Regulation and Inhibition of Deubiquitinase (DUB) Complex
DUBs antagonize the activities of ubiquitin ligases. Although the function of most human DUBs remains to be determined, it has become clear that DUB activities are indispensible for the normal functions of ubiquitin-proteasome pathways. Abnormal cellular expression of DUBs or the loss of function due to mutation in certain DUB genes have been linked to various human diseases. Among the five subfamilies (see the accompanying figure), USPs are emerging as promising targets for pharmacological intervention because of their connection to many human diseases.
The activity of DUBs, in particular USPs, is stringently regulated through their interaction with many other protein partners. A recent global proteomic analysis of human DUBs identified 774 interacting proteins for the 75 DUBs studied. Remarkably, a number of human USPs were found to be associated with WD40-repeat proteins that adopt a beta-propeller structure. Given its widespread occurrence, the interaction between WD40-repeat proteins and USPs likely represents a fundamentally important way of regulating USP activity. We are investigating the catalysis and regulation of DUBs that function in DNA damage response and cell cycle control. We are also engaged in identifying and developing DUB inhibitors through high-throughput screening (HTS) and chemical synthesis.
Develop Chemical Approaches for Protein Ubiquitylation
Enzymatic ubiquitylation usually requires a number of enzymes, including ubiquitin ligase and the associated factors. The requirement of multiple enzymes limits the yield of enzymatic ubiquitylation. Chemical ubiquitylation circumvents the requirement for the ubiquitin cascade enzymes and can be readily generalized for modifying different target proteins. However, several challenges for chemical ubiquitylation also exist. Lysine residue, the site of ubiquitylation, is highly abundant in proteins. Achieving high regioselectivity is thus essential for chemical ubiquitylation. Moreover, different from other forms of post-translational modification (such as phosphorylation and methylation), ubiquitylation utilizes a small protein as modifier that itself contains many reactive groups. This represents another level of complexity for chemical ubiquitylation. We are using PCNA as a model system to develop chemical approaches for efficient protein ubiquitylation. Currently we are exploiting intein chemistry and disulfide crosslink for site-specific monoubiquitylation. The chemically ubiquitylated PCNA will be used as a probe to understand the molecular mechanism of eukaryotic translesion synthesis.
Posttranslational modification of proteins represents a crucial way of regulating cellular functions. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins plays an essential role in a number of biological processes. Ubiquitin (Ub) modification was initially discovered as a signaling mechanism for proteasome-mediated protein degradation. In recent years, ubiquitin has been found to play far broader roles in eukaryotic cells. New pathways regulated by ubiquitin are being discovered at a fast pace, virtually in almost every important aspect of cell biology, including DNA damage repair/tolerance, signal transduction, transcription, nuclear transport and innate immune response. We are investigating the eukaryotic translesion synthesis (TLS) and its regulation by ubiquitylation and SUMOylation of proliferating cell nuclear antigen (PCNA).
We are interested in deciphering the roles of the protein or protein complexes involved in the DNA damage repair/tolerance pathways, in particular the enzymes responsible for the dynamic process of ubiquitylation or SUMOylation, as well as the functional outcomes of the post-translational modification by ubiquitin and ubiquitin-like modifier. We are also trying to discover novel ubiquitin pathways in DNA damage response using DNA microarray, bioinformatics and genetic approaches. Once we identify the target proteins we apply enzymological and structural approaches for the in-depth characterization of the protein with the goal of understanding the catalysis and regulation. Our investigations have important impact on human health, particularly, human cancer. In parallel to our mechanistic studies we are developing the small-molecule and peptidomimetic antagonists that can modulate the essential factors and enzymes in DNA damage response pathway. Some of these molecules will serve as the drug lead for the next generation of anti-cancer therapy.