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Identification and interrogation of ubiquitin and ubiquitin-like protein decoders
Date
2022
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Regulation of cellular processes is multifaceted and complex. Regulation can be achieved through post-translational modification (PTM) of a protein to either up or down regulate a cellular pathway. Ubiquitin is a PTM and codes for an array of signaling events in the cell. Because ubiquitin itself can also be a substrate of ubiquitination, a complex network of modifications exists, thus signaling different fates for the substrate proteins, this is known as the ubiquitin code. Ubiquitin is most celebrated for its role in signaling proteosome degradation of the substrate protein, however it is also known to signal autophagy, DNA damage repair, and cell cycle regulation, to name a few. Deubiquitinases, or DUBs, function to remove the ubiquitin modification from the substrate protein and effectively abrogate the signal. DUB dysregulation has been observed in disease such as cancer and neurodegenerative disease leading to aberrant signaling that is detrimental to the cell. This disease-associated dysregulation makes DUBs attractive therapeutic targets for small molecule intervention. Promising DUB inhibitors in preclinical models encourage the development and pursuit of inhibition of other disease-associated DUBs. ☐ Ubiquitin-like protein, Interferon Stimulated Gene 15 (ISG15), is structurally similar to diubiquitin and also functions as a PTM. ISG15 is unique in that it additionally functions as a free protein, reportedly acting in a non-covalent manner in anti-viral response by sequestering viral proteins and acting extracellularly as a cytokine-like molecule. Literature surrounding ISG15 has primarily co-opted techniques for studying ubiquitin and has thus focused on ISG15’s PTM activity in its analogous process, ISGylation. Targets of ISGylation have been identified by proteomics as well as characterization of the lone human deISGylase, USP18. Despite this work, a rigorous understanding of ISG15’s non-covalent interactions and functions remains elusive. ☐ In this work, the development and utilization of novel protein-based probes was performed to better understand the recognition of branched polyubiquitin chains and to elucidate interactions of ubiquitin-like protein ISG15 in innate immune response in the first and second chapters respectively. The final chapter discusses the development of a cell-based assay in order to advance high-throughput screening in drug discovery efforts to inhibit USP15. In total, this work improves the ability to interrogate critical cellular pathways with novel protein probes, photocrosslinking, and proteomics, while also advancing the physiological relevance and efficiency of drug screening and development efforts using live cell imaging.
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Keywords
Ubiquitin, Protein decoders, Interferon Stimulated Gene 15, Polyubiquitin chains