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Toll-like receptors (TLRs) play central roles in innate immune defense against infection by binding to microbial molecules. Because humans have only a small number of innate immune receptors available to counteract a virtually unlimited number of microbial molecules, these receptors cannot be too restricted in their antigen specificity. Instead, as has been proposed, they recognize common patterns in a large number of microbial molecules and are sometimes referred to as pattern recognition receptors (PRRs).
As of now, 10 TLRs have been reported in humans and 12 in the mouse. They are involved in the recognition of multiple groups of microbial molecules which are not usually found in humans. TLR4 forms heterodimers with MD-2 that are activated by lipopolysaccharide (LPS) from gram-negative bacteria. TLR2 forms heterodimeric complexes with either TLR1 or TLR6 that can bind to lipopeptides or lipoproteins from bacterial membranes. TLRs 3, 7, 8, and 9 are localized in intracellular organelles and recognize diverse types of microbial nucleic acids. TLR5 can be stimulated by the protein, bacterial flagellin. Therefore, the spectrum of TLR ligands is unusually broad for a single family of proteins; they can form stable complexes with molecules ranging from hydrophilic nucleic acids to hydrophobic lipids, which vary in size from small-molecule drugs to large macromolecules.
Structures of TLRs extracellular domains
The extracellular domains of TLRs belong to the leucine-rich repeat (LRR) family. This is a relatively large protein family with 19,556 entries in the InterPro database that includes 561 human proteins. These proteins are involved in diverse physiological functions, mostly via protein-protein interactions. LRR family proteins with known structures have 2∼25 LRR modules, each of which contains a 20∼30–amino acid sequence that includes the LxxLxLxxN motif (Figure 1a).
The conformation of the central β-sheet of an LRR family protein can be defined by its twist, tilt, rotation angles, and radii. Most LRR proteins have uniform radii and β-sheet angles throughout the protein. Unexpectedly, the structures of some TLRs show structural transitions that divide the proteins into three subdomains: N terminal, central, and C terminal. TLRs 1, 2, 4, and 6 belong to this three-domain subfamily (Figure 1b). All the central domains of TLRs 1, 2, 4, and 6 lack the asparagine networks that have an important role in stabilizing the overall shape of the horseshoe-like structures. Therefore, structural distortions are allowed, at least in part because these asparagine networks are interrupted in the central domains. Interestingly, the domain boundaries fulfill important functions in these TLRs. The ligand-binding pockets of TLRs 1, 2, and 6 coincide with the structural transitions between the C-terminal and central domains (Figure 1c).
Figure 1. Arrangement of Toll-like receptor (TLR) domains.
Engagement of TLRs activates multiple signaling cascades leading to the induction of genes involved in innate immune responses. Binding of ligands followed by dimerization of TLRs recruits TIR domain-containing adapter proteins such as myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein-inducing IFN-β (TRIF), TIR-associated protein (TIRAP), and TRIF-related adaptor molecule (TRAM). Individual TLRs recruit specific combinations of these adapter molecules to elicit specific immune responses tailored to infectious pathogens. MyD88 is recruited to all TLRs except for TLR3 and associates with IL-1R-associated kinases (IRAKs) and TNFR-associated factor 6 (TRAF6), resulting in activation of canonical inhibitor of kappa light polypeptide gene enhancer in B-cells, kinases (IKKs) (IKKα and IKKβ) and nuclear factor (NF)-κBs (Figure 2). In contrast, TRIF is recruited to TLR3 and TLR4, leading to activation of NF-κB as well as noncanonical IKKs (TRAF-family-member-associated NF-kB activator (TANK) binding kinase 1 (TBK1) and IKKι) and interferon (IFN) regulatory factor (IRF 3 via TRAF proteins. TIRAP functions as a sorting adapter that recruits MyD88 to TLR2 and TLR4, whereas TRAM functions as a bridge adapter between TLR4 and TRIF.
Figure 2. MyD88-dependent TLR pathway and its negative regulators.
TLR ligands and related diseases
TLRs are the most characterized PRRs. The members form homo- or heterodimers and recognize various pathogen-associated molecular patterns (PAMPs) (Table 1). TLR signaling leads to production of proinflammatory cytokines and type I IFNs and these responses are crucial for host defensive responses against pathogens. However, the aberrant activation of TLR signaling may be responsible for the pathogenesis of autoimmune, chronic inflammatory and infectious diseases (Table 1). Furthermore, increasing evidence has indicated that TLRs respond to endogenous molecules, most of which are released from dead cells, and are often referred to as damage-associated molecular patterns (DAMPs), suggesting that TLRs can survey danger signals and are associated with sterile inflammation.
Table 1. TLR ligands and related diseases.
|TLR1||(w/TLR2) triacyl lipoprotein||n.d.|
(w/TLR1) triacyl lipoprotein
(w/TLR6) diacyl lipoprotein, LTA, zymosan
|(w/TLR6) HMGB1, HSPs, ECM||Candidiasis|
|TLR4||LPS, viral envelop proteins||HMGB1, HSPs, ECM,|
Ox-phospholipids, b-defensin 2
(w/TLR6) Amyloid-b, Ox-LDL
|TLR6||(w/TLR2) Diacyl lipoprotein, LTA, Zymosan||(w/TLR2) HMGB1, HSPs, ECM|
|TLR7/hTLR8||ssRNA||ssRNA (immune complex)|
|TLR9||DNA, hemozoin||DNA (immune complex)||Malaria, SLE|
Negative regulation by specific regulators is crucial for immune homeostasis and its collapse often causes various diseases. As the importance of inhibitory TLR regulation is unveiled, the therapeutic manipulation of TLR signaling for the treatment of diseases that are derived from overactivation of innate immunity. TLR antagonists, structural analogs of TLR ligands that interact with receptors but fail to initiate signal transductions, are being developed to treat excessive or chronic inflammation and autoimmunity. One evident target example is sepsis characterized by whole-body inflammation caused by microbial infection. The most promising TLR4 antagonist, eritoran can limit excessive inflammatory responses induced by LPS and improve survival in mouse septic models. Structural analysis suggests that antagonism by eritoran results from hydrophilic interactions between TLR4 and MD-2, a TLR4-binding protein on the cell surface and crucial for eliciting TLR4 signaling. Another example of a TLR4 antagonist, ibudilast (AV-411) has the potential for treatment of neuropathic pain. Ibudilast, which had been characterized and used as an anti-inflammatory drug based on phosphodiesterase inhibition, can also suppress glial cell activation by induction of IL-10.
The importance of TLR signaling for both immune homeostasis and for defense mechanisms against pathogens has emerged in the past decade. As such, it is clear that TLR function must be tightly regulated and many negative regulators of TLR signaling have been identified. Therefore, understanding negative regulation of TLR signaling may be helpful to develop methods of artificial manipulation of TLR signaling to restore inflammatory diseases, overcome uncontrolled inflammation, and make countermeasures against infection. Chemical inhibitors that suppress essential components for TLR signal activation have been developed. However, there has been little progress in therapeutic applications that target negative regulators of TLRs.