MAGEL2 regulates leptin receptor internalization through ubiquitination pathways involving USP8 and RNF41

  • Author / Creator
    Wijesuriya, Tishani M.
  • Children with Prader-Willi syndrome have neonatal feeding difficulties, developmental delay and excessive appetite. Loss of MAGEL2 alone causes a related neurodevelopmental disorder (Schaaf-Yang syndrome) and may contribute to obesity in children with Prader-Willi syndrome who lack MAGEL2 and other genes. MAGEL2 is essential in neurons that sense levels of the adipose tissue-derived hormone leptin. The MAGEL2 protein is important for recycling or degradation of proteins in the brain and interacts with and modifies the activity of E3 ubiquitin ligases. RNF41 is a E3 ubiquitin ligase that associates with a ubiquitin-specific protease (USP8). Together with USP8, RNF41 regulates the recycling of the leptin receptor by targeting it either for degradation or for recycling to the cell membrane. I hypothesized that MAGEL2 regulates the interaction between RNF41 and USP8, and that loss of this regulation could impair leptin response pathways in the brain in children with PWS. Human U2OS cells were transfected with recombinant constructs encoding epitope tagged versions of MAGEL2 and wild type and mutant forms of RNF41. Immunofluorescence was used to visualize the co-localization of different forms of RNF41 with MAGEL2 in intracellular compartments. I expressed recombinant MAGEL2, RNF41 and USP8 in combinations in human U2OS cells and examined the relative abundance of each protein in the presence or absence of the other components of the complex. The interactions between MAGEL2 and other proteins were examined by the Bio ID system. I identified interactions among components of the RNF41-USP8 complex that depended either on the activity of the RING domain of RNF41 (RNF-SQ mutant form) or the binding domain of RNF41 (RNF-AE mutant form). Co-expression of MAGEL2 with components of the RNF41-USP8 complex modified the abundance of proteins in the complex. My results suggest that RNF41 enhances USP8 ubiquitylation, and I found that MAGEL2 diminishes the ability of RNF41 to auto-ubiquitinate or to ubiquitinate. Co-expression of MAGEL2 also modified the intracellular localization of components of the RNF41-USP8 complex. Levels of endogenous RNF41 and USP8 level in brain tissues from Magel2 knockout mice were different from levels found in tissues from wild type littermates. My results suggest that MAGEL2 could modify the activity of the RNF41-USP8 ubiquitination complex in leptin sensing neurons, providing a possible mechanism for dysregulation of leptin sensing in neurons in children with Prader-Willi syndrome.

  • Subjects / Keywords
  • Graduation date
    Spring 2017
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • 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.