Year: 2024
Type: dissertation
Abstract: Guanosine-5'-triphosphate (GTP) is an essential molecule for cell survival and function. Although free GTP plays a crucial role in several cellular processes, most studies focus solely on the protein bound GTP and its association with cancer. Efficient methods for determining cellular GTP concentration are currently lacking. Chromatography or capillary electrophoresis are commonly utilized for separation and mass spectrometry (MS) for quantification of GTP in biological samples. For monitoring GTPases and their interactions with ligands, differential scanning fluorimetry, differential scanning calorimetry, or differential static light scattering are typically utilized. However, these methods either lack sensitivity or throughput. In this PhD project, the Protein-Probe method, based on a Eu3+-chelate peptide probe that interacts with the hydrophobic core of the protein, has been developed and applied for studying different factors that affect the thermal stability of small GTPases, such as inhibitors, buffer ions and nucleotides. Furthermore, the method was explored for a better understanding of the binding specificity of the recent Food and Drug Administration approved KRASG12C covalent inhibitors to RAS GTPases. Additionally, a novel dual-labeled Förster Resonance Energy Transfer (FRET)-based peptide probe was introduced and compared to the Protein-Probe method in the thermal stability assay. The FRET-Probe technique was also applied to determine the chemical stability of proteins with different chemical denaturants. For measuring cellular GTP level, a homogenous high throughput assay was developed to measure the amount of GTP in cells utilizing a highly GTP-specific antibody. The assay yielded comparable results to those obtained with CE/MS, demonstrating a similar level of accuracy, while also exhibiting a substantial enhancement in throughput. In conclusion, this thesis work aimed at developing novel and robust methods to address the current limitations in monitoring cellular GTP concentration and to study small GTPases. The focus of the study was free GTP, recognizing its essential role in various cellular processes.
Source: UTUPub (University of Turku)
Author: Mahran, Randa
Institution: University of Turku
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Japan Agency for Medical Research and Development, Keio University, Suomalainen Tiedeakatemia, Emil Aaltosen Säätiö, Turun Yliopisto, Otto A. Malm Lahjoitusrahasto, Swiss Cancer Research Foundation, Turun Yliopistosäätiö, National Institutes of Health, Japan Society for the Promotion of Science, Japan Science and Technology Agency, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, Core Research for Evolutional Science and Technology
Awards: G1404607167, G1688034774, G1902985293, Exploring the molecular mechanism of GTP-driven stress response system, G2521286739, G2576085165, G2972400531, G3941176865, G5514976981, G6718509927, G6936640578, G6940248666, G7150650115, G7606810250, Targeting the Novel PI5P4K Pathway to Induce Glioblastoma Senescence, G8894390220, Mechanistic role of phosphatidylinositol 5-phosphate 4-kinase beta in GTP-dependent lysosomal acidification for stress-resilient cell growth and metabolism, Therapeutic resistance and aggressive malignancy in glioblastomas: the contribution of GTP metabolism through regulation by IMPDH2
OpenAlex ID: W7039587810