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Proteomics Insights. 2019 Mar 3;10:1178641818825268. doi: 10.1177/1178641818825268. eCollection 2019.

Expression and Characterization of Human Fragile X Mental Retardation Protein Isoforms and Interacting Proteins in Human Cells.

Author information

1
Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
2
OriGene Technology, Inc., Rockville, MD, USA.
3
Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL, USA.
4
Departments of Biochemistry, Neurological Sciences and Pediatrics, Rush University, Chicago, IL, USA.

Abstract

Fragile X mental retardation protein is an mRNA-binding protein associated with phenotypic manifestations of fragile X syndrome, an X-linked disorder caused by mutation in the FMR1 gene that is the most common inherited cause of intellectual disability. Despite the well-studied genetic mechanism of the disease, the proteoforms of fragile X mental retardation protein have not been thoroughly characterized. Here, we report the expression and mass spectrometric characterization of human fragile X mental retardation protein. FMR1 cDNA clone was transfected into human HEK293 cells to express the full-length human fragile X mental retardation protein. Purified fragile X mental retardation protein was subjected to trypsin digestion and characterized by mass spectrometry. Results show 80.5% protein sequence coverage of fragile X mental retardation protein (Q06787, FMR1_HUMAN) including both the N- and C-terminal peptides, indicating successful expression of the full-length protein. Identified post-translational modifications include N-terminal acetylation, phosphorylation (Ser600), and methylation (Arg290, 471, and 474). In addition to the full-length fragile X mental retardation protein isoform (isoform 6), two endogenous fragile X mental retardation protein alternative splicing isoforms (isoforms 4 and 7), as well as fragile X mental retardation protein interacting proteins, were also identified in the co-purified samples, suggesting the interaction network of the human fragile X mental retardation protein. Quantification was performed at the peptide level, and this information provides important reference for the future development of a targeted assay for quantifying fragile X mental retardation protein in clinical samples. Collectively, this study provides the first comprehensive report of human fragile X mental retardation protein proteoforms and may help advance the mechanistic understanding of fragile X syndrome and related phenotypes associated with the FMR1 mutation.

KEYWORDS:

characterization; fragile X mental retardation protein; interacting proteins; mass spectrometry; post-translational modifications

Conflict of interest statement

Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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