Prions: how I learnt to stop tedious experiments and love the protein aggregation

Proteins fold into elaborate structures to execute their functions, and failure to do so causes many diseases, including neurodegenerative Alzheimer's disease and Parkinson's disease. However, cells can also use protein aggregation to their benefit, forming compartmentalized membrane-less bodies. Tracking these elaborate folding behaviors of proteins has been a challenge. We have developed a simple, yet powerful, tool to track protein folding in the cell with minimal observer interference.

HFSP Long-Term Fellow Erinc Hallacli and colleagues
authored on Tue, 02 January 2018

Protein folding has a complicated energy landscape and a protein’s native fold is usually not the energetically favored one. Right from the first appearance of amino acids in a ribosome’s exit tunnel, protein folding is monitored and chaperoned.  Elaborate protein quality control mechanisms make sure that misfolded proteins are degraded and recycled. However, as with all biological systems, these mechanisms are not perfect and proteins can irreversibly misfold and cause cellular toxicity. This is the basis for many human diseases ranging from, Alzheimer’s disease, ALS, Parkinson’s disease and Huntington’s disease, to name a few.

Figure: yTRAP module is integrated into the yeast genome as a single copy. Soluble yTRAP sensor allows GFP reporter to be expressed, as opposed to the aggregated yTRAP. Shown is a well-known prion PSI in yeast in its soluble (green) and aggregated-prion-form (dark) as sensed by the PSI yTRAP.  We have generated hundreds of sensors for RNA binding proteins to understand their prion-like behaviors.

As always, there is another side of the coin. Sometimes cells can use protein aggregation to their advantage. Proteins can go through phase transitions in the cell and form membrane-less compartments to better execute their functions. These transitions are highly controlled and reversible. An extreme version of this is that proteins can be stably inherited in different conformations, with different functions. Prions, as they are named, are beautiful examples of nature’s ability to use protein folding to transmit information epigenetically.

Traditionally, observing protein aggregation involves complicated biochemical procedures or sophisticated microscopy. Although they can be quite informative, they are laborious and not amenable to high-throughput studies. We have developed a simple quantitative tool to measure protein aggregation by channeling the aggregation status to a transcriptional reporter.  This is achieved by attaching a special tag to the protein under investigation. The tag is composed of a highly specific DNA binding domain, along with a strong transcriptional activator. If the protein is soluble, the tag activates a fluorescent reporter, which can be monitored quantitatively, and if the protein is aggregated, the reporter is shut off.

By using this strategy, we have tracked different prion states in yeast and found determinants of their stability. We created de novo prions, where their folding is deterministically controlled by the experimenter. Furthermore, we have generated a library of sensors to understand the folding properties of RNA binding proteins in yeast. With the help of this library, aggregation prone RNA binding proteins were uncovered. We monitored the consequences of their aggregation and we performed genome-wide screens to see how the folding of a protein influences another one. In summary, we present here a simple, high-throughput, quantitative tool to track complicated behaviors of proteins in vivo.

Reference

Genetic Tool to Track Protein Aggregates and Control Prion Inheritance. Newby GA*, Kiriakov S*, Hallacli E*, Kayatekin C, Tsvetkov P, Mancuso CP, Bonner JM, Hesse WR, Chakrabortee S, Manogaran AL, Liebman SW, Lindquist S, Khalil AS. A. Cell. 2017 Nov 2;171(4):966-979.e18. doi: 10.1016/j.cell.2017.09.041. Epub 2017 Oct 19. *Contributed equally.  

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