CRISPR/Cas9 and cDNA overexpression screens to identify transmembrane transporters involved in PROTAC sensitivity/resistance
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1136866 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29862900.fastq.gz | Fastqsanger.gz | |||
| SRR29862901.fastq.gz | Fastqsanger.gz | |||
| SRR29862902.fastq.gz | Fastqsanger.gz | |||
| SRR29862903.fastq.gz | Fastqsanger.gz | |||
| SRR29862904.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 77 |
Similar Datasets
Project description:Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce selective protein degradation by linking an E3 ubiquitin ligase enzyme to a target protein. Here we used transporter-focused and genome-wide CRISPR/Cas9 as well as a barcoded solute carriere (SLC)-focused cDNA libraray to screen for transporters that are involved in the resistance or sensitivity to BET- and SLC9-targeting PROTACs.
2024-09-23 | GSE272433 | GEO
Project description:Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce selective protein degradation by linking an E3 ubiquitin ligase enzyme to a target protein. This approach allows scope for targeting “undruggable” proteins and several PROTACs have reached the stage of clinical candidates. However, the roles of cellular transmembrane transporters in PROTAC uptake and efflux remain underexplored. Here, we utilized transporter-focused genetic screens to identify the ATP binding cassette transporter ABCC1/MRP1 as a key PROTAC resistance factor. Unlike the previously identified inducible PROTAC exporter ABCB1/MDR1, ABCC1 is highly expressed among cancers of various origins and constitutively restricts PROTAC bioavailability. Moreover, in a genome-wide PROTAC resistance screen, we identified candidates involved in processes such as ubiquitination, mTOR signaling and apoptosis as genetic factors involved in PROTAC resistance. In summary, our findings reveal ABCC1 as a crucial constitutively active efflux pump limiting PROTAC efficacy in various cancer cells, offering insights for overcoming drug resistance.
2025-07-17 | MTBLS11394 | MetaboLights
Project description:<p>Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of SLCs in response to environmental stresses remain poorly elucidated. To infer functional relationships between SLCs and metabolites, we developed a strategy to identify SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9–based transcriptional activation approach to uncover transporters relieving cells from growth-limiting metabolic bottlenecks. Among the transporters identified, we characterized the cationic amino acid transporter SLC7A3 as a gene that, when up-regulated, overcame low availability of arginine and lysine by increasing their uptake, whereas SLC7A5 was able to sustain cellular fitness upon deprivation of several neutral amino acids. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells uncovered functional transporter-nutrient relationships and revealed that transport activity up-regulation may be sufficient to overcome environmental metabolic restrictions.</p>
2022-09-20 | MTBLS4268 | MetaboLights
Project description:Arabidopsis thaliana mutant lines of secondary metabolite transporters microbiome
| PRJEB65466 | ENA
Project description:We report the construction of 5 yeast meiotic cDNA libraries and perform proof-of-principle screens to show that these yeast cDNA libraries can be used to identify genes and gene isoforms that are important for competitive fitness. Samples 1-25 are from different stages of cDNA library construction and deep sequencing was used to characterize gene representation in each yeast cDNA library. Samples 26-175 are from proof-of-principle competitive fitness screens.
2021-05-12 | GSE174268 | GEO
Project description:To identify the possible genes influencing macrophage pro-inflammatory activation, we designed CRISPR screens using a human metabolic sgRNA library containing metabolism-related transcription factors, small molecule transporters, and metabolic enzymes in a Cas9-expressing lentiviral vector.
2023-08-03 | GSE216353 | GEO
Project description:MDM2 inhibitor remarkably induced biomineralization in hDPSCs but it remained the problem that p53 activation is insufficient due to MDM2-p53 autoregulatory feedback loop. To overcome the limitation of the MDM2 inhibitors, we applied proteolysis targeting chimera (PROTAC), a technology that degrades a protein of interest (POI) by intracellular ubiquitin-proteasome system. Hence, we propose a strategy to induce hard tissue regeneration by MDM2-targeting PROTAC technology. We selected Nutlin-3 of POI ligand among various MDM2 inhibitors based on the screening process, and the selected CRBN of E3 ligase ligand. The MDM2-PROTAC synthesis platform was designed by each ligand combination. By performing the degradation test for selected compounds, we evaluated the characteristics of the developed MDM2 PROTAC such as the maximal degradation concentration (DCmax), the half of maximal degradation concentration (DC50), and half-lifetime. To investigate gene expression profiling of MDM2-targeting small molecules, we conducted RNA-sequencing under MDM2 PROTAC and Nutlin-3 (POI ligand) treated conditions. We not only confirmed a robust effect on biomineralization by MDM2-targeting small molecules but also demonstrated the potent osteogenic differentiation ability of MDM2 PROTAC when compared to an MDM2 inhibitor. MDM2-PROTAC significantly increased mRNA levels of osteogenic differentiation marker genes. Also, the significant bone generation effect of MDM2 PROTAC was validated in an ovariectomy (OVX)-induced osteoporosis animal model. Through these results, it is expected that a new therapeutic modality for hard tissue regeneration will be possible, and the application range of the PROTAC system can be expanded.
2024-06-11 | GSE255297 | GEO
Project description:The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation, and survival enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAFV600E have shown great efficacy in the clinic but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC termed P4B displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAFV600E cell lines. In addition, P4B displayed utility in two cell lines harboring alternate BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a rationale for optimizing the drug-like properties of P4B to enable proof of concept studies in vivo.
2020-05-25 | GSE148500 | GEO
Project description:To evaluate how TGF-β regulates human airway basal stem cells (BCs), we used PROTAC to inhibit TGF-β signaling in BCs and exposed Ctrl-BCs and PROTAC-BCs to recombinant TGF-β1 for 24 hours, followed by bulk RNA sequencing analysis. We found that multiple cell cycle related and proliferation related genes were upregulated in PROTAC-BCs compared to Ctrl-BCs, while the expression levels of cell migration related genes were reduced in the PROTAC-BCs group, and genes related to barrier formation and stem cell differentiation were also downregulated.
2025-11-24 | GSE305983 | GEO