Photoactivation of Innate Immunity Receptor TLR8 in Live Mammalian Cells by Genetic Encoding of Photocaged Tyrosine

Abstract

The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll‐like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single‐stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole‐cell environment. Here, we engineer the first light‐controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.

Integrated Transcriptome and 16S rDNA Analyses Reveal That Transport Stress Induces Oxidative Stress and Immune and Metabolic Disorders in the Intestine of Hybrid Yellow Catfish (Tachysurus fulvidraco♀ × Pseudobagrus vachellii♂)

“…Therefore, the decreased transcript levels of tlr9 and tlr13 may be a protection measure to enhance the mucosal barrier and attenuate intestinal injury and inflammatory responses under transport stress. The inconsistent patterns of tlr5 , tlr9 , and tlr13 transcript accumulation under transport stress were considered to reflect the effectiveness of the innate immune response, with its intricate balance between activation and regulation [ 42 ]. In another study, the down-regulation of syngr3 , which encodes a synaptic vesicle protein, was found to alleviate synaptic dysfunction in mouse and fruit fly primary neurons [ 43 ].…”

Extracellular vesicle-associated microRNA-30b-5p activates macrophages through the SIRT1/ NF-κB pathway in cell senescence

“…Peripheral blood mononuclear cell (PBMC) were isolated from murine whole blood with Ficoll reagent following the previously established protocols ( 42 ).…”