Investigation of chemical ubiquitination of PCNA and mechanism, inhibition of USP1/UAF1 & the molecular recognition of RNA by the pseudouridine synthase RluA
Date
2011
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Publisher
University of Delaware
Abstract
Chapter 1: Pseudouridine synthases (Ψ synthases) catalyze the isomerization of uridine to pseudouridine (Ψ) and are present in all domains of life. The Ψ synthases fall into six families based on sequence similarity, and crystal structure of members of each family show that they share the same core fold and have one universally conserved aspartic acid residue in motif II. The cocrystal structures of the E. coli Ψ synthase RluA bound to an RNA oligomer corresponding to the anticodon stem-loop (ASL) of E. coli tRNAPhe has been determined. This structure is only the second of a Ψ synthase bound to an RNA substrate. In the RluA•RNA cocrystal, the RNA has undergone significant conformational changes from its unbound form. The isomerized uridine U32, is everted in the active site of RluA. This conformation is stabilized by the reverse-Hoogsteen base pair formed by U33 and A36 and the hydrogen bond formed between A36 and Pro 36 on RluA. To test the importance of particular interactions in the RluA•RNA cocrystal structure, kinetic studies were undertaken. RNA containing 5-fluorouridine, F5U, has been used as a mechanistic probe to distinguish the two proposed Ψ synthases’ mechanisms. RluA forms heat-sensitive and apparently covalent adduct with [F5U]RNA. Upon heating, hydrated F5U products are also formed by the collapse of the adduct of RluA. Thus, the reactivity between RluA and two altered [F5U]ASLs (A36C and U33C) was investigated. ☐ Chapter 2 to Chapter 4: The non-proteolytic function of ubiquitination has attracted increasing attention in recent years, including protein trafficking, immune response, transcription regulation and DNA damage response. Monoubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic translesion synthesis (TLS), a mechanism utilized by cells to synthesize past DNA lesion. One obstacle in studying eukaryotic TLS resides in the difficulty of preparing sufficient amount of ubiquitinated PCNA for in-depth biochemical and biophysical investigation. In Chapter 2, we developed a chemical approach that combines the power of intein chemistry and the facile disulfide exchange chemistry for efficient protein ubiquitination and SUMOylation. The chemically ubiquitinated PCNA is functionally equivalent to the native ubiquitinated PCNA in effecting polymerase switch between the replicative and the specialized DNA polymerases. We also demonstrated the strict requirement of PCNA ubiquitination for polymerase switch. Moreover, we probed the effect of the site of ubiquitination by preparing chemically ubiquitinated PCNAs that differ only in the position of modification. Our study revealed a surprising degree of flexibility of ubiquitin modification. ☐ Deubiquitinating enzymes (DUBs) cleave the ubiquitin moiety from mono- and poly-ubiquitinated proteins. Close to 100 DUBs have been identified in the human proteome. Abnormal cellular expression of DUBs or the loss of function due to mutation in certain DUB genes have been linked to various human diseases, including cancer and neurodegenerative diseases. Ubiquitin-specific proteases (USPs) constitute the largest DUB family. There is growing evidence suggesting that 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, 34 human USPs were found to be associated with WD40-repeat proteins that adopt a β-propeller structure comprising up to eight blades. Given its widespread occurrence, the interaction between WD40-repeat proteins and USPs likely represents a fundamentally important way of regulating USP activity. Based on the reported kinetic data for USP1/UAF1 complex and USP1 alone, we hypothesize that the interaction between UAF1 and USP1 may reorganize the catalytic triad into a more productive conformation. To probe the active site conformation, in Chapter 3, cysteine-reactive small organic molecules (H2O2 or iodoacetamide) were used to determine the reactivity of the catalytic sulfhydryl group in USP1/UAF1 complex or USP1 alone. ☐ DUBs are promising targets for pharmacological intervention. The advantage of inhibiting DUB lies in the specificity of therapeutic intervention that can lead to better efficacy and eliminate nonspecific side effects seen in proteasome inhibitors. In Chapter 4, we identified small-molecule inhibitors against the USP1/UAF1 complex through high throughput screening. Two highly selective inhibitors, pimozide and GW7647, inhibit USP1/UAF1 noncompetitively with a Ki of 0.50 and 0.75 μM, respectively. We demonstrated that both compounds are reversible inhibitors that bind the USP1/UAF1 complex at a site different from the active site. Because USP1/UAF1 is involved in DNA damage response through deubiquitinating PCNA and FANCD2 in translesion synthesis and Fanconi anemia pathways, we tested USP1/UAF1 inhibitors as a sensitizer of cancer cells to DNA damaging agent, cisplatin. Our results indicated that the USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting the proliferation of cancer cells.
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Keywords
Pseudouridine synthases, DNA lesion, Polymerase switch, Cancer cells, Organic molecules