RNA-seq of Cdc42 knockout murine cytotoxic T lymphocytes
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ABSTRACT:
PROVIDER: PRJEB80897 | ENA |
REPOSITORIES: ENA
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Project description:The cytotoxic activity of lymphocytes is particularly dependent on the regulated and polarised delivery of lytic granules to infected or malignant cells. Although genetic and mechanistic studies have identified a number of factors that regulate exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Here, using a genome-scale CRISPR knockout screen in human natural killer cells, we characterise a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation among the most prominent pathways. By combining global protein lipidation and membrane lipid composition studies, we further uncover the critical role of ZDHHC17 in SNAP23 palmitoylation and targeting of palmitoylated SNAP23 to the membrane for cytotoxic granule fusion and release. Using ODYA-17 labelling of Ctrl and sgZDHHC17 NK-92 cells followed by pulldown and proteomic analysis, we investigated the palmitoylome of the NK-92 model cell line. By comparing the abundance of ODYA-17 labelled proteins in the lysates of Ctrl and sgZDHHC17 NK-92 cells, we defined the molecules that depend on the activity of the palmitoyl transferase ZDHHC17.
2025-09-18 | PXD045419 | Pride
Project description:Regulated exocytosis controls key cellular functions ranging from neurotransmitter release to the secretion of immune mediators and its disruption is associated with numerous pathologies. The cytotoxic activity of lymphocytes is particularly dependent on regulated and polarized lytic granule delivery towards infected or malignant cells. Although genetic and mechanistic studies have identified factors regulating exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Through a genome-scale CRISPR knockout screen in a human natural killer cell line, we characterized a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation amongst the most prominent pathways. By combining global protein lipidation and membrane lipid composition studies, we found that ZDHHC17 drives palmitoylation of the core SNARE complex protein SNAP23 to target cytotoxic granules to GM1-rich lipid rafts whose assembly is controlled by serine palmitoyltransferase.
2025-09-16 | GSE307513 | GEO
Project description:Cdc42 is a Rho family GTPase known for its central role in cell polarity and cytoskeletal regulation. To understand the role of Cdc42 in polarised secretion from cytotoxic T lymphocytes (CTLs) we used CRISPR/Cas9 gene deletion. While Cdc42-deleted CTLs initially showed reduced secretion for up to two days after CRISPR-mediated deletion, full secretion was rapidly restored and even enhanced while CDC42 protein remained absent. In contrast, chemical inhibition of CDC42 using CASIN consistently decreased secretion in wild-type cells, but had no impact on Cdc42-deleted CTLs, confirming the specificity of this inhibitor. Comparative proteomics and transcriptomics of CTLs after Cdc42-deletion revealed transcriptional changes that could support improved T cell function including compensation via other Rho GTPases. Targeting the promoter region of Cdc42 did not trigger transcriptional adaptation, consistent with a nonsense-mediated decay mechanism of genetic compensation. Our work highlights the importance of taking orthogonal approaches to study protein function, and reveals the remarkable robustness of primary T cells to adapt to loss of an essential gene.
2025-09-08 | PXD063348 | Pride
Project description:Acute kidney injury (AKI) is an unsolved global public health problem, requiring new effective therapies. Cytokinesis cycle 42 (CDC42) is a potent therapeutic target for multiple diseases, whereas its function in AKI remains unknown. Here we report that inhibition of CDC42 protects against AKI via orchestrating kruppel-like factor 2 (KLF2)/HIF-1α/PINK1-mediated mitophagy. We showed Cdc42 was not only abundantly expressed in murine renal tubular epithelial cells (RTECs) but also up-regulated in human and murine RTECs post cisplatin exposure, respectively. Then, administration of a CDC42 inhibitor in vivo and in vitro, conditional knockdown of Cdc42 in murine RTECs, and knockout of CDC42 in human RTECs abated cisplatin-caused mitochondrial dysfunction and kidney injury. Furthermore, by integratively using transcriptome sequencing, bioinformatics analysis, dual luciferase reporter assay, and chromatin immunoprecipitation, we found CDC42 inhibition alleviated mitochondrial dysfunction via KLF2/HIF-1α/PINK1-mediated mitophagy, during which CDC42 inhibition enhanced the promoter activity of KLF2, while KLF2 transcriptionally up-regulated HIF-1α, and HIF-1α subsequently transcriptionally up-regulated PINK1, the crucial regulator of mitophagy. Moreover, the function of KLF2 and HIF-1α in CDC42-mediated RTECs damage and mitochondrial dysfunction was verified. In conclusion, we uncovered the role and underlying mechanism of CDC42 in AKI, providing AKI patients with new therapeutic strategies.
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2021-01-01 | GSE147433 | GEO
Project description:We wished to examine early transcriptional changes that occur after TCR engagement in CD8+ cytotoxic T lymphocytes (CTLs). To this end, we differentiated effector CTLs from OTI TCR transgenic mice for 7 days in vitro and then stimulated them with anti-CD3 for various times before lysing for RNA-seq. Results demonstrated significant transcriptional changes starting from 20 minutes. After 60 minutes, upregulated genes were most enriched for cytokines and transcriptional machinery.
2023-10-15 | E-MTAB-12083 | biostudies-arrayexpress
Project description:We investigated transcriptional changes in MAZR-, Runx3- and MAZR/Runx3-deficient cytotoxic T lymphocytes (CTLs). This analysis revealed that MAZR plays a compensatory role in the Runx3-dependent transcriptional program of CTL differentiation.
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