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Functional roles of acidic residues in the juxta-transmembrane region of Toll-like receptor 3 and 9

Jihee Kim

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Toll-like receptors (TLRs) are involved in sensing conserved molecules derived from microbial and viral origin and mediating induction of innate immunity and inflammation. So far, 10 and 12 functional TLRs have been identified in humans and mice, respectively. Each TLR appears to recognize distinct ...
Toll-like receptors (TLRs) are involved in sensing conserved molecules derived from microbial and viral origin and mediating induction of innate immunity and inflammation. So far, 10 and 12 functional TLRs have been identified in humans and mice, respectively. Each TLR appears to recognize distinct microbial ligands such as fungal and bacterial outer membrane components, bacterial lipoproteins, and bacterial and viral nucleic acids. In addition to distinct signaling induction, they have been known to localize in different compartments within cells. TLRs sensing extracellular ligands are expressed at the cell surface, whereas other TLR family members responsible for recognition of nucleic acids are mostly found intracellularly. Over-activation of the nucleotide-sensing TLRs is linked to autoimmune diseases such as systemic lupus erythematosus and psoriasis. The nucleotide-sensing TLRs including TLR3, TLR7, and TLR9 traffic from the ER to endolysosomes where they recognize their cognate ligands and initiate signaling. It has been shown that a polytopic membrane protein, UNC93B plays an essential role in delivering the intracellular TLRs from the ER to endolysosomes by binding to the transmembrane domain of the receptors. Indeed, 3d mice having a single point mutation in UNC93B1 (H412R) have defects in TLR3, 7, and 9 signaling. However, the detailed molecular mechanism underlining the UNC93B1-mediated activation of the nucleotide-sensing TLRs is poorly understood. To identify the specific amino acid sequences of the nucleotide-sensing TLRs that are important for interaction with UNC93B1, we compared the putative transmembrane domain and neighboring regions of various TLRs and found that all intracellular TLRs share a common characteristic, they contain acidic amino acids in the luminal side juxta-transmembrane region. Based on this observation, we investigated the importance of these acidic amino acid residues for the activity of the nucleotide-sensing TLRs by using mutant TLRs carrying mutation at the acidic residues. We first discovered that the acidic residues (D812 and E813) in the juxta-transmembrane region of TLR9 are crucial for interacting with UNC93B1. The mutant TLR9 poorly bind UNC93B1. The defect of the mutant TLR9 to interact with UNC93B1 resulted in the altered TLR9 localization. TLR9 could not reach the endolysosomes and remained in the endoplasmic reticulum even after CpG stimulation. Consequently, the cells expressing the mutant TLR9 did not response to the CpG stimulation. To extend our observation with TLR9 to other nucleic acid-sensing TLRs, we further generated TLR3 mutant proteins having mutation at D699 and E704 in the juxta-transmembrane region. In consistent with TLR9, the mutation of acidic residues in the juxta-transmembrane region of TLR3 not only prevented TLR3 from interacting with UNC93B1 but also rendered it incapable of inducing signaling in response to Poly I:C. Surprisingly, however, the mutant TLR3 properly translocalized to the CD63-positive endolysosomes in bone marrow-drived dendritic cells. Our data suggest that the localization of TLR3 is regulated by a distinct mechanism that does not require the interaction with UNC93B1. In summary, we demonstrate that the acidic residues in the juxta-transmembrane region of TLR3 and TLR9 possess important reguratory roles for the physical binding with UNC93B1 and the subsequent TLR signaling. Although the exact role of UNC93B1 in mediating TLR3 signaling still remains to be fully elucidated, the identification of specific amino acid residues in TLR3 and TLR9 that are crucial for the UNC93B1 binding may help to find a strategy for regulating activity of these receptors.
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Abstract …………………………………………………………………………………............... i
Contents …………………………………………………………………………………………... iii
List of Figures …………………………………………………………………………………..… iv
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Abstract …………………………………………………………………………………............... i
Contents …………………………………………………………………………………………... iii
List of Figures …………………………………………………………………………………..… iv
Abbreviations …………………………………………………………………………………….. v



Chapter . Functional roles of acidic residues in the juxta-transmembrane region of Toll-like receptor 3 and 9

I. Introduction ………………………………………………………………………………….... 2
II. Materials and Methods …………………………………………………………………….. 4
III. Results ……………………………………………………………………………………….. 7
IV. Discussion ………………………………………………………………………………….. 12
V. References ………………………………………………………………………………….. 16
VI. Figures …………………………………………………………………………………….. 19



Summary ……………………………………………………………………………………….. 30
Acknowledgement ..…………………………………………………………………………… 32
Curriculum Vitae ……………………………………………………………………………….. 33