptotic cell death is induced. Structure function analysis showed that both the specific localization and induction of apoptosis depend on the TCRβ TM domain and associated residues at the COOH-terminus of TCR.228 Considering the structural assembly of TCR (Fig. 11), one can hypothesize that incomplete peptide versions of TCRβ containing the TCRβ TM domain act through the SCHOOL-like mechanisms and functionally disconnect CD3εγ from the remaining TCR complex. Thus, upon stimulation, this results in the lack of the activation signal provided by CD3γ (Bγ), which is known to contribute specialized structural and signaling functions229231 and may improve certain mature T-cell responses such as specific adhesion and activation-induced cell death.228 This suggests that within the SCHOOL platform, the corresponding TCRβ TM peptides can be successfully used to functionally “dissect” TCR signaling in both fundamental and clinical research.
Thus, together, these experimental data directly prove that we can selectively “disconnect” specific signaling subunits in MIRRs using the SCHOOL platform-driven TM strategy. This provides us with a novel powerful tool to study MIRR functions and immune cell signaling as well as to rationally design novel inhibitors and/or modulators of the immune response.3034,54,132,133
Similar molecular mechanisms of action are suggested by the SCHOOL model for other MIRR TM peptides to describe or predict their inhibitory/modulatory effect on receptor-mediated cell activation (Table 1). Recently, the SCHOOL model-driven TM-targeted strategy has been successfully applied to develop a novel concept of platelet inhibition and resulted in the invention of a new class of platelet inhibitors.134,138,232 This topic will be covered in more detail below.
In summary, the first results in man for TCRα CP (Table 2) and in vitro and animal data obtained for other TM peptides (Table 1) demonstrate the high therapeutic potential of the MIRR TM peptides. This proves the SCHOOL platform-driven TM strategy as not only representing an invaluable tool for fundamental research but also as providing a highly promising drug discovery platform for the development of novel therapies.
Cytoplasmic interactions. Concept. As mentioned, the CYTO domains of the MIRR signaling subunits, including CD3ε, CD3δ, CD3γ, ζ, Igα, Igβ, DAP12, DAP10, FcεRIβ and FcRγ, represent a new class of IDPs.35,52,53,55,131 Interestingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling.233235 In addition, intrinsically disordered regions of human plasma membrane proteins have been recently demonstrated to preferentially occur in the cytoplasmic segment.236 Finally, it has been suggested that protein phosphorylation, one of the critical and obligatory events in cell signaling, occurs predominantly within intrinsically disordered protein regions.237 Within the SCHOOL platform, the intrinsically disordered state of the MIRR signaling subunit CYTO domains plays an important role in MIRR triggering and TM signaling.30,31,34,35,5255,131 It also suggests that the CYTO domains of those MIRR signaling subunits that have not been studied so far, are IDPs as well. Future studies will prove or disprove this hypothesis.
Surprisingly, all intrinsically disordered CYTO domains studied exist under physiological conditions as specific oligomers (mostly, dimers), as I discovered in 2001 and published in 2004, providing first evidence for the existence of specific dimerization interactions for IDP species.52 Even more interestingly, these IDPs do not undergo a transition between disordered and ordered states upon dimerization or interaction with well-ordered protein partner.35,52,53,55,131,238 The observed specific dimerization IDPs is distinct from non-specific aggregation behavior seen in many systems and opposes the generally accepted view on the behavior of IDPs. This opens a new line of fundamental research in the new and quickly developing field of IDPs.
The unexpectedness, unusualness and uniqueness of the discovered biophysical phenomenon that was found to be a general phenomenon with all CYTO domains studied in this work,52 lead me to hypothesize that the homointeractions between MIRR signaling subunits represent the key missing piece in the puzzle of MIRR triggering and TM signal transduction and provide biophysical background for the SCHOOL model of MIRR signaling.30,31,3335,54,55,133,137,138
Since it was first published in 2004,30 the model has revealed the inter-MIRR CYTO homointeractions as important therapeutic targets as well as points of fundamental importance to study molecular mechanisms underlying the MIRR-mediated cell response in health and disease (Figs. 4 and and1414).30,31,3335,54,55,133,137,138 Within the SCHOOL platform, formation of competent signaling homooligomers in CYTO milieu is necessary and sufficient to trigger receptor activation (Fig. 3). This suggests that specific blockade of the interreceptor CYTO homointeractions between MIRR signaling subunits by CYTO-targeted agents or site-specific point mutations within the dimerization/oligomerization interfaces prevents formation of competent signaling oligomers (Fig. 14) and initiation of a MIRR-mediated cell response. Similar to the intra-MIRR TM interactions (Fig. 10), modulation of the inter-MIRR homointeractions between particular signaling cytoplasmic domains allows us to modulate the ligand-induced cell response, including partial and complete inhibition. In addition, our ability to selectively prevent the formation of signaling oligomers of particular subunit(s) can also be an important tool in functional studies of MIRRs.
Figure 14
Figure 14
SCHOOL platform and therapeutic inhibition of cytoplasmic interactions in multichain receptor signaling. Within the SCHOOL model of multichain immune recognition receptor (MIRR) signaling, specific blockade of cytoplasmic homointeractions between signaling (more ...)
Similar to other specific protein-protein interactions, the MIRR CYTO interactions can be affected by peptides and their derivatives, small molecule disruptors of protein-protein interactions, site-specific mutations, and by other similar agents/modifications. As mentioned above, methods of computational design, synthesis and optimization of modulatory peptides and peptidomimetics as well as HTS techniques to search for the relevant mutations or small molecule disruptors are currently developed and well-established,127 thus making the proposed CYTO-targeted approach technologically feasible.
Importantly, in contrast to TM-targeted agent-affected MIRRs that can be still activated by specific antibodies that trigger cell activation (Fig. 10), antibody stimulation of CYTO-targeted agent-affected MIRRs does not result in MIRR triggering and generation of the activation signal (Fig. 14).
Thus, the interreceptor CYTO homointeractions between MIRR signaling subunits represent important points of control in MIRR triggering and cell activation. The relevant CYTO-targeted agents for any particular member of the MIRR family can be readily designed using our current knowledge about structural organization of the receptor and molecular mechanisms of its signaling. Since now we can use the SCHOOL model-driven CYTO strategy for rational design of clinically and fundamentally important agents effective in inhibition and/or modulation of MIRR-mediated TM signaling (Fig. 14). This gives us a powerful and well-controlled influence upon MIRR-mediated cell activation, thus controlling the immune response.
Evidence. Since homooligomerization of the MIRR signaling subunit CYTO domains was discovered52 and these CYTO homointeractions were suggested to represent an important therapeutic target,30,31,54 no direct experimental evidence has been reported yet to support the SCHOOL model-driven CYTO strategy for modulation of MIRR signaling.
One of the reasons is that the unusual biophysical phenomenon of IDP homooligomerization has been discovered very recently.52 Despite it has become of more and more interest to biophysicists and biochemists,239,240 at the current state of our knowledge, the molecular mechanisms of IDP homooligomerization are not well understood. As a result, dimerization/oligomerization interface(s) are still not characterized at the residual level, thus impeding design of specific inhibitors for these protein-protein interactions. A strong complication is that IDP homooligomerization is accompanied by a new, previously unknown nuclear magnetic resonance (NMR) phenomenon—the lack of significant changes in chemical shift and peak intensity upon a specific protein complex formation.35,52,53,55,131,238 Considering that NMR is unparalleled in its ability to provide detailed structural and dynamic information on IDPs and that NMR has emerged as the most important tool for studies of IDP interactions at the residual level,241,242 novel NMR strategies need to be developed. One can expect that further multidisciplinary studies will shed light on the possible structural basis of these interesting IDP features. This will allow us to apply currently developed and well-established methods of computational design, synthesis and optimization of modulatory peptides and peptidomimetics as well as HTS techniques to search for the relevant mutations or small molecule disruptors.127
Importantly, the recent success in using CYTO-targeted agents to modulate FcγRIIA signaling (Fig. 9),174 clearly demonstrates the technological feasibility of the SCHOOL platform-driven MIRR CYTO strategy of receptor modulation (Fig. 14) as well as its fundamental and clinical importance.
In general terms, viral pathogenesis is the process by which viral infection leads to disease. The consequences of a viral infection depend on a number of viral and host factors that affect pathogenesis. Infection of host cells by enveloped viruses requires fusion of the viral membrane with the host cell membrane. This fusion is mediated by viral glycoproteins (gp), the proteins that are anchored to the viral membrane. The fusion glycoproteins of enveloped viruses, typically type-I integral membrane proteins, are known to contain in their sequences a short region called the “fusion peptide” (FP), which is required for mediating membrane fusion.243,244 This region interacts with the host cell membrane at an early stage of the membrane fusion process. Despite advances in our understanding of the major principles of viral fusion mediated by the fusion glycoproteins,243248 little is known about their role in functional modulation of MIRR-mediated TM signal transduction.
This section is focused on viral immunomodulatory activity as related to MIRR signaling. Within the SCHOOL platform, in some scenarios, the molecular mechanisms underlying this activity affect MIRR TM interactions and represent a general viral strategy to modulate the immune response.31,32,133,140 Importantly, this not only proves the SCHOOL model-driven TM strategy of receptor modulation, but together with the lessons learned from viral pathogenesis, also significantly advance our ability to develop novel therapeutic approaches.
HIV pathogenesis.
CD4+ T cells are the main targets of HIV-1 in the host. Interestingly, the magnitude of viral replication in these cells is closely linked to their activation state: in activated memory CD4+ T cells, HIV-1 readily undergoes multiple rounds of replication whereas resting helper T cells are largely refractory to productive infection.249,250 Indeed, several steps in the life cycle of HIV-1 have been identified where potent blocks in virus propagation occur when ample T-cell activation is lacking.
Fusion peptide. The FP found in the N terminus of the HIV envelope gp41 functions together with other gp41 domains to fuse the virion with the host cell membrane.251,252 Surprisingly, this peptide has been recently shown to have not only a fusogenic activity but also a T cell-targeted immunomodulatory activity: it colocalizes with CD4+ and TCR molecules, coprecipitates with the TCR, and inhibits antigen-stimulated T-cell proliferation and proinflammatory cytokine secretion in vitro.205 These effects are specific, T-cell activation via PMA/ionomycin or mitogenic antibodies to CD3 is not affected by FP and FP does not interfere with antigen-presenting cell function.205 In mice, HIV FP shows immunosuppressive activity, inhibiting the activation of arthritogenic T cells in the autoimmune disease model of adjuvant-induced arthritis and reducing the disease-associated interferon-γ (IFNγ) response.205 The close match between these findings205 and the experimental data generated for TCRα CP197,201,202,204,224 suggests a mechanistic similarity underlying the TCR-targeted HIV FP and TCRα CP activities.
However, as with TCRα CP, despite ongoing studies of HIV gp41 FP,206 the molecular mechanisms of immunomodulatory action of this peptide have not been elucidated until 2006 when the SCHOOL model was first applied to this area.31 Considering the close similarity in patterns of inhibition of T-cell activation and immunosuppressive activity observed for FP205 and CP,197,204,224 the SCHOOL model reasonably suggests a similar molecular mechanism of action for TCR TM peptides and HIV gp41 FP (Fig. 12 and Tables 1 and and33).30,31,54,133,137 Primary sequence analysis of these two peptides (Fig. 12 and Table 4) shows different primary sequences but a similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 4 (CP) or 8 (FP) amino acids. For CP, Arg and Lys residues are known to mediate the interaction between recognition TCRα subunit and signaling CD3δε and ζ subunits.78 Importantly, for FP, both arginines are located in the C-terminal half, suggesting that this sequence could be important for the interaction with the TCR. Figure 12 shows a potential mode of action of CP and FP as proposed by the SCHOOL model (Tables 1 and and33). Briefly, CP and FP compete with the TCRα chain for binding to CD3δε and ζζ, thus resulting in TM pre-dissociation/functional disconnection of the affected signaling subunits from the remaining receptor complex (Fig. 12). This mechanism of FP action suggests the existence of an interaction interface in the C-terminal half of the peptide. Within the model,31,133,137 the peptide prevents formation of CD3δε and ζ signaling oligomers and thus inhibits antigen-dependent T cell activation (Fig. 12 and Table 3), acting similarly in this respect to TCRα CP (Fig. 12 and Table 1).30,31,54,133,197
Table 3
Table 3
Selected viral agents reported to modulate the immune cell response and suggested or predicted by the SCHOOL model to affect MIRR transmembrane interactions
Importantly, according to the model (Fig. 10), stimulation with anti-CD3 antibodies in the presence of FP still should result30,31,34,54,133,137 and results205,206 in TCR triggering and cell activation. This suggests that clinically relevant antibodies (anti-CD3 antibody, OKT3) could be used during HIV infection to modulate the affected T-cell response in a similar manner as anti-CD3 monoclonal antibody can be used to overcome potential suppression of the proliferative capacity by TCRα CP.196 Recently, OKT3 antibodies have been used successfully in HIV therapy to augment immune activation.253 More recent studies206 have confirmed the predicted molecular mechanism of immunomodulatory activity of the HIV FP. Finally, it should be noted that the proposed mechanism is the only mechanism consistent with all experimental data on immunomodulatory action of HIV gp41 FP reported up to date.205,206
Interestingly, a highly specific natural inhibitor of HIV-1 gp41 FP has been recently reported to block HIV-1 entry.254,255 This agent that has been isolated from human hemofiltrate and designated VIRus Inhibitory Peptide (VIRIP),255 represents a 20-residue peptide, corresponding to the C-proximal region of α1-antitrypsin. Importantly, it has been shown that VIRIP directly interacts with the gp41 FP and a few amino acid changes increase its antiretroviral activity potency by two orders of magnitude, thus demonstrating the usability and efficiency of rational peptide design approaches.255
Thus, according to the SCHOOL model, the TCR TM interactions represent not only important therapeutic targets for immune-mediated diseases but also a point of HIV intervention. The molecular mechanisms revealed by the model can be used in rational antiviral drug design and the development of novel antiviral therapies.
HIV Nef protein. Another interesting application of the SCHOOL model to HIV pathogenesis relates to the molecular mechanisms of action of the HIV pathogenicity factor Nef, a key protein in viral replication and progression of disease. Several studies have shown that this protein interacts with the TCR ζ chain and mediates downmodulation of TCR-CD3 complex.256258 Notably, Nef lowers the threshold of CD4+ T-cell activation.259,260 Other study showed that Nef induces transcription of an array of genes almost identical to that triggered upon exogenous stimulation of TCR.261 Nef has been also reported to affect T-cell activation events through its interactions within the lipid raft microenvironment,262 induce signal transduction via the recruitment of a signaling machinery, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade,263 and finally, to form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T-cell activation.264 Thus, the extent of T-cell activation imprinted by expression of Nef is a matter of controversy. In addition, although we know that Nef binds the TCR ζ chain,238,257,265 the role of this interaction and the mechanism used by Nef to modulate T-cell activation remain unknown.
Importantly, similar to ζ,52,53 Nef exists in several discrete oligomeric species, namely monomers, dimers and trimers.266 Within the SCHOOL model,30,31,54 natively oligomeric Nef may crosslink homodimeric ζ chains, leading to formation of multivalent TCR complexes that have been shown to be responsible for sensing low concentrations of antigen.127 Thus, this mechanism could explain the observed activation sensitization in T cells by Nef.259,260 On the other hand, Nef dimers may crosslink ζ homodimers in the “permissive” relative orientation and promote formation of competent signaling ζ oligomers, thus generating an activation signal A (Fig. 11)30,31,33,34,54 and resulting in dissociation of the ζ signaling oligomers from the remaining receptor complex with its subsequent internalization. The SCHOOL model thus suggests that the oligomer interfaces of ζ and/or Nef are involved in the molecular mechanisms underlying the immunomodulatory effects of Nef.30,31,54 Indeed, as recently shown,179 a Nef mutant carrying a mutation targeted to the conserved residue D123, in addition to losing the ability to oligomerize, is defective for MHC class I downmodulation and enhancement of viral infectivity, suggesting that the oligomerization of Nef may be critical for its multiple functions.
Possibly, both proposed mechanisms may take place in vivo, and selection between these two alternative pathways may possibly depend on the type of cells infected and/or on the cell membrane lipid content. Thus, CYTO heterointeractions at the Nef-ζ interface and CYTO homointeractions in Nef and ζ oligomers may represent attractive targets for the design of antiviral agents.
SARS-CoV pathogenesis.
The coronavirus SARS CoV is the etiological agent of SARS that represents the life-threatening disease associated with a mortality of about 10%.267 Recent studies, in which a total of 38 patients with SARS were enrolled, have shown that CD4+ and CD8+ T lymphocyte levels were reduced in 100% and 87% of patients, respectively.268 Thus, one can suggest that the virus can have an immunomodulatory activity and this activity is TCR-targeted.
Importantly, in the traditional view of HIV disease course, acute HIV infection is characterized by massive and rapid CD4+ T-cell loss, whereas chronic infection is characterized by persistent immune activation that drives viral replication and further CD4+ T-cell depletion.269,270 Thus, HIV infection has been thought of as a relatively indolent disruption of CD4+ T cells eventually leading to collapse of immune function. As with SARS CoV,268 this notion has been largely based on measurements of CD4+ T-cell counts in peripheral blood.269,270
Thus, despite the lack of direct evidence, it is reasonable to suggest that SARS-CoV has a TCR-targeted immunomodulatory activity. More specifically, as with HIV, this activity might be especially important during virus entry to suppress the host response to virus infection. Like other enveloped viruses encoding class I viral fusion proteins such as HIV271 and Ebola and avian sarcoma viruses,272 SARS-CoV is presumed to use membrane fusion mechanisms for viral entry.273275 It has been shown that the SARS-CoV viral spike (S) protein 2 (S2) is a class I viral fusion protein and is responsible for driving viral and target cell membrane fusion.276 Recently, inhibitory peptides derived from the membrane-proximal heptad repeat region (HR2) of the S2 protein have been suggested as an attractive basis for the development of therapeutics for SARS.277 The putative SARS-CoV FP has also been identified at the N terminus of the SARS-CoV S2 subunit.278 Interestingly, as shown by using synthetic peptides,278 the fusogenic activity of the SARS-CoV FP appears to be dependent on its amino acid sequence, as scrambling the peptide renders it unable to partition into large unilamellar vesicles (LUVs), assume a defined secondary structure or induce both fusion and leakage of LUV.
Primary sequence analysis of the SARC-CoV FP and TCRα TM domain (or TCRα CP) shows different primary sequences but reveals a similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 4 amino acids (Table 4). As mentioned, these two positively charged residues are critical for TCR assembly and function. Within the SCHOOL model,30,31,54,133,137 the RILLLK and RSMTLTVQAR motifs in TCRα CP and HIV FP (Table 4), respectively, play an important role in mimicking the TCRα TM region and therefore in an inhibitory activity of these peptides (Fig. 12 and Tables 1 and and33). Intriguingly, SARS CoV FP has a structural motif KTPTLK that is strikingly similar to that of TCRα CP (Table 4). Considering the common structural features of three peptides (TCRα CP, SARS-CoV FP and HIV FP) as well as functional similarities between HIV FP and SARS FP in the context of their T cell-targeted activities, one can be suggested that like TCRα CP and HIV FP, SARS-CoV FP should mimic the TCRα TM domain and therefore exhibit an inhibitory effect on the antigen-mediated TCR TM signaling (Table 3).32,133,140 In the context of the SCHOOL model, molecular mechanisms of this inhibitory action of SARS-CoV FP are similar to those suggested for TCRα CP and HIV FP.3032,54,133,137,140
Table 4
Table 4
Primary sequences of MIRR transmembrane domains involved in viral pathogenesis and viral fusion proteins and peptides suggested or predicted by the SCHOOL model to affect MIRR transmembrane interactions
Thus, as hypothesized, the TCR TM interactions might represent a point of SARS-CoV intervention. Future studies will experimentally prove or disprove this hypothesis.
HTLV-1 pathogenesis.
HTLV-1 is a type C complex retrovirus. It infects and immortalizes human CD4+ T cells in vitro and is associated with the development of adult T cell leukemia/lymphoma (ATL).279282 Recent observations demonstrate an immunomodulatory ability of the HTLV-1 regulatory protein p12283,284 and suggest roles that T-cell activation may play in the pathogenesis of HTLV-1-induced disease.279,283285
Below, similarities between the HIV gp41 and HTLV-1 gp21 FPs and the Nef and HTLV-1 p12 proteins, respectively, are considered. Structural and functional predictions related to potential TCR-targeted activities of HTLV-1 FP and p12 will be also described.
Fusion peptide. Similarly to HIV gp41 protein,251,252 the ectodomain of HTLV-1 TM protein (gp21) contains an N-terminally located fusion peptide, a sequence that inserts into target cellular membranes and is well-known to be critical for membrane fusion activity.286,287 However, in contrast to the HIV FP, there has been no report to date of an immunomodulatory activity of the HTLV-1 FP.
Primary sequence analysis of these two FPs (Table 4) indicates different sequences but reveals an interesting similarity in charged or polar residue distribution patterns with two positively charged residues spaced apart by 8 (HIV FP) or 7 (HTLV-1 FP) amino acids. Considering the structural similarities of both FPs and the fact that T cells are main target for both viruses, it is reasonable to suggest a TCR-targeted immunomodulatory activity for the HTLV-1 FP. As proposed by the SCHOOL for HIV FP and TCRα CP (Fig.12; Tables 1 and and33),30,31,54,133,137 a potential mode of action of HTLV-1 can involve competition with the TCRα subunit for binding to CD3δε and ζ subunits in TM milieu, thus resulting in disconnection/pre-dissociation of the affected signaling subunits from the remaining receptor complex. As with the HIV FP, this mechanism of HTLV-1 FP action suggests the existence of an interaction interface in the C-terminal half of the peptide. Within the model, the peptide should prevent formation of signaling-competent CD3δε and ζ oligomers and thus inhibit antigen-dependent T-cell activation, acting similarly in this respect to both TCRα CP and HIV FP (Fig. 12 and Tables 1 and and33).30,31,54,133,137,197 However, stimulation with anti-CD3 antibodies of these “pre-dissociated” TCRs should still result in receptor triggering and cell activation (Fig. 12).30,31,54,133,137 Thus, as with HIV infection, the model suggests that clinically relevant antibodies (i.e., OKT3) could be used during HTLV-1 infection to modulate the affected T-cell response.
In summary, this new hypothesis considers the largely unexplored immunomodulatory role of the FP in the HTLV-1 infection and pathogenesis of ATL. If true, this hypothesis will generate new therapeutic targets and opportunities. One can also suggest that our current and future clinical knowledge, experience and therapeutic strategies can be potentially transferred in this respect between the HIV- and HTLV-1-related medical conditions.
HTLV-1 p12 protein. The p12 protein of HTLV-1 is a small oncoprotein that has been shown to have multiple functions. Expression of p12 has been demonstrated to induce nuclear factor of activation of T cells (NF-AT), increase calcium release and transcriptional factor Stat 5 activation in T cells suggesting that p12 may alter T-cell signaling.288290 Interestingly, p12 is important for viral infectivity in quiescent human peripheral blood lymphocytes (PBLs) and the establishment of persistent infection in rabbits.291,292 Thus, despite the distinct structures, both retroviral accessory proteins HTLV-1 p12 and HIV Nef are able to modulate TCR-mediated signaling and play a critical role in enhancing viral infectivity in primary lymphocytes and infected animals. Interestingly, it has been recently reported that p12 could complement for effects of Nef on HIV-1 infection of Magi-CCR5 cells, which express CD4+, CXCR4 and CCR5 on the surface or macrophages.284 Also, the clones of Jurkat cells expressing the highest levels of p12 have been found to exhibit a more rapid rate of cell proliferation than the parental cells.284 Similarly to HIV Nef, the p12 protein, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse (IS).283 Thus, both Nef and p12 are recruited to the IS, but Nef potentiates TCR signaling293 while p12 dampens it.283
In summary, targeting TCR-mediated signaling seems to be a shared feature of both HIV and HTLV-1 viruses, reflecting probably their similar evolutionary pathway towards their adaptation to the host immune response. Thus, it is possible that similar molecular mechanisms may be involved in TCR-targeting strategies used by Nef and p12 to modulate TCR-mediated signaling pathways. If true, this hypothesis will generate new therapeutic targets (i.e., protein-protein interactions at the interface of p12 and its potential TCR-related partners) and opportunities, similar to those suggested for HIV Nef.
CMV pathogenesis.
To escape from NK cell-mediated surveillance, human CMV interferes with the expression of NKG2D ligands in infected cells. In addition, the virus may keep NK inhibitory receptors engaged by preserving human leukocyte antigen (HLA) class I molecules that have a limited role in antigen presentation.294 Despite considerable progress in the field, a number of issues regarding the involvement of NK receptors in the innate immune response to human CMV remain unresolved.
Recently, a direct interaction between the human CMV tegument protein pp65 and the NK cell activating receptor NKp30 has been reported.295 It has been shown that the binding of pp65 to NKp30 is specific and functional. Surprisingly, the recognition of pp65 by NKp30 does not lead to NK cell activation but instead results in a general inhibition mediated by the dissociation of the signaling ζ subunit from the NKp30-ζ receptor complex.295 This results in the diminishing of activating signals and loss in the ability of NK cells to kill normal, tumor and virus-infected cells.295
Within the context of SCHOOL model,30,31,54,133 the reported action of the human CMV pp65 protein may be due to its potential impact on the TM interactions between NKp30 and ζ, leading to disconnection and dissociation of the ζ subunit.133 This would prevent formation of signaling-competent ζ oligomers upon ligand stimulation and consequently, inhibit NK cell cytolytic activity (Fig. 10 and Table 3) in a manner similar in this respect to the inhibitory action of TCRα CP (Fig. 12 and Table 1). Interestingly, primary sequence analysis of the N-terminal end of pp65 shows the existence of multiple positively and negatively charged amino acid residues (Table 4). Thus, this pp65 region possibly contains the sequence that mimics the NKp30 or ζ TM domain with the Arg or Asp residues, respectively, that are known to mediate the interaction between recognition NKp30 chain and signaling ζ subunit.82,296 However, further experimental studies are needed to confirm the proposed mechanism.
Lessons from viral pathogenesis.
There are several important lessons that we can learn from the SCHOOL model-revealed molecular mechanisms of viral activity important for viral immune escape.31,32,133,140 One can also consider the striking similarities of the molecular mechanisms and basic structural principles that are suggested by the SCHOOL model to explain immunomodulatory effects of viral fusion and accessory proteins as well as of synthetic agents that affect intra- or inter-MIRR protein-protein interactions in TM or CYTO milieus, respectively.
In general, viruses seem to use TM-targeted immunomodulatory activity of their fusion proteins mostly during virus entry to suppress the host immune response, whereas modulation of CYTO interactions by using accessory proteins such as HIV Nef and HTLV-1 p12 plays a role in viral replication and enhancing viral infectivity in the host. Thus, our improved understanding of MIRR signaling-targeted immunomodulatory viral activity might allow us to reveal novel targets at these stages of viral pathogenesis.
The lessons that we can learn from viral pathogenesis in the context of the SCHOOL platform of immune signaling are believed to be very important for our further understanding of the molecular mechanisms that viruses use to infect the host and escape its immune response. These lessons are also believed to be of not only fundamental but also clinical importance.
  • The SCHOOL model30,31,54,133,138 suggests the molecular mechanisms of inhibitory action of MIRR TM peptides such as TCR TM peptides,196,198,201,204,208,218,221,223 NK TM peptides,217 and GPVI TM peptide.134,138 Within the model, the same mechanisms are likely used in vivo by HIV gp41 FP and fusion proteins of other viruses, such as SARS-CoV and HTLV-1 FPs, to suppress the host immune response.31,32,133,137,140 Importantly, viral agents are highly specific and efficient in inhibition of immune receptors. Together with our knowledge of the protein-protein interactions underlying this process, this allows us to use modern well-established computational, bioinformatic and synthetic methodologies58,209216 to design and produce highly specific and effective TM-targeted agents that are able to affect specific TM interactions of a targeted MIRR and suppress and/or modulate the receptor-mediated immune response. These agents would be of great fundamental and clinical value.
    Similar conclusions can be drawn for our ability to use the SCHOOL model of immune signaling and our current knowledge of the CYTO-targeted viral strategies in order to design and produce efficient and specific CYTO-targeted agents.
  • According to the SCHOOL model, TCRα CP, HIV gp41 FP and as predicted, SARS-CoV FP and HTLV-1 FP, affect similar TCR TM interactions (Fig. 12 and Tables 1 and and33).3032,54,133,137,140 Primary sequence analysis of these peptides (Table 4) shows different primary sequences but a striking similarity in charged or polar residue distribution patterns. This suggests that a computational approach combined with our knowledge of the molecular mechanisms of action, can and should be used for the rational design of effective immunomodulatory TM-targeted peptides. General well-established principles of designing TM peptides with an ability to insert into the membrane might be readily used at this stage.212
  • The SCHOOL model suggests that TM-targeted agents inhibit MIRR-mediated cell activation induced only by antigen/ligand but not antibodies to MIRR signaling subunits (Figs. 10 and and1212). Indeed, it has been shown for TCRα CP197 and HIV FP205 that these TM peptides inhibit only antigen-mediated T-cell activation, whereas stimulation with anti-CD3 antibodies in the presence of the peptides still result in functional cell response. Entirely similar considerations can be applied for other viral FPs such as SARS-CoV FP and HTLV-1 FP. Thus, the SCHOOL model suggests that antibodies to MIRR signaling subunits can be used during viral infection as promising therapeutics to modulate the affected immune cell response.
  • As shown for TCR, we are able to functionally disconnect any particular CD3 and/or ζ signaling subunits from the remaining receptor complex by using the relevant TM peptides and basic principles of the SCHOOL model of TCR signaling (Figs. 11 and and1313). This means that we can design, synthesize and use these peptides as a powerful tool to dissect fine molecular mechanisms of viral pathogenesis in the context of TCR signaling.
  • Two unrelated enveloped viruses, HIV and human CMV, use a similar mechanism to modulate the host immune response mediated by two functionally different MIRRs-TCR and NKp30. As predicted by the SCHOOL model, SARS-CoV and HTLV-1 can also use similar mechanisms during virus infection. Intriguingly, as shown in Table 4, similar positively charged residue distribution pattern with two Arg and/or Lys residues spaced apart by 8 amino acids is observed for the FPs of seemingly unrelated viruses such as HIV, Lassa virus (LASV),297 lymphocytic choriomeningitis virus (LCMV),297 Mopeia virus (MOPV),297 and Tacaribe virus (TACV).297 Thus, it is very likely that similar general immunomodulatory mechanisms can be (or are) used by other viral and possibly non-viral pathogens.31,32,133,140 In addition, as with HIV gp41 FP,254,255 it is promising to apply a similar strategy to block viral entry by using the agents able to interact directly with FPs of other viruses.
In conclusion, rather than targeting virus-specific proteins or processes, it would be advantageous to transfer therapeutic strategies that target redundant processes found among a number of viruses. In addition, as demonstrated by the similar function of natural HIV FP and synthetically derived clinically relevant TCRα CP, viral immune evasion strategies can be transferred to therapeutic strategies that require similar functionalities. Viruses represent years of evolution and the efficiency and optimization that come along with it. Therefore, viral functions should not only be studied as foreign processes but as efficient strategies that we can use in our own attempts at immune evasion or immunomodulation.
Revealing new points of intervention that can be used for therapeutic inhibition and/or modulation of a variety of functionally unrelated receptors expressed on various cells, a general SCHOOL platform therefore suggests novel therapies for disorders that are mediated by these receptors. Despite this section is focused on multiple MIRR-mediated diseases with unmet needs, novel therapies for SR-mediated disorders can be also developed using the SCHOOL platform-driven inhibitory strategy.
Thrombotic disorders.
Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix, which triggers platelet adhesion and aggregation.298300 The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels.299 Despite intensive research efforts in antithrombotic drug discovery and development,301 uncontrolled hemorrhage still remains the most common side effect associated with antithrombotic drugs that are currently in use on the about $11 billion market.
The major physiological function of platelets is hemostasis, prevention of bleeding, and the effect of aspirin has established that they are also involved in its pathological variant, thrombosis.302 Platelets also play a critical role in coronary artery disease and stroke, as evidenced by the well-documented benefits of antiplatelet therapy.301,303
Platelet adhesion, aggregation and activation induced by collagen is critically dependent upon the engagement and clustering of GPVI, a type I transmembrane platelet glycoprotein of about 62 kDa and the major collagen receptor on platelets.83,299,304307 GPVI has no intrinsic signaling capacity and signaling is achieved through the association with its signaling partner, the FcRγ chain.83 The selective inhibition of GPVI and/or its signaling is thought by most experts in the field to inhibit thrombosis without affecting hemostatic plug formation, thus providing new therapeutical strategies to fight platelet-mediated diseases.83,307312 As illustrated in Figure 15, GPVI-deficient platelets adhere to the subendothelium of denuded rabbit vessel but most of them adhere as single cells or very small aggregates, whereas the normal platelets form large aggregates on the subendothelium under these conditions.83 Thus, in contrast to current antithrombotics, GPVI receptor-specific inhibitors represent an ideal class of clinically suitable antithrombotics.
Figure 15
Figure 15
Adhesion of normal and GPVI-deficient platelets under flow conditions. Blood from a normal individual (A) and GPVI-deficient blood (B) were flowed over denuded rabbit vessel for 5 min and the adhered platelets were fixed with glutaraldehyde after washing (more ...)
However, despite intensive studies of the GPVI-FcRγ receptor complex,83,305,313,314 the mechanism of GPVI signaling was not known until recently when the SCHOOL model was introduced and applied to GPVI triggering and TM signal transduction.30,31,54,133,134,138 This resulted in the development of a novel concept of platelet inhibition and the invention of new platelet inhibitors within this promising antithrombotic strategy.134,138,232 The invented inhibitors are proposed to be useful in the prevention/treatment of thrombosis and other medical conditions involving collagen-induced platelet activation and aggregation as well as in the production of drug-coated medical devices (including stents) to prevent device thrombosis.134,138,232
Within the SCHOOL model, GPVI-mediated platelet activation is a result of the interplay between GPVI-FcRγ TM interactions that maintain receptor integrity in platelets under basal conditions and are provided by the association of two TM Asp residues in the FcRγ homodimer with the TM Arg residue of GPVI,85 and homointeractions between FcRγ subunits that lead to initiation of a signaling response (Fig. 16A). Binding of the multivalent collagen ligand to two or more GPVI-FcRγ receptor complexes pushes the receptors to cluster, rotate and adopt an appropriate orientation relative to each other (Fig. 16A, step 1), at which point the trans-homointeractions between FcRγ molecules are initiated (Fig. 16A, step 2). Upon formation of FcRγ signaling oligomers, the Src-family kinases Fyn or Lyn phosphorylate the tyrosine residues in the FcRγ ITAM (Fig. 16A, step 2). This leads to TM transduction of the activation signal and dissociation of FcRγ oligomers and downmodulation of the engaged GPVI subunits (Fig. 16A, step 3). Later, the dissociated oligomeric FcRγ chains can interact with FcRγ subunits of the non-engaged GPVI-FcRγ complexes, resulting in formation of higher-order signaling oligomers and their subsequent phosphorylation, thus providing lateral signal propagation and amplification (not shown).
Figure 16
Figure 16
Novel concept of platelet inhibition. (A) The SCHOOL model of collagen-stimulated GPVI-FcRγ transmembrane (TM) signaling, proposing that the homooligomerization of the FcRγ signaling subunit plays a central role in triggering the GPVI-FcRγ (more ...)
Interestingly, for the preformed oligomeric GPVI receptor complexes,114,125 the model suggests that under basal conditions, the overall geometry of the receptor dimer keeps FcRγ chains apart, whereas stimulation by collagen results in breakage of GPVI-GPVI extracellular interactions and reorientation of signaling FcRγ homodimers, thus bringing them into a close proximity and an appropriate relative orientation permissive of initiating the FcRγ homointeractions (Fig. 16A, step 1). Importantly, this highlights a striking similarity between the data on the coexistence of mono- and multivalent TCRs127 or GPVIs114,125 in resting T cells or non-stimulated platelets, respectively. The SCHOOL model suggests a similar molecular explanation to answer an important and intriguing question raised in these studies: why does the observed basal TCR or GPVI oligomerization not lead to receptor triggering and subsequent T-cell or platelet activation, respectively, whereas agonist-induced receptor crosslinking/clustering does?3034,54,133,138
Suggesting how binding to collagen triggers the GPVI-mediated signal cascade at the molecular level, the SCHOOL model of collagen-induced GPVI signaling reveals GPVI-FcRγ TM interactions as a novel therapeutic target for the prevention and treatment of platelet-mediated thrombotic events.31,132134,138 Specific blockade or disruption of these interactions causes a physical and functional disconnection of the subunits (Fig. 16B, Table 1). Antigen stimulation of these “pre-dissociated” receptor complexes leads to clustering of GPVI but not FcRγ subunits. As a result, FcRγ signaling oligomers are not formed, ITAM Tyr residues do not become phosphorylated and the signaling cascade is not initiated. Agents that target GPVI-FcRγ TM interactions may thus represent a novel class of platelet inhibitors. These include, but are not limited to, peptides, peptide derivatives and compositions and non-peptide small molecule inhibitors. Preliminary experimental results134,138,232 provided support for this novel concept of platelet inhibition. These and other data (Sigalov AB, Barnard MR, Frelinger AL, Michelson AD, unpublished results) demonstrated that depending on donor, incubation of whole blood samples with a peptide corresponding to the TM domain of GPVI (Gly-Asn-Leu-Val-Arg-Ile-Cys-Leu-Gly-Ala-Val) at a final concentration of 100 µM prior to addition of collagen (10 and 20 µg/ml) or convulxin (10 ng/ml) leads to a 30%–60% reduction in both the percentage of P-selectin-positive platelets and the expression of the platelet activation markers, P-selectin and PAC-1. This effect is specific: platelet activation via ADP (20 µM) is not affected by the peptide. As assumed by the SCHOOL model, this peptide inserts into the platelet membrane and competes with GPVI for the binding sites on the FcRγ TM domain. This results in “pre-dissociation” of the GPVI-FcRγ receptor complex and prevents formation of competent FcRγ signaling oligomers upon collagen binding (Fig. 16B). Importantly, a control peptide containing a single amino acid substitution (Arg to Ala) does not display inhibitory activity, a phenomenon predicted by the SCHOOL model since this peptide cannot compete with GPVI for the binding sites on FcRγ in the TM milieu.
In conclusion, a combination of basic principles of the SCHOOL model with well-known approaches to computationally design, synthesize and optimize TM peptides and peptidomimetics that target TM helixes in a sequence-specific manner 112,58,199202,207216 as well as with other well-established techniques to search for the relevant TM mutations or small molecule disruptors makes the proposed strategy both feasible and of great fundamental and clinical value. Combining breakthrough scientific ideas and advances in different fields30,31,52,53,133,134,137,138,232 and the high market potential,315 the SCHOOL technology opens exciting new avenues in innovative antithrombotic drug discovery and development.
Atopic dermatitis.
Atopic dermatitis (AD) is an increasingly common, chronic, relapsing, inflammatory skin disease characterized by impaired epidermal barrier function and cutaneous inflammation. The prevalence of AD has steadily increased during the past few decades. AD affects 1–3% of adults and up to 20% of children, in 85% of them the disease onset is before the age of 5 years.316 Besides the search for reliable and meaningful diagnostic tools, novel therapeutic approaches are required, as most of the treatments of AD are limited to symptomatic therapies.
Activation and skin-selective homing of peripheral-blood T cells and effector functions in the skin, represent sequential immunological events in the pathogenesis of AD.317 Numerous studies have pointed to the role of activated CD4+ T cells in AD. Topical immunomodulators that affect T cells and block T-cell activation may therefore have a role in the treatment of AD.
In the context of the SCHOOL platform-driven inhibitory strategy (Figs. 4 and and1010), blocking of T cell activation is best exemplified by studies on the immunomodulatory effect of TCRα CP (or TCR mimic peptide).196201,208,217,219,225,318 This peptide efficiently abrogates T cell-mediated immune responses in mice and man in vitro and in vivo (Table 2).196 When applied to murine ear skin prior to application of a contact allergen, TCRα CP efficiently blocked ear swelling responses in mice.196 In patients with AD, psoriasis or lichen planus, the peptide being applied topically one time a day on 3 consecutive days196 inhibited inflammatory reactions equally well as betamethasone in the same patient. These data clearly indicate that the SCHOOL TCR inhibitors can be a promising therapy for human T cell-mediated dermatoses and a substitute for corticosteroids.
Rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that causes chronic inflammation of the joints. About 1% of the world's population is afflicted by RA, women three times more often than men. The economic burden created by RA is enormous with direct costs per patient of about $2,500–14,500 annually and indirect costs of about $1,500–45,000 annually. For this reason, new methods of treatment are eagerly sought and developed.319 Typically, new approaches to RA involve the use of monoclonal antibodies, peptides, antigen mimetics, T-cell vaccinations, infusion/injection of anti-inflammatory cytokines or cytokine inhibitors.318,319
Similar to AD, therapeutic inhibition of TCR can represent a promising strategy to treat RA. Recently, using the Mycobacterium tuberculosis (MTB) adjuvant-induced arthritis model in Wistar rats, TCRα CP was tested as a potential therapeutic agent for the treatment of acute onset arthritis.318 The peptide itself and the peptide-lipid conjugate (PLC) administered either subcutaneously or intraperitoneally were effective in the treatment of acute arthritis.318 The effectiveness of intraperitoneally administered PLC has been shown to be comparable to cyclosporin.318
These findings demonstrate that despite potential caveats, the use of the SCHOOL TCR inhibitors to treat human autoimmune, allergic and other T-cell mediated diseases can provide an exciting alternative to current therapies.
Sepsis and hemorrhagic shock.
Septic shock is characterized by massive release of proinflammatory mediators and leads not only to tissue damage, but also to haemodynamic changes, multiple organ failure and ultimately death. 750,000 cases of sepsis occur annually in the US and intensive medical care of these patients includes antibiotics, intravenous fluids, blood transfusions, kidney dialysis, nutritional and respiratory support and sometimes surgery to remove the source of an infection. However, even with this regimen, about 215,000 sepsis patients die annually in the US.
Initial findings established TREM-1, a member of the MIRR family (Fig. 6), as an amplifier of the systemic inflammatory response syndrome associated with sepsis.320,321 Blockade of TREM-1 has been shown to protect mice against lipopolysaccaride (LPS)-induced shock, as well as microbial sepsis caused by live Escherichia coli or caecal ligation and puncture.320,322 By demonstrating a critical role of TREM-1 in acute inflammatory responses to bacteria, these results implicate TREM-1 as a promising therapeutic target for septic shock.
Hemorrhagic shock is primarily caused by traumatic injury, from automobile accidents, bullet or knife wounds and falls. Trauma causes approximately 150,000 deaths per year, and is the leading cause of death in the population under age 45 in the US. Recently, early inhibition of the TREM-1 pathway has been shown to be useful during severe hemorrhagic shock in rats in preventing organ dysfunction and improving survival.323
The SCHOOL model-driven inhibitory strategy suggests a novel approach to therapeutic inhibition of TREM-1 and allows us to design and synthesize TREM-1-specific inhibitors that can represent highly promising therapeutics to lessen morbidity and mortality by protecting against septic shock and to improve survival after hemorrhagic shock. Importantly, these agents can be used for other TREM-1-related disorders and conditions.324
Cancer.
Non-small cell lung cancer (NSCLC), which accounts for 85 to 90% of diagnosed lung cancers, has a five-year survival rate of just 15 percent and kills approximately 1 million people annually. Only in the US, there are more than 200,000 new lung cancers patients every year, and about 160,000 will die every year due to a lack of effective therapy.
Despite it is known that tumor-associated macrophages can release growth factors, cytokines and inflammatory mediators and, by doing so, facilitate tumor progression or metastasis, the role of TREM-1, which can trigger and amplify the inflammatory response, in cancer progression is still unknown. Recently, TREM-1 expression in tumor-associated macrophages has been intriguingly shown to be associated with cancer recurrence and poor survival of patients with NSCLC, thus suggesting that TREM-1 and the inflammatory response may play an important role in cancer progression.325 Besides this, these findings suggest that inhibition of the TREM-1 pathway may be a promising target for the development of new, targeted anticancer therapy to block the specific inflammatory response and stop the cancer progression.325
Thus, the TREM-1-specific inhibitory agents designed and synthesized in line with the SCHOOL model-driven inhibitory strategy may represent a novel therapy for NSCLC.
Inflammatory bowel diseases.
Inflammatory bowel diseases (IBDs)326 affect millions of people worldwide with 2.8 million currently diagnosed in the United States alone. Patients with IBDs can be divided into two major groups, those with ulcerative colitis and those with Crohn's disease. The incidence of IBD is rising in developing countries around the world. Currently the global market is estimated at $16 billion and this is set to continue to rise. Despite the identified advances in all major areas relevant to IBD pathogenesis, there is a great need for additional targets and therapeutic agents for effectively reducing these disorders.327
Recently, NKG2D receptor expressed on CD4+ T cells has been reported as a novel target for the treatment of IBDs.328330 Briefly, intestinal inflammation in colitic severe combined immunodeficiency (SCID) mice has been recently shown to be characterized by significant increase of CD4+NKG2D+ T cells.329,330 As also demonstrated,329,330 neutralizing anti-NKG2D mAb treatment prevents or ameliorates the development of colitis primarily by inhibiting the expansion and/or infiltration of pathogenic T cells in the colon and secondarily by inhibiting the development of pathogenic Th1 cells. The authors concluded that targeting of NKG2D signaling in NKG2D-expressing pathogenic CD4+ T cells may be a useful strategy for the treatment of Th1-mediated chronic intestinal inflammation such as Crohn disease.329,330
Importantly, in addition to IBDs, other diseases and conditions including but not limiting to celiac disease, type I diabetes, hepatitis, rheumatoid arthritis and solid organ allograft rejection, have been also shown to involve NKG2D-mediated cell activation and benefit from NKG2D inhibition.331335
NKG2D is a member of the MIRR family (Fig. 6). Thus, the NKG2D-specific inhibitors including TM-targeted agents (Table 1) designed and synthesized in line with the SCHOOL model-driven inhibitory strategy can represent a superior alternative to large protein therapeutics in the prevention and treatment of IBDs and other NKG2D-related disorders.
Allergic diseases.
Antibodies repress invasions of bacteria or viruses but also have the potential to induce inflammation that is harmful to humans. Fc receptors (FcRs) that recognize the Fc portion of antibodies are present on many immune cells and provide an essential link between humoral and cellular branches of the immune system.80 FcRs exist for every antibody class: FcγR bind IgG, FcαR bind IgA, FcεR bind IgE, FcµR bind IgM and FcδR bind IgD. The deregulated antibodies attack the body, affecting our quality of life, and also sometimes leading to autoimmune diseases.
Excessive production of IgE causes allergic diseases such as atopic dermatitis, food- or insect allergy, allergic rhinitis and bronchial asthma. These diseases affect around 20% of Americans and nearly a third of the population worldwide, with an increasing incidence.336 The worldwide market for allergy remedies market continues to grow rapidly with annual sales of $36 billion.
The inhibition of FcεRI function represents a promising anti-allergic strategy and has already led to various therapeutic approaches for allergies, including monoclonal antibodies specific for IgE.39,336 However, as therapeutic agents, antibodies pose serious disadvantages.51 The FcεRI-specific inhibitors including peptides, peptidomimetics and small molecules designed and synthesized in line with the SCHOOL model-driven inhibitory strategy can provide an effective alternative to current therapies of allergic and other FcεRI-related diseases.
Considering growing interest in targeting cell surface receptor signaling as a potential treatment strategy for different diseases, the development of novel pharmacological approaches critically depends on our improved understanding of the molecular mechanisms underlying receptor-mediated transmembrane signal transduction.
My central hypothesis is that within the single- and multichain receptor families, the similar structural architecture of the receptors dictates similar mechanisms of receptor triggering. This suggests the existence of similar therapeutic targets in seemingly unrelated diseases and makes possible the development of global pharmacological approaches as well as the transfer of our clinical knowledge, experience and therapeutic strategies between these diseases.
Discovery of an unusual and unique biophysical phenomenon, the existence of specific homointeractions between signaling-related intrinsically disordered proteins, defined the last key piece in the puzzle of receptor triggering and led to the development of a novel general platform for receptor signaling, the signaling chain homooligomerization (SCHOOL) platform. Within the platform, homooligomerization of receptor intracellular signaling domains is considered as a necessary and sufficient condition for receptor triggering. The platform therefore suggests that receptor oligomerization induced or tuned upon ligand binding outside the cell is translated across the membrane into protein oligomerization inside the cell. Assuming that the molecular principles underlying transmembrane signaling and cell activation mediated by single-chain and multichain receptors are similar, the SCHOOL platform can readily describe molecular signaling of any particular receptor. In doing so, the platform suggests molecular mechanisms for the vast majority of unexplained observations accumulated to date and reveals key points of control in receptor triggering as novel universal therapeutic targets for a diverse variety of receptor-mediated disorders. Also, the platform significantly improves our understanding of the immunomodulatory activity of many human viruses.
Excitingly, the SCHOOL platform unravels the striking similarity of the molecular mechanisms underlying immunomodulatory activities of TCR transmembrane peptides and viral fusion peptides. It appears that different viruses use their fusogenic peptides not only to fuse their membranes to their target host cells but also to modulate and escape the host immune response. These findings strongly support the feasibility, utility and both fundamental and clinical importance of the SCHOOL platform-driven modulatory strategy, which is now “approved” by nature. This also suggests the possibility of design and synthesis of novel therapeutics that can work as specific and effective as viruses do.
Within the SCHOOL platform, similar approach can be applied to any particular receptor of the single- and multichain receptor families and therefore to any disease or medical conditions mediated by this receptor. Application of this approach to the major collagen receptor on platelets, GPVI, has already resulted in the development of a novel concept of platelet inhibition and the invention of innovative platelet inhibitors. Importantly, the similar mechanistic principles were used to explain immunomodulatory effects of clinically relevant TCR transmembrane peptides and to design, synthesize and apply new GPVI-targeted platelet inhibitors. Thus, this is not only a comprehensive example of the usability and predictive power of the SCHOOL model but also supports the central hypothesis in the context of our ability to develop global therapeutic strategies to treat seemingly disparate diseases.
In summary, the SCHOOL platform provides a set of basic molecular principles underlying receptor-mediated signaling that can be readily used for rational drug discovery and design. Considering the multiplicity and diversity of receptors involved in the pathogenesis of numerous human diseases, the platform, together with the lessons learned from viral pathogenesis, can contribute significantly to the improvement of existing therapies and the development of novel therapeutic strategies for malignancies, thrombotic diseases, inflammatory diseases, diverse immune disorders, including those with infections caused by various viruses and other receptor-mediated medical conditions.
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