ABSTRACT Ubiquitination, the post-translational attachment of ubiquitin or ubiquitin chains, controls the stability, interaction or activity of numerous key regulatory proteins in eukaryotic cells. Consequently, misregulation in protein ubiquitination can result in various human diseases, such as metabolic disorders, cancers, muscle and nerve degeneration. At the core of the ubiquitination process is the E3 ligase, which brings ubiquitin and the target protein together, and enables the transfer of the ubiquitin to its target. My lab investigates the largest family of E3 ligases, known as Cullin-RING ligases (CRLs). These enzymes are modular protein complexes, featuring a common cullin scaffold and an interchangeable substrate receptor that recruits specific target proteins for CRL- dependent ubiquitination and subsequent degradation. Seven cullins (Cul1-7) exist in human cells, each of which interacts with different sets of substrate receptors, yielding ~250 CRLs. We use a variety of approaches including biochemistry, biophysics, molecular genetics, quantitative proteomics, and mathematical modeling to study how CRLs work, how their activities are regulated, and what critical roles they play in cells and organisms. Given that a large number of substrate receptors compete for access to the same cullin, our current research focus is to uncover how the cellular repertoire of diverse CRLs is controlled to ensure ubiquitination of various CRL substrates at the right time. Using Cul1 based CRL1, we previously reported that CRL1s constantly undergo cycles of assembly and disassembly, which allows rapid recycling of Cul1 and timely formation of new CRLs when their target proteins emerge and demand ubiquitination. A crucial player in this highly dynamic process is Cand1, a protein exchange factor that promotes the exchange of substrate receptors associated with the same Cul1 core. Eliminating the Cand1 activity leads to impaired degradation of CRL1 substrates in human cells and severe developmental defects in multicellular organisms. In this application, we ask, how are the dynamics of other CRLs regulated? What role does Cand2, a homologue of Cand1 in human cells, play in regulating CRLs? What advantage does this evolutionarily conserved dynamic exchange mechanism provide for the CRL system? To answer these questions, we will use in vitro biophysical assays to quantify kinetic parameters for CRL and Cand1/2 interactions. We will apply our updated quantitative immunoprecipitation-mass spectrometry assay to characterize the impact of Cand1 and Cand2 on the cullin-associated proteome. We will employ genome-editing techniques such as CRISPR to examine the biological role of Cand1/2, using cultured human cells and the model plant Arabidopsis as our experimental systems. We will continue developing our mathematical model of CRL assembly and activity, to help understand the CRL network in different cell types or under changing cellular environment. Our efforts ...