Background: MAGEL-2 is a gene frequently deleted or mutated in individuals affected with PWS. Furthermore, mice lacking MAGEL-2 display symptoms similar to those seen in PWS children. However, a critical barrier to our understanding of MAGEL-2’s link to PWS has been determining its function within cells. Recently, my group has solved this enigmatic question. We showed that MAGEL-2 functions to prevent aberrant degradation of proteins that normal reside in the plasma membrane. It does so through the specific modification of another protein, called WASH. This modification, termed ubiquitination, activates the WASH protein that facilitates recycling of proteins sparing them from aberrant degradation in the lysosome, the garbage can of a cell. In this proposal, we aim to determine how disruption of MAGEL-2’s function in protein recycling contributes to PWS. We believe that our novel characterization of MAGEL-2 provides innovative new conceptual advances to the PWS field that have direct implications into not only understanding the cellular origins of PWS, but also provide new therapeutic avenues.
Hypothesis: We propose that disruption of MAGEL-2 results in PWS because of the aberrant degradation of specific, but unidentified, proteins that are critical for function of neurons in the hypothalamic region of brain that control processes affected in PWS, such as feeding, behavior, and fertility.
Aim 1: Create a suitable neuronal cell culture system to study MAGEL-2 cellular function.
MAGEL-2 is highly expressed in the hypothalamus region of the brain, which has been strongly implicated in PWS. Thus we will use genome editing technology to mutate MAGEL-2 to mimic the genome of PWS patients in hypothalamic neurons. Known functions of MAGEL-2 in regulating protein recycling will be validated in these cells.
Aim 2: Identify the specific proteins in hypothalamic neurons whose recycling and abundance depends on MAGEL-2. We will utilize our hypothalamic neurons created in Aim 1 to identify those proteins regulated by MAGEL-2 using quantitative proteomics to identify proteins whose abundance is altered upon mutation of MAGEL-2. Those proteins identified will be validated by our established biochemical and microscopy techniques assaying protein recycling.
Aim 3: Determine the relevance of those proteins recycled by MAGEL-2 to PWS using mice lacking MAGEL-2. First, we will determine if those proteins identified in Aim 2 are indeed regulated by MAGEL-2 in animals by examining their localization and abundance in the hypothalamus of mice in which MAGEL-2 is present or absent. Next, we will determine whether rescuing the activity of any of these proteins with drugs can ameliorate the PWS symptoms of mice lacking MAGEL-2, such as increased fat mass, feeding behavior, and infertility.
In summary, these studies will provide new insights into how MAGEL-2 contributes to PWS and identify novel therapeutic approaches to alleviating symptoms of this devastating disease.
Cellular and disease functions of the Prader-Willi Syndrome gene MAGEL2. Tacer KF, Potts PR. Biochemical Journal .2017 Jun 16;474(13):2177-2190.
A Comprehensive Guide to the MAGE Family of Ubiquitin Ligases. Lee AK, Potts PR. Journal of Molecular Biology. 2017 Apr 21;429(8):1114-1142.
USP7 Acts as a Molecular Rheostat to Promote WASH-Dependent Endosomal Protein Recycling and Is Mutated in a Human Neurodevelopmental Disorder. Hao YH, Fountain MD Jr, Fon Tacer K, Xia F, Bi W, Kang SH, Patel A, Rosenfeld JA, Le Caignec C, Isidor B, Krantz ID, Noon SE, Pfotenhauer JP, Morgan TM, Moran R, Pedersen RC, Saenz MS, Schaaf CP, Potts PR. Molecular Cell. 2015 Sep 17;59(6):956-69.
Ryan Potts, PhD
University Texas Southwestern