Ubiquitination is a major posttranslational modification of proteins which governs the fates of modified substrates, i.e., changes in stability, subcellular localization and enzymatic activities. Ubiquitination is a triple-step process which involves activation, conjugation and ligation of a ubiquitin moiety mediated by E1, E2, and E3 enzymes, respectively. E3 ligases play crucial roles in recruiting substrates, and they primarily determine substrate specificity. Aberrant regulation of E3 ligases disrupts important cellular homeostasis such as cell cycle control, DNA damage response, proteastasis, and viability determination, and accounts for pathophysiology of various human diseases including cancer and neurological disorders.
The human genome encodes more than 600 E3 ligases which are classified into three major families: RING, RBR and HECT domain E3s. Each E3 has its specific substrates, and they are susceptible to marked changes in their stability or activities when the E3 is mutated or overexpressed in pathogenic conditions. It has been a major challenge to identify direct substrates of each E3, because of the complexity of the enzyme cascades and crosstalk among numerous E3s. Our lab in collaboration with Dr. Jun Yin’s lab at Georgia State University developed a novel technique named OUT (Orthogonal Ubiquitin Transfer), which enables of profiling only direct substrates of each E3. The animation explains how OUT works in cultured cells. Using OUT, our collaborative team has identified novel substrates of the HECT E3 E6AP/UBE3A, and the Ubox E3s CHIP/STUB1 and E4B. We also used OUT to reveal that the two E1 enzymes in mammals, UBA1 and UBA6, initiate ubiquitination of partially overlapping yet distinct pools of cellular proteins.
E6AP/UBE3A plays important roles in not only viral and non-viral oncogenesis but also neurological disorders such as Angelman syndrome (AS) and autism spectrum disorders (ASD). Our OUT screens identified a number of novel E6AP substrates and we are investigating to define pathophysiological roles of several key substrates in cancer. Also, to obtain a new perspective about pathogenic changes in the ubiquitylome of AS and ASD neurons, we are developing induced pluripotent stem cell (iPSC) lines engineered for OUT screens to determine E6AP substrates in neural progenitor cells and mature neurons.
CHIP/STUB1 is a chaperone-associated E3, controlling stability of numerous proteins, especially in coordination of protein folding. Our OUT screens identified various oncoproteins as novel CHIP substrates, which is consistent with the notion that CHIP functions a tumor suppressor and this action is diminished in breast cancer. We are also studying how this CHIP-dependent control of proteostasis prevents development of Alzheimer’s disease.
We also demonstrated that another HECT E3, Smurf2, plays an essential role in mitotic progression (see movie) and functions as a tumor suppressor against breast cancer development. The immunohistochemical figure on the right shows the expression of Smurf2 protein is downregulated specifically in triple-negative breast cancer tissues. We are preparing to apply the OUT technology to identify previously undefined substrates of Smurf2 and reveal downstream pathways controlling breast cancer development. We are preparing to apply the OUT technology to identify previously undefined substrates of Smurf2 and reveal downstream pathways controlling breast cancer development.
Finally, our unique work that revealed the overlapping yet distinct functions of UBA1 and UBA6 provided important insight into in vivo regulation of the mammalian dual-E1 ubiquitination system. As small molecule inhibitors of E1 enzymes have been developed and tested as therapeutic agents, impact of each E1 inhibitor on the entire proteome should be carefully assessed. We are continuing to characterize representative substrates of ubiquitination initiated exclusively by UBA1 and UBA6, so that some biomarkers could be developed for patients that will undergo anti-E1 therapies in the near future.