Chapter 4 │ Page 119 receptors [29]. To investigate these e ects, computational molecular dynamics (MD) simulations have proven a valuable tool [30]. For example, Yusupov et al. [31] showed that oxidation of cell adhesion protein CD44 and its ligand, hyaluronan, significantly decreased their binding a inity, and found indications that the breaking of important disulfide bonds near the binding groove plays an important role. Lin et al. [32] reported that the disruption of salt bridges of immune checkpoint CD47 connecting it to its ligand, caused by oxidation of Lys residues, was associated with conformational changes, and decreased the binding a inity of the CD47 – SIRPα complex. On the other hand, oxidation does not necessarily disrupt normal protein function in all cases. Indeed, while Ghasemitarei et al. [33] found that oxidation of cysteine inside the protein channel of the xC- antiporter severely impaired cysteine uptake by the channel, a similar oxidation did not have a strong e ect on transport through the AQP1 channel, as shown by Yusupov et al. [34]. In this study, we employed non-reactive MD simulations to investigate the e ect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Umbrella sampling (US) was used to determine the binding energy of these MHC-I complexes to their NK cell receptor, respectively KIR2DL1 and NKG2A/CD94. To complement these simulations with experiments, we analyzed the expression of these ligands on three head and neck squamous cell carcinoma (HNSCC) cancer cell lines, a cancer type known for NK cell enrichment [35]. Our data reveals limited e ects of therapy-induced oxidation in both the in silico and in vitro approach. Expanding our focus, we performed flow cytometric analyses on the expression of various NK-regulating ligands, including the T cell immunoreceptor with Ig and ITIM domains (TIGIT) receptor ligands CD155 and CD112 and immune checkpoint CD73, and discovered a rapid reduction of these targets upon NTP exposure. Furthermore, the NK cell activating MICA/B proteins were upregulated on the cellular membrane 24h post-treatment. Our study unravels appealing targets for therapy-induced oxidation and sheds light on the complex chemical and biological
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