Search

Post-translational modifications are chemical changes that occur to proteins after their synthesis, which are essential to their function and regulation. Ubiquitination is a post translation modification that serves key roles in the regulation of proteins. USP7 is a deubiquitinase that has several critical substrates important for human health and disease, including the cancer relevant proteins PTEN, p53, MDM2 and DMNT1. Most of these substrates have been identified by cell biology or proteomics experiments, but a detailed biochemical and structural analysis is lacking, likely due to the challenge of generating site-specific and stoichiometrically ubiquitinated proteins. Therefore, we leveraged our expertise in protein semi-synthesis to generate these ubiquitinated substrates to study USP7’s ability to recognize and hydrolyze the ubiquitin, which will reveal key details on how USP7 selects its substrates. In our investigation, we generated several mono-ubiquitinated peptides, that could be functionalized later to install on the protein, and assayed USP7’s ability to hydrolyze the ubiquitin.

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.