Chapter 4 │ Page 135 discussed above, a few additional salt bridges were found to be transiently present in our simulations. Except for the salt bridge Glu21KIR2DL1 – Arg69HLA-Cw4, these additional salt bridges are the result of promiscuous interactions between the amino acids that form salt bridges in the crystal structure. A special case is the salt bridge Asp183KIR2DL1 – Lys146HLA-Cw4. These amino acids did not interact in any of the simulations, as their relative distance was too large, except in replicate 2 of the oxidized system, where a salt bridge formed with a persistence of 92% in the equilibrated part of the simulation. This replicate underwent the largest pivot of HLA-Cw4 with respect to KIR2DL1, also evident from its RMSD (see Figure 3a), allowing formation of this new interaction. In summary, our simulations indicate that NTP-induced oxidations alter the structure of the investigated ligand-receptor complexes, but do not a ect their binding strength. To complement these computational results experimentally, we monitored the expression status of these protein complexes following plasma treatment of cancer cells in vitro. 3.3. MHC-I Complexes HLA-C And HLA-E Are Moderately Altered After NTP Application To determine the immediate oxidative e ects of NTP on the MHC-I complex molecules HLA-C and HLA-E in an experimental setting, di erent HNSCC cell lines were exposed to NTP and immediately analyzed for ligand expression (0h analysis). While no significant di erences in mean fluorescence intensity (normalized ΔMFI) were reported for HLA-E across all cell lines (Figure 6b), detection of HLA-C decreased significantly compared to untreated controls in the SCC61 cell line (Figure 6a, middle panel). At an NTP regime of 200Hz, HLA-C expression was reduced by 1.4-fold, which further diminished to more than a 2-fold reduction in expression levels with a 500Hz regime (0.69 and 0.44, respectively; p ≤ 0.0317).
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