Mechanoreceptors play a key role in many cellular processes, such as protein expression, and at the same time allow cells to respond to changes in their environment. Recent research has shown that T and B cell receptors can also act as mechanoreceptors. We designed a new mechanoreceptor, based on the CAR protein (chimeric antigen receptor) and the INSRTR ngGFP construct. The INSRTR system is based on inserting a peptide which forms a coiled coil in the protein in a way that does not change the function of the protein. The addition of a peptide which forms a coiled coil with the insert (formation of a dimer) leads to a change in structure and thus affects the function of the protein. We prepared fusion proteins by inserting the sequence for autoinhibited ngGFP into the sequence for CAR. The fusion proteins were expressed in HEK293T cells, and the success of the expression was demonstrated by western blotting. The function of the mechanoreceptor was tested by addition of Raji cells to the transfected cells. Raji cells express the CD19 antigen, a protein against which the scFv of our fusion protein was directed. The interaction between the CAR receptor and the antigen expressed on Raji cells would result in activation of the mechanosensor (increased fluorescence), which was monitored by confocal microscopy. Before the addition of Raji cells, we observed fluorescence on the membrane of cells transfected with the autoinhibited and constitutively active constructs. The addition of Raji cells had no effect on the change in fluorescence, which led us to conclude that the mechanosensor was not functional. We prepared new constructs based on the previous ones, in which we removed the variable part of the receptor. The autoinhibited constructs were activated by adding a solution of a peptide that forms a coiled helix with the inserted peptide, and observed the fluorescence localized on the cell membrane. We tested the effect of different lengths of the connecting sequence between the helices. In addition to the length of the linker sequence, we also wanted to determine the effect of removing the autoinhibitory loop. For this purpose, we added a cleavage site for the TEV protease to the linker sequence. Upon addition of TEV protease to the cells, we observed a change in fluorescence.
We prepared several different fusion proteins and used them to demonstrate a new possibility of using coiled helices and a way to modify mechanosensors.
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