The Role of Pirh2 E3 Ligases in Ubiquitination and p73 Regulation

• Author / Creator

  Abou Zeinab, Rami M

• Cancer and tumor suppressors have been highly associated with many cancer researches. The main function of these proteins is to detect any error in DNA that might eventually leads to abnormal cell division and repair these errors; hence eliminating the risk of cancer development. Many researches focused on the p53 family protein consisting of three proteins: p53, p63, and p73. p53 was the first to be discovered, and many scientists refer to this protein as the “master switch”, or the “guardian of genome” because of its critical role in coordinating cellular processes. p53 acts as a checkpoint in the cell cycle monitoring all the cellular responses. At this level, should any stress signal occur affecting the cellular mechanism, p53 will be highly expressed and will trigger a variety of programs including a series of repressing and activating responses. Unfortunately, 50-60% of human tumors show mutations in the p53 proteins and the remainder, despite bearing wild type (WT) proteins, shows a dysfunctional system. More attention later on also included p73 proteins after revealing their role in cell cycle arrest and apoptosis. The exact mechanism for the tight regulation of these proteins is not fully understood; however ubiquitination has been shown to be the master regulatory process. Several ubiquitin E3 ligases such as MDM2, Pirh2, AIP4, UBE4B, etc.; are shown to act as regulators. Interestingly among all E3 ligases, Pirh2 is the only one over-expressed in a wide range of human tumors: 41, 64, 61, 70 and 82% in primary breast cancer, hepatocellular carcinoma, head and neck, prostate, and lung cancer respectively. Knowing that Pirh2 is over-expressed, the first objective of this thesis was focused on examining the self-regulatory process of Pirh2 including the optimal conditions and the role of E2 enzymes. Also we mapped the domains of Pirh2 to reveal that in addition to the RING domain, commonly known for the catalytic activity, portions of the C terminal domain are also essential for Pirh2 self-ubiquitination mechanism. Besides, lysine chains (K48, K63,and KO), known for their role in determining the substrate’s fate, were analyzed showing lack of significance on the contrary to p53 ubiquitination, where K48 lysine chains are utilized. The second objective of this thesis was to analyze Pirh2 and p73 correlation knowing that p73 share up to 80% homology with p53 at the DNA binding sequence. Interestingly, Pirh2 has been shown to bind, ubiquitinate and down-regulate p73 tumor suppressor function without altering p73 protein level. Lack of degradation was explained with the use of lysine chains 63 that do not trigger proteosomal degradation post ubiquitination. The third objective of this thesis focused on Pirh2-p73 regulatory pathway especially when studies have shown that the same substrate, p73 in our case, has been shown to be regulated by other ligase: AIP4. In this study, we reveal for the first time a novel correlation between two ligases within the same signalling pathway where Pirh2 physically interacts with AIP4 and significantly down-regulates its expression. This down-regulation is shown to involve the ubiquitination of AIP4 by Pirh2. At the p73 level, we showed that Pirh2 ceased the AIP4-p73 negative regulatory pathway. In-vivo and in-vitro ubiquitination analyses regarding p73 isoforms (α & β) confirmed the decrease of p73-AIP4 induced ubiquitination when Pirh2 is introduced. At the translation level and regarding p73 cell cycle arrest function in specific, Pirh2 secured p73 G1 arrest role despite over-expressing AIP4. Overall, our findings added valuable knowledge over the E3 ligase self-regulation and also the substrate regulation. Also we introduced a novel concept correlating two independent signalling pathways. This opens a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins as it is the case for p53 and p73 proteins; hence, providing a new framework for the development of novel anti-cancer targeting strategies.

• Subjects / Keywords

  • Pirh2
  • P73
  • Ubiquitination

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3610VX4F

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.