Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Greg Kelly

Abstract

Ubiquitination plays a fundamental role in determining protein fate. Once ubiquitinated, the cargo is directed to the proteasome for partial or complete degradation or lysosome for complete degradation. Failing to eliminate these cargos results in the accumulation of toxic proteins that contribute to neurodegenerative and immunological diseases, cancer and other human maladies. Thus, identifying proteins subject to ubiquitin-mediated degradation and characterizing the mechanisms governing these processes underscores their importance to human health. The calcium/calmodulin-dependent serine protein kinase (CASK) is one such protein that is required for brain development. However, mutations that cause CASK to accumulate are correlated to X-linked mental retardation and autism spectrum disorder in humans. I have investigated CASK protein turnover and found that the protein is constantly degraded. This degradation, however, is only partial suggesting that the peptides generated have functions distinct from the full-length polypeptide. Subsequent analyses revealed that these peptides form as a result of CASK being first phosphorylated and then ubiquitinated prior to its limited degradation in the proteasome. During these investigations I identified poly ADP-ribosyl transferase-like 2 (PARP-2) as a protein also degraded through the ubiquitin-proteasome system. PARP-2 degradation occurs when cells are grown in the presence of serum and Ro52 was identified as a candidate E3 ubiquitin ligase required for ubiquitination. Interestingly, when cells are serum-starved, PARP-2 was sequestered to an SDS-insoluble fraction by a yet-to-be identified mechanism. Finally, further investigations with Ro52 revealed that when ectopically expressed, cells develop large circular structures, which I identified to be autophagosomes, the intermediate organelles in autophagy that selectively target ubiquitinated cargo for lysosomal degradation. The RING finger domain of Ro52 and its E3 ligase activity are, however, not required for this process and the deletion of the RING domain does not affect the inclusion and targeting of ubiquitinated proteins to the autophagosomes. Together, these results from studying two disparate proteins, CASK and PARP-2, not only highlight the selective ability of ubiquitination to specify the limited or complete degradation of proteins, but also shed new light on Ro52 as an adaptor involved in the autophagic turnover of proteins in the lysosome.


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