Abstract
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two late onset neurodegenerative diseases, have been shown to share overlapping cellular pathologies and genetic origins. Studies suggest that a hexanucleotide repeat expansion in the first intron of the C9orf72 gene is the most common cause of familial FTD and ALS pathology. The C9orf72 protein is predicted to be a differentially expressed in normal and neoplastic cells domain protein implying that C9orf72 functions as a guanine nucleotide exchange factor (GEF) to regulate specific Rab GTPases. Reported studies thus far point to a putative role for C9orf72 in lysosome biogenesis, vesicular trafficking, autophagy and mechanistic target of rapamycin complex1 (mTORC1) signaling. Here we report the expression, purification and biochemical characterization of C9orf72 protein. We conclusively show that C9orf72 is a GEF. The distinctive presence of both Rab- and Rho-GTPase GEF activities suggests that C9orf72 may function as a dual exchange factor coupling physiological functions such as cytoskeleton modulation and autophagy with endocytosis.
Highlights
Frontotemporal dementia and amyotrophic lateral sclerosis (ALS) are two rapidly progressive debilitating neurodegenerative disorders
The C9orf72 gene was initially cloned into a variety of vectors such as pTriEx-4 Ek/LIC (N-terminal His6 Tag), pET-SUMO (N-terminal SUMO-His6 tag), pGEX-6p-1 (N-terminal glutathione S-transferase (GST) tag), as well as a modified version of pMAL-p2X (N-terminal His6-maltose-binding protein (MBP) tag)
Both the GST-tagged and His6-MBP-tagged C9orf72 expressed in soluble form, the protein was unstable
Summary
Frontotemporal dementia and amyotrophic lateral sclerosis (ALS) are two rapidly progressive debilitating neurodegenerative disorders. These two neurodegenerative diseases have long been linked both clinically as well as pathologically (Andersen & Al-Chalabi, 2011; Lomen-Hoerth, Anderson & Miller, 2002). One of the unifying pathogenic-signatures, mechanistically linking the two diseases, is a hexanucleotide repeat expansion (GGGGCC) in the first intron of the C9orf gene (DeJesus-Hernandez et al, 2011; Renton et al, 2011). In persons with the repeat expansion mutation there can be hundreds or even thousands of these repeat units (DeJesus-Hernandez et al, 2011; Renton et al, 2011). The repeat expansion has been shown to be unstable and repeat numbers have been shown to increase over generations (Vance et al, 2006)
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