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The aggregation of cellular proteins into insoluble clumps is a hallmark of many diseases, including Alzheimer’s, Parkinson’s, systemic amyloidosis, prion diseases, and type 2 diabetes. Protein agglomeration can also be a feature of normal cellular functions, such as signal transduction, synapse modification, and the regulation of RNAs during cellular stress.
Tools for studying such physiological and pathological protein aggregations, however, are limited, explains biomedical engineer Ahmad Khalil of Boston University. The principal options for researchers, he says, are either to destroy cells and analyze their innards for protein aggregates, or append a fluorescent tag to the proteins of interest within cells and view the formation of clumps (bright spots) with a microscope.
While this second option maintains the protein’s normal physiological surroundings, Khalil says, “inherently it is not a very high-throughput way of studying this phenomenon.”
Khalil and colleagues’ new approach, called yeast transcriptional reporting of aggregating proteins (yTRAP), allows for high-throughput analysis and doesn’t destroy cells. An aggregation-prone protein of interest is first fused to a synthetic transcriptional activator, which can drive gene expression from a synthetic promoter only when the protein is not aggregated. Linking the synthetic promoter to a fluorescent reporter allows easy identification and, if desired, sorting of cells with and without aggregates.
The team has used yTRAP to detect accumulations of prions and other proteins in yeast; to perform a high-throughput screen for aggregation-preventing mutants; and to identify cells that “remembered” an environmental stimulus (heat)—using a yeast strain engineered to express a stably aggregating prion under the control of a heat-responsive promoter.
The assay “can inform us about a very important cellular process,” says Madan Babu of the MRC Laboratory of Molecular Biology in Cambridge, U.K., who was not involved with the project. But it also “can be applied to a number of different questions,” he says. “It’s a bit of a tour de force.” (Cell, 171:966–79, 2017)
|AGGREGATION DETECTION TECHNIQUE||HOW IT WORKS||ASSAY EQUIPMENT||HIGH THROUGHPUT CAPACITY||CELL TYPES|
Fluorescent tagging of aggregation-prone protein
Proteins are fluorescently tagged—either by being engineered as fluorescent fusion proteins, or by applying fluorescent antibodies. Soluble proteins appear as diffuse fluorescence, while aggregates appear as bright spots.
|yTRAP||Proteins are fused to a synthetic transcriptional activator. The fusion protein is then expressed in cells where corresponding synthetic promoters drive expression of reporters. Soluble proteins activate the reporter, while aggregated proteins do not.||
Fluorescence microscope, flow cytometer, fluorescence-
activated cell sorter (FACS), or fluorescence microplate reader
Limited to yeast so far, but future versions are planned
for other cell types