Impairments in protein synthesis and signaling in Fragile X human cells Open Access

Annam, Jayabhargav (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/w0892b702?locale=en
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Abstract

Fragile X Syndrome (FXS) is the most common monogenic cause of an intellectual disability. The disease is caused by an inherited expanded trinucleotide repeat, which leads to methylation of the promoter a subsequent loss of the expression of the Fragile X Mental Retardation Protein (FMRP). FMRP is an mRNA binding protein that acts a negative regulator of approximately 4% of the mRNAs produced in the brain. Previous work done in FXS animal models has indicated that in the absence of FMRP, there is an increase in PI3K enzymatic activity and PI3K-regulated protein synthesis. We hypothesize that the same increases in PI3K activity and protein synthesis would be seen in FXS patient-derived cells, which could be a very useful insight into the molecular mechanisms underlying FXS in a human cellular model. To measure nascent protein synthesis in control and FXS cells, we used two methods: metabolic labeling of newly synthesized proteins via fluorescent non-canonical amino acid tagging (FUNCAT) combined with either fluorescence microscopy or flow cytometry. To measure PI3K activity, we used western blots for p110β, the catalytic subunit of PI3K. Our results indicate that there were significant increases in both in the FXS patient-derived cells, as was predicted by previous studies in animal models. In the use of flow cytometry to measure protein synthesis in FXS patient-derived cells, we developed a new application of the technique and a novel assay that can be used for future studies attempting to measure levels of abnormal protein synthesis. Furthermore, through our finding that FMRP regulates PI3K signaling through p110β and that this pathway is dysregulated in patients, a new potential therapeutic drug target and human biomarker for FXS has been found.

Table of Contents

Introduction……………………………….……………………………………………………1

Methods……………………………....………………………….……………………………..9

Results……………………………….……………………………..……………………….....14

Discussion……………………………………………………………………………..……....19

References………………………………………………….…………………………………..25

Figures…………………………………….…………………………………………………….29

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