NextGen RiBiomics · Next Generation Proteomic Analysis of Pre-Ribosomal Proteome Dynamics Coupled to Glucose Metabolism in Caner Cells

Next Generation Proteomic Analysis of Pre-Ribosomal Proteome Dynamics Coupled to Glucose Metabolism in Caner Cells

This project will focus on the relationship between the process of ribosome biogenesis (RiBi) and glucose metabolism in human cells. Solid tumour cells are continuously exposed to glucose deprivation and hypoxia microenvironments because of the inadequate vascular supply. Cancer cells in these tumours have to limit energy expenditure to survive under such energy-deprived conditions. Meanwhile, the most energy-consuming process in eukaryotic cells is protein synthesis by the ribosome and ribosome subunit biogenesis, so these steps are tightly regulated in response to metabolic changes. Therefore, characterising the mechanisms involved in these processes may lead to novel cancer treatment strategies, for example inducing deregulation of either protein synthesis, or RiBi, from metabolic control. The ribosome, which consists of four ribosomal RNAs (rRNAs) and ∼80 ribosomal proteins (RPs), is essential for protein synthesis in the cell. The mammalian ribosome is composed of a large (60S) subunit and a small (40S) subunit. Its biogenesis takes place in a dedicated subnuclear compartment, the nucleolus, where a transcribed rRNA precursor (47S pre-rRNA in human) is assembled with proteins to form a large 90S pre-ribosomal particle (PR). Then the 47S pre-rRNA is processed to separate the 90S particle into pre-40S and pre-60S particles. These separated particles independently undergo maturation steps further and move from the nucleolus to the nucleoplasm then eventually to the cytoplasm. However, the detailed molecular mechanisms involved in RiBi in human cells has not been fully elucidated. Moreover, the proteins involved in this process have also not been fully identified. A major reason for this is that the isolation of PRs from human cells is very challenging. Several biochemical subcellular fractionation steps, coupled with sucrose density gradient centrifugation (SDGC), is required. In contrast, a simpler co-immunoprecipitation approach allows efficient extraction of PRs from yeast cells, but this is not effective for analysis of extracts from human cells. Particularly, SDGC, which is the most widely used method for the isolation of ribosomes and PRs since the ribosome was discovered in 1960s, is widely recognized to have limitations, including limited reproducibility and the relatively long time required to set up and perform the analysis. Therefore, a first major aim for this project was to establish a new, more efficient method for isolating ribosomes and PR complexes. Next, we aimed to optimise this new method for isolating PRs and to identify their components using Mass Spectrometry (MS)-based proteomic analysis. Finally, the aim was to compare the components of PRs extracted either from normal cells, or from glucose-starved cells, to try to identify proteins which play a role in the regulation of RiBi under conditions of glucose starvation.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз