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.
