Molecular mechanisms underlying the assembly of the human proteasome and endogenous protein complexes

点击翻译按钮获取中文摘要

Project Summary Advances in structural biology techniques including single particle cryogenic electron microscopy (cryo-EM) have enabled unprecedented molecular insights into the function of biological macromolecules. However, the study of many proteins and ribonucleoprotein complexes remains challenging due to current limitations in sample preparation approaches. Traditionally, proteins of interest are produced in over-expression systems within bacterial, insect, and mammalian cell lines. While this approach can allow the production and purification of proteins with high yields, it often requires substantial optimization that can limit the study of biomedically important membrane proteins and large protein complexes, especially where specific chaperones and cellular conditions are required that are difficult to replicate in vitro. To overcome these limitations, we are developing methodology to efficiently tag and purify endogenous proteins by leveraging advances in CRISPR/Cas gene editing. This approach enables us to investigate macromolecular complexes and their intricate assembly pathways under native and context-specific conditions that are relevant to human health and disease. We are interested in developing and applying the approach in three major areas of study: constitutive protein complexes, cell-type specific macromolecular assemblies, and cell state dependent membrane protein complexes. Using the proteasome as a model system, we will investigate the assembly pathway of proteasomal complexes by cryo-EM and mass spectrometry. This work will provide mechanistic insights into critical protein degradation machinery and help to establish important methodology for the study of endogenous protein assemblies. Next, we will expand our approach to the study of protein complexes in different cell types, including hematopoietic and epithelial cells. This goal will be achieved by developing efficient strategies to optimize CRISPR/Cas gene editing in specialized cell types, which will constitute an important step towards the study of proteins in their native states. Finally, we will examine the conditional assembly of protein complexes and membrane protein assemblies. For this direction, we will investigate proteins involved in nutrient sensing at the lysosome in conjunction with mTOR signaling. This work will provide molecular insights into the mechanisms regulating key metabolic pathways and shed light on how mTOR integrates different signals to promote cell growth and proliferation. Additionally, we will establish protocols for screening cellular and biochemical conditions to acquire context-specific protein assemblies. Altogether, these studies will provide mechanistic insights into remarkable molecular machines and develop important methodology that can be applied to the study of other biological systems. These methods will enable us to unravel the molecular mechanisms underlying the function of proteins in specific cellular environments and help advance structural biology towards understanding how biological macromolecules work in their native contexts.