Development of ubiquitinated protein probes for identifying reader and effector proteins in DNA damage tolerance
Date
2022
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Publisher
University of Delaware
Abstract
Post-translational modification (PTM) by ubiquitin is a tightly controlled and complex modification on substrate proteins for their proper maintenance within the cell. Ubiquitin (Ub) PTM has roles in many pathways encompassing protein trafficking, DNA damage response, and its most well-known function protein degradation. Ubiquitin is attached to a protein by a three-enzyme cascade (E1/E2/E3) and removed by the aptly name deubiquitinase enzymes (DUB). Investigation into proteins involved with the DNA damage repair and tolerance pathways as well as ubiquitin pathways will lead to a deeper understanding of the cellular processes. This could lead to therapeutic advancements targeting the dysregulated pathways in disease states. In fact, inhibiting a cancer cell’s ability to repair DNA damage by small molecules, such as the FDA approved drug Olaparib, has proven to be an effective strategy for treatment. A better understanding of the cellular mechanisms of DNA damage repair and tolerance would reveal new potential targets for therapeutic applications, particularly for cancer treatment. My research is aimed at uncovering proteins involved in the eukaryotic DNA damage tolerance pathway and investigating their function and regulation, which will be discussed in the following three chapters. ☐ Chapter 1: Proper maintenance of DNA is critical for cell survival. DNA damage lesions inhibit replication and could cause cellular death if not resolved. DNA damage tolerance pathways work to resolve this issue and continue replication allowing for progression through the cell cycle. Ubiquitination of proliferating cell nuclear antigen (PCNA) controls the two branches of this pathway. Error prone pathway signaled by monoubiquitination of PCNA is well understood but the error free pathway, signaled by polyubiquitination of PCNA remains largely unknown. Protein interactors after the polyubiquitination of PCNA have not been well studied. Uncovering the proteins involved in this branch of the pathway will shed light on how this process occurs and how the cell ensures stability of its genome when DNA damage is encountered during replication. ☐ Expanding our chemical ubiquitination methods, we sought to generate a polyubiquitin PCNA probe to try and uncover these interactors. Utilizing our previous linker technology, we developed a semi-synthetic strategy to produce a probe for this purpose. Our HA tagged probe constructed of non-cleavable linkages and a pBpa photo-inducible crosslinker aimed to capture protein interactors in DNA damage induced yeast cell lysate. Through protein pulldown and orbitrap MS/MS analysis we identify proteins captured by our probe which may be involved with the error free DNA pathway. This construction of polyubiquitin probes for identification of protein interactors could easily be adapted to other cellular pathways and processes. ☐ Chapter 2: Translesion synthesis, the error prone DNA damage tolerance pathway is an important pathway to maintain DNA stability during replication. Recently Ubp10 has been identified as a yeast enzyme responsible for deubiquitination of PCNA post translesion synthesis. In the study, other potential DUBs were identified via activity-based probe but were pulled down less strongly. Still, we do not know how Ubp10 achieves its Ub-PCNA substrate specificity. Employing pBpa photoactivatable crosslinking methodologies we sought to elucidate the interactions between Ub-PCNA and Ubp10. We crosslinked recombinant Ub-PCNA probe with recombinant Ubp10 and performed in gel digestion and orbitrap MS/MS analysis to map interaction sites. We have found that Ubp10 is captured in potentially multiple conformations based on different identified crosslinking sites. Further crosslinking identification will help to uncover more about the interactions taking place. Determination of Ubp10’s specificity could help to understand more broadly the underlying mechanisms which contribute to DUB specificity. DUBs have more recently become targets for drug development due to the role Ub dysregulation plays in many diseases. Thus, a better understanding of DUB-substrate interactions could lead to improved drug development. ☐ Chapter 3: Ubiquitin dysregulation is present in many diseases such as neurodegenerative diseases. Diagnosis and treatment are paramount to the survival of patients suffering from disease. As such, antibody development is quickly evolving as a powerful therapeutic tool for the treatment of disease. Ubiquitinated proteins make good targets for antibody development as they would be highly specific to dysregulation occurring in some diseased states. Antibody development for ubiquitinated substrates is new but an emerging avenue as an antibody specific for modified H2B was published more recently. We sought to develop an antibody specific for ubiquitinated alpha synuclein which may be implicated in the Lewy body formation known to cause neurological disorder found in Parkinson’s disease. Through a selection process using a yeast display library we selected for ScFv’s specific for our constructed ubiquitinated alpha synuclein. We have sequenced and expressed full length antibodies for validation from our library of hits. Our antibody could prove to be a diagnostic or therapeutic tool for Parkinson’s disease patients. This development process is flexible and can be adapted for other ubiquitinated targets, further improving the therapeutic capabilities of antibodies.
Description
Keywords
Ubiquinated protein probes, Reader proteins, Effector proteins, DNA damage