Multiplexed Real-Time NMR GTPase Assay for Simultaneous Monitoring of Multiple Guanine Nucleotide Exchange Factor Activities from Human Cancer Cells and Organoids.
Teklab GebregiworgisChristopher B MarshallTadateru NishikawaNikolina RadulovichMaría-José SandíZhenhao FangRobert RottapelMing-Sound TsaoMitsuhiko IkuraPublished in: Journal of the American Chemical Society (2018)
Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.
Keyphrases
- single cell
- rna seq
- induced pluripotent stem cells
- induced apoptosis
- high throughput
- endothelial cells
- cell cycle arrest
- signaling pathway
- magnetic resonance
- endoplasmic reticulum stress
- high resolution
- poor prognosis
- oxidative stress
- pi k akt
- electronic health record
- big data
- solid state
- binding protein
- mass spectrometry
- genome wide
- young adults
- cell proliferation
- protein protein
- artificial intelligence
- dna methylation
- deep learning