Investigating the inhibition of USP1 in DNA damage response and developing chemical approach to study DUB specificity and PCNA ubiquitination
Date
2015
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Publisher
University of Delaware
Abstract
Chapter 1 and 2: Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Ubiquitin specific protease 1 (USP1) plays critical roles in DNA damage responses, including DNA translesion synthesis (TLS), Fanconi anemia (FA) and homologous recombination (HR). In addition, USP1 also functions in preserving the stem cell-like characteristics in osteosarcoma cells by stabilizing inhibitor of DNA binding (IDs) proteins. The catalytic activity of USP1 is stimulated through its interaction with the WD40-repeat protein USP1-association factor 1 (UAF1). ☐ Through quantitative High-Throughput Screening (qHTS) followed by sequential medicinal modification and optimization, we developed ML323, a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against other human DUBs, deSUMOylase, deneddylase and unrelated proteases in Chapter 1. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum- based anticancer drugs. ☐ Both USP1 and UAF1 are large proteins with 785 or 677 amino acids, respectively. Neither USP1 nor UAF1 structure is available, which raises the difficulty to identify the binding site of ML323 in USP1-UAF1 complex. To address the question, in Chapter 2, we made inhibitor ML323 attach to USP1-UAF1 complex from noncovalently to covalently through photoactivated cross-linking chemistry. Then we tried to utilize isotope-labeled strategy coupled with the mass spectrometry to find out the ML323-modified peptides in USP1-UAF1 complex. ☐ Chapter 3: Ubiquitination refers as the post-translational modification of target proteins by covalent attachment of one ubiquitin or ubiquitin moieties to the amine group of lysine on the target proteins through isopeptide bond. Ubiquitin has seven lysine residues, K6, K11, K27, K29, K33, K48 and K63, which allow the formation of polyubiquitin chain with different linkages. The structures and functions of polyubiquitin vary with different chain linkages. DUBs cleave ubiquitin moiety from mono- or poly-ubiquitinated proteins. To date, close to 100 DUBs, which can be grouped into five subfamilies, have been identified in human genome. Studies have shown that some DUBs are specific for particular substrates and ubiquitin chain linkages. One obstacle in studying the DUBs’ specificity is the difficulty of preparing ubiquitin substrates with defined chain linkages. In Chapter 3, we developed a chemical approach to generate diUb substrates with different chain linkages, and characterized the kinetics of DUBs with the chemically synthesized diUbs. We also generated the first activity-based diUb probes in the filed and demonstrated the diUb probe strategy could be applied to DUB specificity profiling. ☐ Monoubiquitination of proliferating cell nuclear antigen (PCNA) plays important roles in TLS by recruiting the TLS polymerase Polη to carry out the DNA lesion by-pass synthesis. PCNA forms a head-to-tail ring sharp as a homotrimer complex. It remains unclear the relevance of homotrimer ubiquitination in response to DNA damage. To investigate the problem, in chapter 3, we generated a fusion PCAN trimer with head-to-tail linked by two reported flexible linkers and introduced monoubiquitination to one, two, or three subunits, respectively by chemical approach. With these constructs, we will be able to reveal the relevance of monoubiquitinated PCNA in TLS.
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Keywords
USP1, DNA damage response, DUB specificity, PCNA ubiquitination