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Proteomic Analysis for Cul5 E3 Ligase Complex as a Key Negative Feedback Regulator of TCR/Il2 Signaling and Anti-Tumor Activity in Cd8+ T Cells

ABSTRACT: To investigate the mechanism by which Cul5 regulates CD8+ T cell cytokine-dependent differentiation and TCR-dependent activation, we performed quantitative mass spectrometry (MS) by data-independent acquisition (DIA)-MS of total proteins in the Cul5 KO and NC primary CD8+ T cells in the following conditions: 1) Cytokine dependent expansion and differentiation (T0); 2) 8 hours cytokine withdraw prior to TCR stimulation (T8); and 3) 16 hours TCR stimulation post 8-hour cytokine withdraw (T16). Principal component analysis (PCA) and correlation analysis revealed that replicates in each condition clustered together while different conditions separated from each other, suggesting significant proteomic changes among different conditions of the same T cells as well as between Cul5 KO and NC T cells in each condition. Together with similar total protein quantities among all detected samples, DIA-MS analyses were of high quality. Additionally, the strong reductions of Cul5 abundances in the Cul5 KO cells from all three conditions compared to the NC cells confirms high KO efficiency. Of note, the proteomic analysis showed that the Cul5 protein level was significantly increased upon TCR stimulation post cytokine starvation in the NC cells, suggesting a potential negative feedback regulatory role of Cul5 in CD8+ T cell activation. Consistent with this idea, we observed more markedly upregulation of Cul5 expression upon of TCR stimulation of na•ve primary CD8+ cells. To identify Cul5 interacting proteins in CD8+ T cells, we overexpressed Cul5 with a C terminal HA-tag (Cul5-HA) in mouse primary CD8+ T cells by retroviral transduction. The cells were subjected to TCR stimulation for 12 hr, and Cul5-HA was immunoprecipitated by anti-HA, followed by DIA-MS analysis (co-IP-MS). Compared to the negative control samples (cells transduced with the empty vector), 65 proteins were enriched (p value <0.05 and fold change >1.5) in the anti-HA IP samples. Altogether, we report that Cul5 KO alters CD8+ T cell proteome and the Cul5 interactome.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, T Cell

SUBMITTER: Wenxue Li

LAB HEAD: Yansheng Liu

PROVIDER: PXD036793 | Pride | 2024-01-03

REPOSITORIES: Pride

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Proteomic Analysis for Cul5 E3 Ligase Complex as a Key Negative Feedback Regulator of TCR/Il2 Signaling and Anti-Tumor Activity in Cd8+ T Cells

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