טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBukin Elena
SubjectThe Role of Degringolade (Dgrn), a SUMO-Targeted Ubiquitin
Ligase, in IMD Signaling and Innate Immunity
In Drosophila Melanogaster
DepartmentDepartment of Medicine
Supervisor Professor Amir )Oryan )Orian
Full Thesis textFull thesis text - English Version


Abstract

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Abstract

The NF-kB family of transcription factors plays a major role in immunity, and is highly conserved in Drosophila melanogaster. In Drosophila, NF-kB proteins regulate the host response against infection and pathogens, and are activated by one of two evolutionary conserved signaling pathways: Toll and IMD. An extensive crosstalk between post-translational modifications (PTMs) such as ubiquitin and ubiquitin-like (UbL) proteins regulates NF-kB signaling and gene expression. In this regard, the recently discovered SUMO-Targeted Ubiquitin ligases (STUbLs) bind to SUMOylated proteins and facilitate their ubiquitylation. Degringolade (Dgrn) is the sole STUbL protein in Drosophila. Dgrn is greatly involved in the NF-kB pathway, regulating both the Toll and the IMD pathways.

In this work, I focused on the IMD pathway, and characterized the impact of Dgrn on the IMD pathway. I specifically focused on its interaction with the co-activator Akirin in S2 Schneider cells. My results demonstrate that Dgrn is a negative regulator of the IMD pathway. Molecularly, using pathway-specific reporter gene assays in S2 Schneider cells I found that Dgrn is essential and enhances Toll-dependent gene expression while negatively regulates the IMD-dependent gene expression. Mechanistically, Dgrn interacts with the IMD specific co-factor Akirin. Furthermore, I found that Dgrn physically interacts with Akirin, to mediate its ubiquitylation in an in vitro reconstituted system. Thus it affects Akirin's protein level, at its basal level implying on a role in Akirin translation.

In addition, in order to shed more light on Dgrn’s function in the Drosophila immunity I identified proteins that associate with Dgrn both in unchallenged cells and upon activation of the IMD pathway. To this end I identified Dgrn-associated proteins using affinity purification in S2 Schneider cells followed by mass spectrometry analyses. I identified putative Dgrn-interacting proteins. Collectively, my work suggests that Dgrn is involved in the IMD pathway. In addition, Dgrn activity is likely aimed at several distinct substrates.