Characterization of the Protein Interaction Networks of Necdin and MAGEL2: Insight into How Loss of These Proteins Contributes to Neurodevelopmental Disease

• Author / Creator

  Sanderson, Matthea R

• Prader-Willi Syndrome (PWS) is a neurocognitive developmental disorder that is caused by the deletion or inactivation of paternal genes in the chromosomal region 15q11-q13. The MAGEL2 and NDN (encoding necdin) genes are within the deleted region. Individuals with PWS tend to be obese due to hyperphagia, are hypotonic, have sleep apnea, have some degree of intellectual disability, and exhibit problematic behaviors such as temper tantrums and sudden outbursts. Protein truncating mutations in the MAGEL2 gene result in a disorder known as Schaaf-Yang syndrome (SYS), which has overlapping phenotypes with PWS. Phenotypes recapitulating PWS phenotypes have been observed in gene-targeted MAGEL2 knockout mice. However, there is still much unknown about the function that MAGEL2 plays in the cell. MAGEL2 has a “MAGE homology domain” (MHD) that is shared with ~40 other mammalian MAGE proteins, including necdin. Identifying interacting proteins can help to determine the pathways in which MAGEL2 and necdin participate. Identifying the pathways that these proteins are involved in allows for a better understanding of the role that they play in both SYS and PWS, as well as how mutations impact protein function. I examined the cellular role and impact of mutations in both MAGEL2 and necdin by investigating protein-protein interactions using proximity-dependant biotinylation (BioID).
  I first examined the necdin interactome and the effect of amino acid substitutions on necdin protein interactions. I used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. I identified necdin-proximate proteins that function in both RNA metabolism and cellular stress response, which are novel functions for necdin. I then used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from the interactome of wildtype necdin.
  Next, I investigated the MAGEL2 interactome using BioID-MS. Until this point, all molecular experiments done with MAGEL2 have characterized the C-terminus of the protein, which contains the MHD. I examined interactions for the full-length MAGEL2 protein as well as interactions for the N-terminus and C-terminus of the protein. This process yielded novel MAGEL2-proximate proteins that cluster into protein networks, as well as the discovery of protein interactions that were specific to different regions of the MAGEL2 protein. I identified interactions between MAGEL2 with several RNA metabolic proteins. Most notable was the interaction between MAGEL2 and YTHDF1/2/3, which function in RNA metabolism and cellular stress response. I also found that MAGEL2 regulates YTHDF2 in response to heat shock, indicating that MAGEL2 has a role in cellular stress response.
  
  Finally, I examined the impact of mutations on MAGEL2 protein interactions. A series of mutations have been identified in MAGEL2 in individuals with SYS. There are differences in the severity of the phenotypes seen in SYS, which appears to be dependent on the location of the truncating mutations. We previously showed that mutations in the MHD disrupt MAGEL2 interactions in the cell. However, there are currently no functional assays to evaluate the impact of mutations on MAGEL2. I used BioID-MS to define the interactomes of two MAGEL2 proteins carrying coding variants that disrupt MAGEL2 function. Two mutations are synthetic mutations modeled on disease-causing variants in other MAGE proteins. I found that the mutant MAGEL2 proteins had altered proximity to proteins initially identified as proximal to wildtype MAGEL2.
  My thesis contributes to the understanding of the cellular role of both MAGEL2 and necdin. I identified novel interactions and biological pathways for both MAGEL2 and necdin. I also have also found evidence of a functional role for the N-terminus of MAGEL2, which has not been previously described. My work demonstrates that BioID could be used to evaluate the clinical relevance of mutations in MAGEL2 identified in individuals diagnosed with Schaaf-Yang syndrome. More broadly, changes in protein-protein interactions secondary to variations in protein sequence can point to motifs important for protein structure or function and assist in decisions about the probability that protein variants are pathogenic in human genetic disease.

• Subjects / Keywords

  • Schaaf-Yang Syndrome
  • BioID
  • Prader-Willi Syndrome
  • Medical Genetics

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-xbsk-a878

• License

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