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 (, and ), 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 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.12,58,67–69, 120,142–150
In RTKs, divalent ligand binding is believed to stimulate monomeric receptor dimerization and trans-autophosphorylation at defined tyrosine residues through intrinsic kinase activity.62–64
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.58,145,147,151,153–158
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 (). Importantly, peptide drugs possess several advantages over large protein molecules (). 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
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 ().30,33–35,54
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 ().
There is a growing line of experimental evidence supporting the SCHOOL platform-driven CYTO-targeted strategy of receptor inhibition/modulation (). 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.167–174
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 () 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 (). 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 ().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 () 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 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 ().171,172,178
Dimerization of MyD88 favors the recruitment of downstream signaling molecules such as two IL-1R-associated kinases (IRAKs): IRAK1 and IRAK4 (). 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 ().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
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.169,184–186
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,30–35,54,55
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 (). 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 (). 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 (). This finding directly proves the SCHOOL platform-driven CYTO-targeted strategy for therapeutic inhibition of SRs ().
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 () 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 ( and ).
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 ( and
Importantly, the model has provided a mechanistic explanation at the molecular level for specific processes behind “outside-in” MIRR signaling that were unclear.30–32,34,54,132–134,137,138
Examples include molecular mechanisms of action of the therapeutically important TCR TM peptides196–203
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 ( and
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 ( and
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.1,4–22
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 (). Ligand stimulation of these “pre-dissociated” receptors leads to reorientation and clustering of the recognition but not the TM agent-affected signaling subunits (). As a result, the corresponding signaling oligomers are not formed, ITAM Tyr residues do not become phosphorylated and the signaling cascade is not initiated (). 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 () 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,1–11,58,202,203,207–216
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 ( and
) and to have a powerful and well-controlled influence upon MIRR-mediated cell activation, thus controlling the immune response.30–35,54,55,132–134,137,138
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,196,197,199–204,208
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.32,133,138–140
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 (), 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 ().30,32–35,54,55,132
Importantly, the model suggests intrareceptor TM interactions not only as promising therapeutic targets but also as an important point of viral attack ().31–33,132,133,139,140
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 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 ().218
The peptide might be used also as a treatment for rheumatoid arthritis and other T cell-mediated disorders.198–200,202,204
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 ().30
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 (). This leads to inhibition of antigen- but not antibody-mediated TCR triggering and cell activation (). 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 (, and
). 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.134,138,196–198,201–203,218–225
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 ().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 (). Thus, upon antigen stimulation, these subunits do not participate in signaling, resulting in the lack of the Bδ and Bγ activation signals () 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 ().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 (), 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 functions229–231
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.30–34,54,132,133
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 (). 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 () and in vitro and animal data obtained for other TM peptides () 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.233–235
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.30,31,34,35,52–55,131
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.30,31,33–35,54,55,133,137,138
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 ( and
Within the SCHOOL platform, formation of competent signaling homooligomers in CYTO milieu is necessary and sufficient to trigger receptor activation (). 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 () and initiation of a MIRR-mediated cell response. Similar to the intra-MIRR TM interactions (), 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 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,1–27
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 (), antibody stimulation of CYTO-targeted agent-affected MIRRs does not result in MIRR triggering and generation of the activation signal ().
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 (). This gives us a powerful and well-controlled influence upon MIRR-mediated cell activation, thus controlling the immune response.
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.1–27
Importantly, the recent success in using CYTO-targeted agents to modulate FcγRIIA signaling (),174
clearly demonstrates the technological feasibility of the SCHOOL platform-driven MIRR CYTO strategy of receptor modulation () as well as its fundamental and clinical importance.