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Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

Laine Lysyk, Raelynn Brassard, Elena Arutyunova, Verena Siebert, Zhenze Jiang, Emmanuella Takyi, Melissa Morrison, Howard S. Young, View ORCID ProfileMarius K. Lemberg, Anthony J. O’Donoghue, View ORCID ProfileM. Joanne Lemieux
doi: https://doi.org/10.1101/2020.07.27.224220
Laine Lysyk
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Raelynn Brassard
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Elena Arutyunova
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Verena Siebert
2Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
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Zhenze Jiang
3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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Emmanuella Takyi
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Melissa Morrison
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Howard S. Young
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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Marius K. Lemberg
2Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
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  • ORCID record for Marius K. Lemberg
Anthony J. O’Donoghue
3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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M. Joanne Lemieux
1Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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  • ORCID record for M. Joanne Lemieux
  • For correspondence: mlemieux@ualberta.ca
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Abstract

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min−1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated β-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

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Posted July 29, 2020.
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Insights into the catalytic properties of the mitochondrial rhomboid protease PARL
Laine Lysyk, Raelynn Brassard, Elena Arutyunova, Verena Siebert, Zhenze Jiang, Emmanuella Takyi, Melissa Morrison, Howard S. Young, Marius K. Lemberg, Anthony J. O’Donoghue, M. Joanne Lemieux
bioRxiv 2020.07.27.224220; doi: https://doi.org/10.1101/2020.07.27.224220
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Insights into the catalytic properties of the mitochondrial rhomboid protease PARL
Laine Lysyk, Raelynn Brassard, Elena Arutyunova, Verena Siebert, Zhenze Jiang, Emmanuella Takyi, Melissa Morrison, Howard S. Young, Marius K. Lemberg, Anthony J. O’Donoghue, M. Joanne Lemieux
bioRxiv 2020.07.27.224220; doi: https://doi.org/10.1101/2020.07.27.224220

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