PhD student, University of Wuerzburg/ Rudolf Virchow Centre for Experimental Biomedicine
Structural and biochemical characterisation of the ubiquitin activating enzyme.
Ubiquitin is one of the most versatile post-translational modifications present throughout eukaryotes which is known for its well-established role for proteasomal degradation but plays an essential role in the myriad of cellular processes including DNA repair, immune response and cell cycle progression to name a few. Three classes of enzymes namely E1, E2 and E3 are required for the transfer of the free ubiquitin onto the target protein via an isopeptide bond. In humans, there are two E1 enzymes, around fifty E2 enzymes and more than six hundred E3 enzymes that facilitate this process. Ubiquitin tagging of the protein leads them to the proteasome, a 2.5 MDa complex for degradation. Since the approval of Bortezomib, a proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma, rigorous research has been carried out to inhibit novel therapeutic target upstream of the proteasome. UBA1 and UBA6 are the two E1 enzymes that activate ubiquitin in humans and thus are the master regulators of ubiquitylation. UBA1 is the only dedicated E1 for ubiquitin as UBA6 can activate both ubiquitin and FAT10, another ubiquitin-like modifier. UBA1 plays a major role in ubiquitylation and research in the last decade has established its inhibition as a promising target for cancer therapy. Two adenosyl sulfamate inhibitors which target UBA1 are currently in phase I clinical trials for the treatment of solid tumours. We have crystallised three inhibitors of this class including the ones that are in clinical trials in the complex with ubiquitin and UBA1 and have solved high-resolution crystal structures of these complexes. These structures serve as a platform for developing highly specific and potent inhibitors that can target individual E1 enzymes responsible for the activation of ubiquitin as well as sixteen other ubiquitin-like modifiers. With the help of in vitro inhibition assays as well as molecular dynamics simulation, we confirmed the structural interpretation of specificity of the inhibitors bound in our complex structures. In addition to targeting the ubiquitin activating enzyme (UBA1) for cancer treatment, we are interested in understanding the molecular mechanism of E1 enzyme catalysis. For this purpose, we have utilized biochemical tools in addition to X-ray crystallography to learn the structural and mechanistic insights of UBA1 enzymatic activity. We recently published the findings of the adenosyl sulfamate inhibitors of UBA1 in Structure.
Abstract: Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of plasma cells in the bone marrow (BM). The success of the proteasome inhibitor bortezomib in the treatment of MM highlights the importance of the ubiquitin proteasome system (UPS) in this particular cancer. Despite the prolonged survival of MM patients, a significant amount of patients relapse or become resistant to therapy. This underlines the importance of the development and investigation of novel targets to improve MM therapy. The UPS plays an important role in different cellular processes by targeted destruction of proteins. The ubiquitination process consists of enzymes that transfer ubiquitin to proteins targeting them for proteasomal degradation. An emerging and promising approach is to target more disease specific components of the UPS to reduce side effects and overcome resistance. In this review, we will focus on different components of the UPS such as the ubiquitin activating enzyme E1, the ubiquitin conjugating enzyme E2, the E3 ubiquitin ligases, the deubiquitinating enzymes (DUBs) and the proteasome. We will discuss their role in MM and the implications in drug discovery for the treatment of MM.
Pub.: 24 Dec '15, Pinned: 27 Jul '17
Abstract: Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy-lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies.
Pub.: 01 Sep '15, Pinned: 27 Jul '17
Abstract: Genomic alterations may make cancer cells more dependent than normal cells on mechanisms of proteostasis, including protein folding and degradation. This proposition is the basis for the clinical use of proteasome inhibitors to treat multiple myeloma and mantle cell lymphoma. However, proteasome inhibitors have not proved effective in treating other cancers, and this has called into question the general applicability of this approach. Here, I consider possible explanations for this apparently limited applicability, and discuss whether inhibiting other broadly acting components of the ubiquitin-proteasome system - including ubiquitin-activating enzyme and the AAA-ATPase p97/VCP - might be more generally effective in cancer therapy.
Pub.: 12 Nov '14, Pinned: 27 Jul '17
Abstract: Targeting the activating enzymes (E1) of ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) has emerged as a promising anti-cancer strategy, possibly overcoming the ineffectiveness of proteasome inhibitors against solid tumors. Here, we report crystal structures of the yeast ubiquitin E1 (Uba1) with three adenosyl sulfamate inhibitors exhibiting different E1 specificities, which are all covalently linked to ubiquitin. The structures illustrate how the chemically diverse inhibitors are accommodated within the adenylation active site. When compared with the previously reported structures of various E1 enzymes, our structures provide the basis of the preferences of these inhibitors for different Ub/Ubl-activating enzymes. In vitro inhibition assays and molecular dynamics simulations validated the specificities of the inhibitors as deduced from the structures. Taken together, the structures establish a framework for the development of additional compounds targeting E1 enzymes, which will display higher potency and selectivity.
Pub.: 06 Jun '17, Pinned: 27 Jul '17
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