Qualitative and quantitative proteomic analysis of cancer-PD1 interactome in osteosarcoma cells

ABSTRACT: The PD-1 immune checkpoint is the target for cancer immunotherapy, which has recently revolutionized cancer treatment approaches. Interestingly, PD-1 is not only expressed by immune cells but also by cancer cells, however contradictory reports are available regarding its significance. The role and PD-1 protein-protein interactions have been well characterized in the immune cells, yet little is known regarding the cancer-expressed PD-1 interactome. Considering the distinct role PD-1 plays in these types of cells and high mutational burden in cancer, the PD-1 interactome may differ significantly from our current understanding. We used U2OS cells as a cellular model of osteosarcoma to investigate the PD-1 protein-protein interactions in cancer. Apart from the wild-type PD-1, the study included two phosho-tyrosine PD-1 mutants (Y223 and Y248), where tyrosine was substituted with phenylalanine. PD-1 phosphorylation at residues Y223 and particularly Y248, was reported critical for the PD-1 downstream signaling in immune cells. Therefore, including these protein variants in the study enabled identification of phospho-PD-1 dependent protein interactions. The PD-1 interacting proteins were isolated through PD-1 pull-down performed in U2OS cells overexpressing PD-1 protein fused with V5 and Twin-Strep tags. The interacting proteins were purified on StrepTactin magnetic beads and eluted into SDS reducing buffer. Quantitative bottom-up mass spectrometry analysis and bioinformatic processing identified 52 interacting protein candidates (FDR<1%) for the WT PD-1 and 79 proteins for each of the phospho-tyrosine mutants. 36 proteins were shared across all PD-1 protein variants, 8 proteins were unique for the WT PD-1, implicating that the latter require the functional phosphorylation of PD-1 tyrosine residues.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tibia, Cell Culture

DISEASE(S): Osteosarcoma

SUBMITTER: Jakub Faktor  

LAB HEAD: Sachin Kote

PROVIDER: PXD050950 | Pride | 2025-03-06

REPOSITORIES: Pride