Regulatory Mechanism of Cullin-RING Ubiquitin Ligases - ABSTRACT The primary aim of my research program is to elucidate the fundamental mechanisms and regulation of protein ubiquitination, a post-translational modification that controls the degradation or functions of numerous key regulatory proteins in both plants and animals. Dysregulation of protein ubiquitination can result in various human diseases, such as neurodegenerative disorders, cancers, and muscular atrophy. Central to this process are E3 ligases, which recognize target proteins and tag them with ubiquitin or ubiquitin chains. My lab investigates cullin- RING ligases (CRLs), the largest family of E3 ligases. CRLs use a common cullin-RING scaffold coupled with interchangeable substrate receptors to direct specific proteins for ubiquitination. In human cells, multiple cullins (CUL1-9) associate with different sets of substrate receptors, creating up to 250 unique CRL complexes. Using a variety of approaches—including biochemistry, biophysics, molecular genetics, quantitative proteomics, and cell biology—we investigate the function, regulation, and role of CRLs in cellular processes and organismal development. Our research has focused on understanding how cells manage the repertoire of diverse CRLs to ensure that the right proteins are ubiquitinated at the right time. Previously, we uncovered that CRL1 complexes undergo rapid cycles of assembly and disassembly, allowing CUL1 to be constantly recycled and timely reassembled into new CRL1s as their substrates emerge. This dynamic process relies on CAND1, a cullin- binding protein that enables the exchange of CUL1 substrate receptors. Loss of CAND1 reduces CRL1 activity, impairing substrate degradation and leading to developmental defects in multicellular organisms. Recently, we expanded our study to CRL2 complexes and discovered a distinct regulatory mechanism. Unlike CRL1, where CAND1 promotes protein ubiquitination, CAND1 inhibits CRL2-catalyzed ubiquitination by accelerating CRL2 disassembly without facilitating the formation of new CRL2s. This CAND1-mediated disassembly prevents or slows the degradation of low-affinity substrates, while minimally affecting higher-affinity substrates, thereby enhancing the substrate specificity of CRL2. Building on these findings and prior research, we will next explore CRL regulatory mechanisms across different CRLs and cellular contexts. We will further investigate how CAND1 influences CRL2 and CRL5, with a focus on the effects of CAND1 post-translational modifications and the role of adaptor proteins that link substrate receptors to cullins. Like CAND1, CAND2 and GLMN also bind the cullin- RING core, and we will explore how these proteins impact CRL activities. Given that CAND2 is genetically associated with abnormalities in cardiac and skeletal muscles and CAND1 is essential for plant male gametophyte development, we will extend our studies to systems such as stem cell-derived muscle cells and Ceratopteris richardii plants to uncover the role and dynamic regulation of CRLs in development and disease. Our long-term goal is to advance the understanding of CRL functions and regulation across different biological systems, informing therapeutic strategies that modulate CRL activity for disease treatment.
