A cell cycleregulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages

Animesh Dhara; Anthony P. Sinai

doi:10.1128/mSphere.00085-16

A cell cycleregulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages

Animesh Dhara, Anthony P. Sinai

Microbiology, Immunology, and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G₁ parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii "ubiquitin code."

Original language	English
Article number	e00085-16
Journal	mSphere
Volume	1
Issue number	3
DOIs	https://doi.org/10.1128/mSphere.00085-16
State	Published - May 1 2016

Bibliographical note

Funding Information:
We acknowledge John Boothroyd (Stanford University) and Gary Ward (University of Vermont) for their gifts of antibodies and E. Charles Snow (University of Kentucky) and Peter Bradley (UCLA) for useful discussions during the course of this study. This work was supported by NIH/NIAID grant R21AI099509 and Kentucky Science and Engineering Foundation awards (KSEF-2624-RDE-015 and KSEF-3439-RDE018). All grants were awarded to A.P.S. This work, including the efforts of Anthony P. Sinai, was funded by HHS | National Institutes of Health (NIH) (R21AI099509). This work, including the efforts of Anthony P. Sinai, was funded by Kentucky Science and Engineering Foundation (KSEF) (KSEF2624RDE015 and KSEF3439RDE018). (In the version of this article published on 22 June 2016, "Kentucky Science and Engineering Foundation" was incorrectly listed as "Kentucky Science and Energy Foundation." This was changed in the version published on 30 August 2016.)

Publisher Copyright:
© 2016 Dhara and Sinai.

Keywords

Cell cycle
Deubiquitinase
OTU
Polyubiquitin
Toxoplasma gondii

ASJC Scopus subject areas

Microbiology
Molecular Biology

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10.1128/mSphere.00085-16

Cite this

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title = "A cell cycleregulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages",

abstract = "The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii {"}ubiquitin code.{"}",

keywords = "Cell cycle, Deubiquitinase, OTU, Polyubiquitin, Toxoplasma gondii",

author = "Animesh Dhara and Sinai, {Anthony P.}",

note = "Funding Information: We acknowledge John Boothroyd (Stanford University) and Gary Ward (University of Vermont) for their gifts of antibodies and E. Charles Snow (University of Kentucky) and Peter Bradley (UCLA) for useful discussions during the course of this study. This work was supported by NIH/NIAID grant R21AI099509 and Kentucky Science and Engineering Foundation awards (KSEF-2624-RDE-015 and KSEF-3439-RDE018). All grants were awarded to A.P.S. This work, including the efforts of Anthony P. Sinai, was funded by HHS | National Institutes of Health (NIH) (R21AI099509). This work, including the efforts of Anthony P. Sinai, was funded by Kentucky Science and Engineering Foundation (KSEF) (KSEF2624RDE015 and KSEF3439RDE018). (In the version of this article published on 22 June 2016, {"}Kentucky Science and Engineering Foundation{"} was incorrectly listed as {"}Kentucky Science and Energy Foundation.{"} This was changed in the version published on 30 August 2016.) Publisher Copyright: {\textcopyright} 2016 Dhara and Sinai.",

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AU - Sinai, Anthony P.

N1 - Funding Information: We acknowledge John Boothroyd (Stanford University) and Gary Ward (University of Vermont) for their gifts of antibodies and E. Charles Snow (University of Kentucky) and Peter Bradley (UCLA) for useful discussions during the course of this study. This work was supported by NIH/NIAID grant R21AI099509 and Kentucky Science and Engineering Foundation awards (KSEF-2624-RDE-015 and KSEF-3439-RDE018). All grants were awarded to A.P.S. This work, including the efforts of Anthony P. Sinai, was funded by HHS | National Institutes of Health (NIH) (R21AI099509). This work, including the efforts of Anthony P. Sinai, was funded by Kentucky Science and Engineering Foundation (KSEF) (KSEF2624RDE015 and KSEF3439RDE018). (In the version of this article published on 22 June 2016, "Kentucky Science and Engineering Foundation" was incorrectly listed as "Kentucky Science and Energy Foundation." This was changed in the version published on 30 August 2016.) Publisher Copyright: © 2016 Dhara and Sinai.

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N2 - The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii "ubiquitin code."

AB - The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii "ubiquitin code."

KW - Cell cycle

KW - Deubiquitinase

KW - OTU

KW - Polyubiquitin

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A cell cycleregulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

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