Characterizing the SUMOylation of APC4 and the Function of APC4 Within Neurons
by Jennifer L. Day
Date of Examination:2021-06-02
Date of issue:2021-09-23
Advisor:Prof. Dr. Nils Brose
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Holger Bastians
Persistent Address:
http://dx.doi.org/10.53846/goediss-8826
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Abstract
English
The Anaphase Promoting Complex (APC/C) is an E3 ubiquitin ligase that is involved in multiple molecular processes in eukaryotic cells. It is regulated by an intricate system of post-translational modifications that enables the complex to quickly target different sets of proteins for degradation by the proteasome. The APC/C is best known for its function in regulating the cell cycle, but it is also expressed in non- dividing cells like neurons, where its function is less clear. In the present study, I first sought to elucidate the function of the SUMOylation of APC4, a component of the APC/C, in HEK293 cells and in neurons. In the second part of my study, I tried to determine the function of APC4 in neurons. In regards to the first research focus, I show that APC4 is SUMOylated, and I found that this SUMOylation does not influence the subcellular localization of APC4 or the assembly of the APC/C. I show further that endogenous APC4 is localized to the nucleus and to the cytoplasm of HEK293 cells and neurons, and I found that APC4 is more heavily SUMOylated in the nucleus of HEK293 cells and in the cytoplasm of cortical neurons obtained from a mouse brain. My attempts to determine the role of APC4 SUMOylation in the regulation of the neuronal APC/C were confounded by the fact that my experimental results challenged the earlier notions of the current list of candidate neuronal substrates of the APC/C. In regards to my second research direction, I knocked out the gene encoding APC4, ANAPC4, in cultured neurons and conducted biochemical and morphological analyses of the mutant neurons. In contrast to prior publications, I found that FEZ1, ID1, and NEUROD2 are not substrates of the cortical APC/C. ID1 levels were instead depleted in ANAPC4 knockout cultures, indicating that ID1 is downstream of a substrate of the APC/C. I further show that ANAPC4 knockout neurons have an increase in the number of primary neurites, but they had no changes in the length of their neurites or in their number of branches. Altogether, these ANAPC4 knockout data indicate that many of the previously proposed neuronal substrates of the APC/C are in fact not targeted by the APC/C. Future studies must focus on elucidating the function of the APC/C in neurons and in determining how the SUMOylation of APC4 influences the ubiquitylation of these substrates. In general, the information contained in the present thesis may be important for the development of novel drug targets to treat cancer and possibly diseases of the nervous system.
Keywords: SUMOylation, APC4, APC/C, Anaphase Promoting Complex, Cyclosome, ubiquitylation, ID1
