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bioRxiv · 10.1101/2020.05.05.078998

Ubiquitin Substrates Dramatically Increase Ataxin3 Deubiquitinating Activity: Allosteric crosstalk connects three distinct sites

Rao, M. V.·Grasty, K. C.·Mehetre, P. D.·Loll, P. J.
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Abstract

Ataxin3 is the founding member of the MJD family of deubiquitinating enzymes, and plays important roles in maintaining protein homeostasis and promoting DNA repair. The enzyme also contains a polyglutamine tract of variable length, and in its expanded form the protein becomes the causative agent of a neurodegenerative disorder known as Machado-Joseph disease. In vitro, ataxin3 displays low catalytic activity, prompting questions about how the enzyme is regulated and what signals might lead to its activation. Recently, it has been demonstrated that ataxin3 activity can be stimulated by either mono-ubiquitination or high concentrations of free ubiquitin. Here, we show that ubiquitin conjugates with cleavable bonds can stimulate ataxin3 activity much more strongly than free ubiquitin, with physiological levels of these conjugates increasing activity up to 60-fold over basal levels. Our data are consistent with a model in which ubiquitin conjugates activate the enzyme allosterically by binding in a site adjacent to the catalytic center, known as Site 1. We further show that two additional ubiquitin-binding sites in the enzyme work in concert to modulate enzyme activation, and we propose a model in which ubiquitin conjugates bridge these two sites to drive the enzyme into a high-activity conformation. SignificanceUbiquitin signaling networks modulate almost all aspects of eukaryotic biology, and their outputs reflect the dynamic balance between ubiquitin attachment and removal. The latter process is catalyzed by deubiquitinating enzymes (DUBs), which must be carefully regulated to ensure that their activities are applied appropriately. Ataxin3 is a DUB that participates in quality-control pathways that support cellular health; however, the regulation of its activity has remained poorly understood. Here, we show that ataxin3 can be dramatically activated by naturally occurring ubiquitin species, and that this activation involves a previously uncharacterized interplay between three distinct sites on the enzyme. Our improved understanding of ataxin3 regulation provides insights into allosteric mechanisms that may prove applicable to other enzymes in the ubiquitin universe.

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Rao, M. V., Grasty, K. C., Mehetre, P. D., Loll, P. J.. 2020-05-06. Ubiquitin Substrates Dramatically Increase Ataxin3 Deubiquitinating Activity: Allosteric crosstalk connects three distinct sites. https://doi.org/10.1101/2020.05.05.078998

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