Keywords

Apoptosis, ubiquitination, mitophagy, oxidative stress

Abstract

Omi/HtrA2 is a mitochondrial serine protease with a dual and opposite function depending on its subcellular localization. Most of the previous studies focused on Omi/HtrA2’s pro-apoptotic function when the protein is released to the cytoplasm. It is becoming apparent that the main function of Omi/HtrA2 is within the mitochondria, where it has a pro-survival role. However, its mechanism is still poorly understood. To this end, we used the yeast two-hybrid system to dissect the Omi/HtrA2 pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a deubiquitinating (DUB) complex. In addition we found that Omi/HtrA2 participates in mitophagy by regulating Mulan E3 ubiquitin ligase, which recruits GABARAP (gamma-amino-butyric acid receptor-associated protein) to the mitochondria. Abro1 is the scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex) that is specific for Lys-63 deubiquitination. This complex is similar to the BRCA-1 complex, a known and important player in DNA damage response. Using the yeast two-hybrid screen and a bait consisting of the unique carboxy-terminus of the Abro1 protein, we identified three transcription factors which are members of the activating protein 1 (AP-1) family, namely ATF4, ATF5 and JunD. The AP-1 family member ATF4 is ubiquitously expressed, like Abro1, and important in cell cycle regulation and survival, thus we further analyzed this interaction. Abro1’s interaction with ATF4 was specific and present only when cells are under cellular stress. When Abro1 protein level is increased it provides protection against stress-induced cell iv death, but interaction between Abro1 and ATF4 is necessary to achieve this protection. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex. Under normal conditions Omi/HtrA2 is localized in the intermembrane space (IMS) of the mitochondria. We have recently identified that the mitochondrial Mulan E3 ubiquitin ligase is a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death and autophagy/mitophagy. In addition, we showed that Omi/HtrA2, through regulation of the Mulan protein level, controls mitophagy, especially during mitochondrial stress. To understand Mulan’s function and its control by Omi/HtrA2, we set out to identify E2 conjugating enzymes that form a complex with Mulan E3 ligase. We isolated four specific interacting E2’s, namely Ube2E2, Ube2E3, Ube2G2 and Ube2L3. To identify substrates for each unique Mulan-E2 complex we used fusion baits in a second yeast two-hybrid screening. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP protein, a member of the Atg8 (autophagy) family. The mammalian Atg8 family is composed of seven members that have been linked to important roles in autophagy/mitophagy. We characterized this interaction both in vitro and in vivo and its role in mitophagy. Our results suggest that Mulan participates in various pathways, depending on the nature of its partner E2 conjugating enzyme. In addition, we identified the pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria. v I want to dedicate this hard work to the people who mean the most to me, the people who have been more than supportive in these past five years; they are my very small, but very loud and loving, family. My brother Raffaello has always been proud of me and always told his friends how his sister was a “doctor.” My father, Alvaro, always told me I had to be better than him, a man who has a master’s degree in chemistry. Finally, but most importantly to me, I want to dedicate this work and degree to my mother, Maria Aparecida Troncon, a woman who is like no other. She has always supported me, and with small gestures like having snacks ready when I came to visit after work or coming with me to lab on the weekends so I would not be alone, she told me every day that she was proud of me. Every time she met someone she told them I was her doctor and she said it with the biggest smile on her face. Unfortunately she passed away on December 17th, 2012, just months shy of the completion of my degree. I remember how proud she was when I received my bachelor’s and I can only imagine the size of her smile when I walk across the stage this time as a Ph.D. I love you mom, you are my rock and my strength; without your constant support and dedication I would have not reached this point. I know you are smiling at me from above. Last but not least I want to dedicate this to the man who is my better-half. Thank you for all your love and support, Guillermo. I love you.

Notes

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Graduation Date

2013

Semester

Summer

Advisor

Zervos, Antonis

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Molecular Biology and Microbiology

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0004805

URL

http://purl.fcla.edu/fcla/etd/CFE0004805

Language

English

Release Date

August 2014

Length of Campus-only Access

1 year

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Medicine, Medicine -- Dissertations, Academic

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