Structural & Functional Investigations of Arginyltransferases
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Author/Creator ORCID
Date
2022-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Biochemistry
Citation of Original Publication
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Distribution Rights granted to UMBC by the author.
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Subjects
Abstract
Arginylation, or the transfer of the amino acid arginine from a charged tRNA to a substrate protein, is a post-translational modification (PTM) that is catalyzed by an essential eukaryotic enzyme known as arginyltransferase 1 (ATE1). This critical PTM was first discovered through its role in the N-degron pathway, a hierarchical pathway of the ubiquitin proteasome system, although recent exciting results have shown that arginylation also functions to stabilize essential cellular proteins. Through these mechanisms, ATE1 plays a role in the regulation of innumerable diverse biological processes across the eukaryotic domain such as: the stress response in yeast, leaf and shoot development in plants, cardiovascular development in mammals, neurodegeneration in mammals, certain types of cancers in mammals, and even the virogenesis of the human immunodeficiency virus (HIV1) and SARS-CoV2 in cells. Despite this clear importance, little is understood about this essential enzyme at the protein level, including its structure, its macromolecular interactions, and its regulation. This dissertations addresses several of these questions at the molecular level. First, this work demonstrates that ATE1 is an [Fe-S] cluster-binding protein, and that this cofactor is a common feature across evolution. This dissertations also shows that this [Fe-S] cluster regulates arginylation in vitro and in vivo, suggesting that this [Fe-S] cluster could be an O2-senstive regulatory component of post-translational arginylation. Next, the X-ray crystal structure of apo Saccharomyces cerevisiae ATE1 was determined for the first time to 2.8 � resolution, revealing the atomic-level details of ATE1, which were confirmed by solution small-angle X-ray scattering, cryo-EM, and site-directed mutagenesis approaches. Finally, initial experiments aimed at elucidating the macromolecular interactions among protein substrate, tRNA, and ATE1 have hinted at an important order of binding necessary for enzymatic turnover. Combined, these data have provided a mechanistic framework underpinning N-terminal arginylation. This dissertations then concludes by detailing exciting future experiments that should help inform the greater field of arginylation.