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Characterization of the Physiological Role of the Deubiquitinase OTUB1 in Mice


Ruiz Serrano, Amalia. Characterization of the Physiological Role of the Deubiquitinase OTUB1 in Mice. 2020, University of Zurich, Faculty of Science.

Abstract

Protein ubiquitination is a key regulatory process in a plethora of physiological and pathological pathways. During ubiquitination, the protein ubiquitin is covalently attached to a substrate protein forming mono- or polyubiquitin chains. Ubiquitination can be reversed via deubiquitinases (DUBs). OTUB1 (ovarian tumor domain-containing ubiquitin aldehyde binding protein 1) is one of the most highly expressed deubiquitinases in cells with a unique enzymatic activity cleaving Lys48 ubiquitin chains and preventing Lys48 and Lys63 ubiquitin chain formation non-enzymatically. OTUB1 regulates various pathways and processes in vitro, like proinflammatory and profibrotic signaling, DNA damage response, proliferation and apoptosis.
The cellular oxygen sensor factor inhibiting HIF (FIH) is an asparagine hydroxylase controlling the transactivation activity of the hypoxia inducible factor (HIF), key regulator of the transcriptional response to hypoxia. Our group recently demonstrated that FIH hydroxylates OTUB1 on Asn22, regulating cellular energy metabolism. This thesis aims to investigate the interaction between FIH and OTUB1 in more detail and to assess if OTUB1 is a possible physiological target of FIH.
The molecular interaction between FIH and OTUB1 showed a denaturing-resistant, likely covalently linked protein heterodimer, highly sensitive to hypoxia and depending on FIH enzymatic activity. Several studies have investigated the role of OTUB1 in diseases in vivo. However, the physiological function of OTUB1 is unknown. To understand the in vivo role of OTUB1 and to assess if it is a likely physiologically relevant FIH target, we characterized mice with constitutive (Otub1-/-) and conditional whole-body Otub1 (wbOtub1-/-) deletion. Otub1-/-mice died perinatally due to asphyxiation and showed decreased alveolar space, increased lung cell proliferation and cardiac hypertrophy. wbOtub1-/- mice showed pulmonary hyperventilation and cardiac hypertrophy with left ventricular dysfunction. Additionally, adult animals presented with decreased body weight, increased energy expenditure and glucose metabolism. Furthermore, the AKT/mTOR signaling was increased in cells and tissues without Otub1. Overall, these results demonstrate that OTUB1 is essential for development, respiration, cardiac function and energy metabolic homeostasis in vivo. Interestingly, Hif1an (the gene encoding FIH) homozygous knockout (KO) mice display a metabolic phenotype that is virtually identical to the metabolic alterations observed in Otub1 KO mice.
In summary, we found that FIH forms a stable complex with OTUB1 at a cellular level. Interestingly, the metabolic and respiratory phenotypes of Otub1 KO mice support that the FIH-dependent regulation of OTUB1 is physiologically relevant. Our studies of Otub1 KO mice will further help evaluating the potential of OTUB1 as a therapeutic target and possible role in diseases.

Abstract

Protein ubiquitination is a key regulatory process in a plethora of physiological and pathological pathways. During ubiquitination, the protein ubiquitin is covalently attached to a substrate protein forming mono- or polyubiquitin chains. Ubiquitination can be reversed via deubiquitinases (DUBs). OTUB1 (ovarian tumor domain-containing ubiquitin aldehyde binding protein 1) is one of the most highly expressed deubiquitinases in cells with a unique enzymatic activity cleaving Lys48 ubiquitin chains and preventing Lys48 and Lys63 ubiquitin chain formation non-enzymatically. OTUB1 regulates various pathways and processes in vitro, like proinflammatory and profibrotic signaling, DNA damage response, proliferation and apoptosis.
The cellular oxygen sensor factor inhibiting HIF (FIH) is an asparagine hydroxylase controlling the transactivation activity of the hypoxia inducible factor (HIF), key regulator of the transcriptional response to hypoxia. Our group recently demonstrated that FIH hydroxylates OTUB1 on Asn22, regulating cellular energy metabolism. This thesis aims to investigate the interaction between FIH and OTUB1 in more detail and to assess if OTUB1 is a possible physiological target of FIH.
The molecular interaction between FIH and OTUB1 showed a denaturing-resistant, likely covalently linked protein heterodimer, highly sensitive to hypoxia and depending on FIH enzymatic activity. Several studies have investigated the role of OTUB1 in diseases in vivo. However, the physiological function of OTUB1 is unknown. To understand the in vivo role of OTUB1 and to assess if it is a likely physiologically relevant FIH target, we characterized mice with constitutive (Otub1-/-) and conditional whole-body Otub1 (wbOtub1-/-) deletion. Otub1-/-mice died perinatally due to asphyxiation and showed decreased alveolar space, increased lung cell proliferation and cardiac hypertrophy. wbOtub1-/- mice showed pulmonary hyperventilation and cardiac hypertrophy with left ventricular dysfunction. Additionally, adult animals presented with decreased body weight, increased energy expenditure and glucose metabolism. Furthermore, the AKT/mTOR signaling was increased in cells and tissues without Otub1. Overall, these results demonstrate that OTUB1 is essential for development, respiration, cardiac function and energy metabolic homeostasis in vivo. Interestingly, Hif1an (the gene encoding FIH) homozygous knockout (KO) mice display a metabolic phenotype that is virtually identical to the metabolic alterations observed in Otub1 KO mice.
In summary, we found that FIH forms a stable complex with OTUB1 at a cellular level. Interestingly, the metabolic and respiratory phenotypes of Otub1 KO mice support that the FIH-dependent regulation of OTUB1 is physiologically relevant. Our studies of Otub1 KO mice will further help evaluating the potential of OTUB1 as a therapeutic target and possible role in diseases.

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Additional indexing

Item Type:Dissertation (monographical)
Referees:Wenger Roland H, Scholz Carsten, Lutz Thomas, Marti Hugo H
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology

UZH Dissertations
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Place of Publication:Zürich
Date:2020
Deposited On:17 Nov 2020 11:10
Last Modified:17 Nov 2020 11:10
OA Status:Closed

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