Ribosome is the evolutionarily conserved molecular machine responsible for synthesis of proteins. In eukaryotic cells, ribosome is produced in a biophysically distinct subnuclear compartment, the nucleolus, through a cascade of energy-driven events. This process underlies a number of genetic diseases and is a major target for anticancer therapeutics. However, the physical interactions of the network required for this process remain largely uncharacterized. This application will characterize a key molecular complex called R2TP that acts early in ribosome production by facilitating assembly of several ribosome production enzymes. R2TP has a wide client base, collaborates with a general protein folding chaperone, heat shock protein 90 (Hsp90), and delivers clients to the nucleolus. The Li laboratory has identified effective methods to purify, reconstitute, and structurally characterize R2TP and its interaction with a client protein by use of a powerful set of structural biology and biochemical methods. The long- term goal is to understand the molecular basis for R2TP/Hsp90-mediated assembly process and uncover the molecular basis for its function in ribosome production.
Two specific aims are designed to 1) dissect the client binding and release cycle of R2TP and its regulation by nucleotides and the liquid immiscibility of the nucleolus; 2) characterize at a molecular level how R2TP impacts pre-ribosome structure purified from yeast cells. Results of this study promise to reveal new interfacial surfaces for anticancer drugs and to explain the action of those currently in use. The Li laboratory has assembled a team of scientists with complementary expertise in x-ray crystallography, high-throughput electron cryomicroscopy, mass spectrometry, biophysics, and protein biochemistry in order to maximize the chance of successes while mitigating risks. Relevance: Ribosome production is accelerated in multiple cancer cells. Correspondingly, R2TP/Hsp90 and their clients are overexpressed in these cells. More than fifteen types of anticancer drugs have been investigated or in clinical trials that target molecules involved in this pathway. Though effective in cell-death assays, the pharmacological basis of many compounds remains to be explained and some develop resistance in cells. The proposed research provides a platform to test the actions of these drugs outside the cell and to target previously unknown sites or processes.
Ribosome production requires a cascade of molecular events that are often correlated with genetic diseases and targets of anticancer therapeutics. The lack of molecular basis for this process hinders our understanding of the disease basis and the action of the anticancer drugs. A powerful set of biochemical and biophysical methods is employed to uncover the molecular basis of a key ribosome production pathway.
