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Ap<sub>3</sub>A-based Chemoproteomics : Discovery of a Novel Human RNA Ligase

Ap3A-based Chemoproteomics : Discovery of a Novel Human RNA Ligase

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YUAN, Yizhi, 2021. Ap3A-based Chemoproteomics : Discovery of a Novel Human RNA Ligase [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Yuan2021Abase-54146, title={Ap3A-based Chemoproteomics : Discovery of a Novel Human RNA Ligase}, year={2021}, author={Yuan, Yizhi}, address={Konstanz}, school={Universität Konstanz} }

terms-of-use Ap<sub>3</sub>A-based Chemoproteomics : Discovery of a Novel Human RNA Ligase Yuan, Yizhi AMPylation, first discovered in 1967, is a reversible post-translational modification of proteins. In AMPylation machinery, AMPylator transfers the AMP moiety in ATP onto target proteins to regulate downstream signal pathways. AMPylation was embracing its renaissance in the last decade along with the growing number of AMPylators and deAMPylators identified. The global occurrence of AMPylation within cells suggests its crucial roles in versatile cellular processes.<br /><br />Ap<sub>n</sub>As, a class of naturally occurring nucleotides, are present ubiquitously from prokaryotes to eukaryotes, among which Ap<sub>3</sub>A is one of the most abundant and well-studied one. The basal concentrations of Ap<sub>3</sub>A are nM in different cell types. The increase in its concentration under stress conditions makes Ap<sub>3</sub>A an "alarmone" of stress stimuli. To date, although Ap<sub>3</sub>A has been found to induce diverse biological effects, its exact roles and mechanisms are still poorly understood. Given the structure similarity between Ap<sub>3</sub>A and ATP, it is postulated that Ap<sub>3</sub>A might participate in some biological processes in a similar way as ATP does.<br /><br />In this doctoral thesis, it is hypothesized that Ap<sub>3</sub>A might act as a surrogate of ATP to participate in AMPylation. To verify the hypothesis, a series of Ap<sub>3</sub>A derivatives and the corresponding cognate nucleotides bearing alkynyl chemical handles have been successfully synthesized for the first time. Given C2-ethynyl-modified Ap<sub>3</sub>A as the most compatible probe, an activity-based proteomic profiling assay in the context of AMPylation using a novel trypsin-cleavable azide linker has been established. For the first time, this study shows an Ap<sub>3</sub>A-based AMPylome that includes in total 233 significantly enriched proteins in H1299 and HEK293T cell lysates. 11 of these protein candidates, which possess versatile biological functions, have been validated by immunoblotting assays. Above all, this Ap<sub>3</sub>A-based activity-based proteomic profiling assay leads to the enrichment of an uncharacterized protein C12orf29, which has been proven to be a novel human RNA ligase for the first time. Within this doctoral study, the novel RNA ligase has been reconstituted and preliminarily characterized, which has revealed an uncharted field and may unravel a new human RNA repair machinery. Yuan, Yizhi eng 2021-06-30T05:21:18Z 2021-06-30T05:21:18Z 2021

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