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(7f) Mammalian Cell-Based Methods Monitoring Protein Misfolding and Aggregation

Authors: 
Greogire, S., University of Virginia


Protein folding is a complex process that converts a linear strand of amino acids to a functional, three-dimensional complex. Point mutations in the primary sequence that encodes for proteins is known to cause aberrant protein conformations.  An array of “protein-misfolding” diseases such as Alzheimer’s, Amyotrophic Lateral Sclerosis, Parkinson’s, are caused by misfolded proteins that form protein deposits in essential organs within the body. With the development of new treatments to help treat these diseases, there is a need to develop a robust method that can accurately monitor the folding of these proteins in the presence of the therapeutics as well as in a physiologically relevant system. Despites the benefits of mammalian cells to study misfolding and aggregation of human proteins associated with neurodegenerative diseases, mammalian cell-based monitoring methods have not been explored much. In order to quantitatively and qualitatively monitor protein misfolding and aggregation in mammalian cells, we utilized three folding reporter proteins, split GFPs, mammalian folding reporter GFP (mfrGFP), and EGFP as a fusion partner. As a test case, we chose human copper/zinc superoxide dismutase (SOD1) associated with Amyotrophic Lateral Sclerosis, also called as Lou Gehrig’s disease. There are numerous mutant SOD1s with the different severity of misfolding caused by the point mutations in the amino acid sequence. Fusion of both mfrGFP and EGFP were effective in monitoring mutant SOD1 aggregation within cultured cells, whereas split GFP was not. The cellular fluorescence of transfected HEK293T and NSC-34 cells expressing fusion proteins was determined by flow cytometry. Our findings signify that flow cytometric fluorescence measurement of cells expressing GFP variant fused to mutant SOD1 is a simple, but effective way to monitor mutant SOD1 aggregation in mammalian cells and also has a promise for a high-throughput screening for aggregation modulators.