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MISFOLDED PROTEIN? WE HAVE A CHEMICAL FOR THAT
 
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The desire to connect cell biology, protein folding, and small molecules brings us back to that question posed at the start: How do you assess how much unfolded protein is in a cell at any one moment, and does that even really matter? Deniz's chemical chaperone work with alpha-synuclein clearly showed that there was a neutral ratio between the protective and the denaturing osmolytes, but how widespread is this balancing act in nature? A similar question can be asked of Conn's misfolded receptor localization studies as well as Stockwell's PDI studies. In the end, Jeffrey Kelly's new folding probes might just be able to help answer all of these questions more rapidly than ever before.

Kelly's probes work by specifically binding to the folded, functional protein and not to misfolded forms of the protein. Since these probes are also fluorescent, a researcher can use fluorescence-based quantification to determine the amount of folded protein versus misfolded protein in a cell lysate at a specific time point. While the ability to quantitate and monitor protein folding states in cells provides unique biological information, Kelly's probes could go beyond simply quantifying sheer protein levels; they might also enable scientists to explore the impact of cellular protein chaperones and proteostasis networks on folding dynamics—another merging of the worlds of chemical biology and genomics.

In many respects, small molecule studies on protein folding are still in their infancy—much more work is needed to understand the biology and potential clinical applications of these different chaperones that impact a protein's folded state. But with new tools, and the lessons learned from these latest studies, the field is clearly moving in the right direction.

Understanding cell biology with small molecules

“When we started, it was unclear what was going on with these human loss-of-function mutants,” recalls Michael Conn, referring to his work on the GnRH receptor (GnRHR) where a specific mutation, E90K, led to a condition known as hypogonadotropic hypogonadism (HH).

In fact, 14 different mutations that lead to HH had been described in the receptor, but Conn and his colleagues decided to focus on 3, including E90K, that could be both fully and partially rescued through the use of pharmacological chaperones. The curious question for Conn, however, was why did the addition of the wild-type receptor not change the phenotype even though the pharmacological chaperone did?

Using confocal microscopy, the researchers were able to localize the misfolded versions of GnRHR to the endoplasmic recticulum. This observation explained the lack of localization to the plasma membrane and the effect pharmacological chaperones have in enhancing correct localization (the chaperones were promoting proper folding and localization). But a key biological finding came when Conn and his team realized that the misfolded versions of the GnRHR protein were actually forming oligomers with wild-type GnRHR, effectively rendering the wild-type version useless and a target for the cell's quality control network.





Brent Stockwell has also used small molecules to inform his cell biology studies. “Our discovery of a new cell death pathway came out of a small molecule screen for another purpose,” he recalls. For Stockwell, small molecules provide an advantage when screening as they can be more conditional than genetic reporters, have the potential to affect only single protein domains, and are vastly more translatable across species than genetically based tools.

Still, challenges exist for small molecule screens. According to Stockwell, many available small molecule libraries are still not very good at the moment, although this is steadily improving. “We analyzed 3 million compounds and found that only 1% were lead-like and truly worth acquiring.”

Despite the challenges, the potential impact of small molecules is clearly great. “The pharmacological chaperone really informed us about the underlying biology,” says Conn. –N.B.

References
1.) Liu, Y., Y.L. Tan, X. Zhang, G. Bhabha, D.C. Ekiert, J.C. Genereux, Y. Cho, Y. Kipnis, S. Bjelic, D. Baker, and J.W. Kelly. 2014. Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts. Proc Natl Acad Sci U S A. 111:4449-4454.

2.) Ferreon, A.C.M., M.M. Moosa, Y. Gambin, and A.A. Deniz. 2012. Counteracting chemical chaperone effects on the single-molecule a-synuclein structural landscape. Proc Natl Acad Sci U S A. 109:17826-17831.

3.) Janovick, J., D.M. Stewart, D. Jacob, L.D. Martin, J. Deng, C.A. Stewart. 2013. Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy. Proc Natl Acad Sci U S A. 110:21030-21035.

4.) Wisen, G, and J. Gestwicki. 2008. Identification of small molecules that modify the protein folding activity of heat shock protein 70. Anal Biochem. 374:371-377.

5.) Hoffstrom, B.G., A Kaplan, R. Letso, R. Schmid, G.J. Turmel, D.C. Lo, and B.R. Stockwell. 2010. Inhibitors of protein disulfide isomerase suppress apoptosis by misfolded proteins. Nature Chem Biol. 6:900-906.

6.) Ulloa-Aguirre, A., and P.M. Conn. 2014.Intracellular trafficking of G protein-coupled receptors to the cell surface plasma membrane in health and disease. In A. Ulloa-Aguirre, and P.M. Conn (Eds.) Cellular Endocrinology in Health and Disease. Academic Press, Boston, MA:341-364.

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