Difference between revisions of "Part:BBa K2976001"

Revision as of 16:13, 21 October 2019

Toll-like receptor 1

Toll-like receptor 1 (TLR1) is a transmembrane glycoprotein that participates in the innate immune response to microbial agents. TLR1 could form active heterodimers with TLR2 when exposed to some pathogen-associated molecular pattern molecules (PAMPs), and the heterodimers recognizes plenty of substance in lipoarabinomannan (LAM) biosynthesis with the help of CD14.

Usage

In 2019 CPU_CHINA project, TLR1 is expressed along with TLR2 and CD14 to form the TLR1:TLR2:CD14 cluster on the designer cell membrane. As a Mtb sensor, the complex could recognize the substances of Mtb and then stimulate the downstream signaling pathway. Then, activated NF-κB initiates transcription of the gene circuits to express other proteins in our project.

Biology

After being activated with Mtb, the activation cluster TLR1:TLR2:CD14 triggers NF-kappa-B signaling pathways via MYD88 and TRAF6. NF-κB proteins exist in the cytoplasm in an inactive form because of their association with the IκB proteins. IκB proteins mask the nuclear-localization sequences (NLSs) of NF-κB subunits and retain it in the cytoplasm. Activation of TLR1:TLR2:CD14 cluster cause the degradation of IκB proteins by proteasomes. Then, NF-κB subunits could pass through the nuclear pore complex (NPC) and cause the expression of an array of pro-inflammatory cytokines and chemokines. Similarly, NF-κB subunits also can bind the NF-κB induced promoter and initiate transcription of the downstream genes behind these promoters.

Characterization

This year, we 2019 CPU_CHINA attempted to develop a novel strategy for treating tuberculosis based on immune-like cells. Since our immune-like cells should recognize M.tuberculosis, TLR2:TLR1:CD14 cluster is required on the designer cell membrane. And TLR1 forms active heterodimers with TLR2 to recognizes substance in lipoarabinomannan (LAM) biosynthesis with the help of CD14.Thus, we conducted some researches on TLR1, the basic part and obtained valuable results.

Plasmid (TLR2-P2A-TLR1-T2A-CD14) was transfected into HEK293T cells. In order to determine whether TLR1 was successfully expressed on the membrane of the designer cells, we performed Western blotting assay and Flow cytometry analysis

Figure 1.Western blot analysis of TLR1 expression and activation of TLR signaling pathway.

Western blot (Figure 1) result shows that TLR1 was successfully expressed in HEK293T cells after 48h transfection (A). In addition, we also investigated whether NF-κB signaling pathway could be activated in a TLR1/2 dependent pathway. Transfected cells were treated with Pam3Cys-Ser-(Lys)4, a TLR1/TLR2 agonist. According to the results, phosphorylation of IκB was elevated and IκB was down-regulated, which indicate the activation of NF-κB signaling pathway in TLR1/TLR2/CD14 transfected HEK 293T cells, and the successful expression of TLR1 on the cell membrane.

Flow cytometry results (Figure 2) show that TLR1 was expressed on the membrane of artificial HEK293 cells, while strong fluorescence intensity was not observed .Combined with the result of successful activation, it is inferred that TLR1/2 heterodimer formation caused the fluorescence disappearance.

Sequence and Features