Featured Research

from universities, journals, and other organizations

New probes quantify folded and misfolded protein levels in cells

Date:
March 4, 2014
Source:
The Scripps Research Institute
Summary:
Scientists have invented small-molecule folding probes that enable them to quantify functional, normally folded and disease-associated misfolded conformations (shapes) of a protein-of-interest in cells under different conditions. Scientists have long needed better tools for making such measurements in cells, because protein misfolding is a major cause of damage to tissues. Disorders that feature excessive protein misfolding afflict millions of people worldwide and include Alzheimer's and Parkinson's diseases, the systemic amyloidoses and prion ("mad cow-type") infections, as well as common enzyme deficiencies.

Scientists at The Scripps Research Institute (TSRI) have invented small-molecule folding probes that enable them to quantify functional, normally folded and disease-associated misfolded conformations (shapes) of a protein-of-interest in cells under different conditions.

Scientists have long needed better tools for making such measurements in cells, because protein misfolding is a major cause of damage to tissues. Disorders that feature excessive protein misfolding afflict millions of people worldwide and include Alzheimer's and Parkinson's diseases, the systemic amyloidoses and prion ("mad cow-type") infections, as well as common enzyme deficiencies.

"This new probe technology should lead to a better understanding of how to fold misfolding-prone proteins in cells," said Jeffery W. Kelly, chair of TSRI's Department of Molecular and Experimental Medicine, Lita Annenberg Hazen Professor of Chemistry and member of the Skaggs Institute for Chemical Biology at TSRI. "The ability to quantify protein folding in a cell using this simple fluorescence-based technology should speed the development of new therapies."

The study, led by Kelly and his laboratory, is reported in this week's online Early Edition of the Proceedings of the National Academy of Sciences.

Profound Consequences

Misfolded proteins have never been easy to distinguish from their normally folded counterparts, especially within cells, because both have the same sequence of amino acids. Yet the loss of the normally folded shape can have profound consequences -- a misfolded protein typically will lose its function within a cell. Worse, misfolding may expose "sticky," previously concealed parts of a protein that cause it to start aggregating with other copies of itself, leading to dysfunction of tissue that does not easily regenerate.

Both loss-of-function and gain-of-toxic function mutations can lead to disease, often shortening lifespan. Boosting the folding systems or the capacity of the protein homeostasis network in cells can prevent or eliminate protein misfolding. These new probes allow scientists to quantify how adapting the protein homeostasis network enhances the folding of a particular protein.

In the new study, Kelly's team aimed to selectively tag only the folded and functional conformations of a protein-of-interest. In one case, to demonstrate proof-of-principle, the scientists employed a model protein, retroaldolase, a designed enzyme created by collaborator David Baker's laboratory at the University of Washington, Seattle. The team also used transthyretin (TTR), a protein whose misfolding and aggregation is known to lead to several fatal disorders, including cardiomyopathies and polyneuropathies. (The Kelly laboratory recently helped develop the first specific drug therapy for the TTR polyneuropathies.)

The team -- particularly the three lead authors, graduate students Yu Liu and Yun Lei Tan and Research Associate Xin Zhang -- accomplished the tagging reaction by designing and making "folding probes" that covalently labeled the properly folded, functional forms of the proteins, but not the misfolded forms. When the scientists added a solution of probe molecules to the soluble contents of cells containing the target proteins, they were able to quantify the folded target proteins from the light emitted by the probes' fluorescent beacons.

Toward Better Screens for New Drugs

Probes that covalently react with folded and functional protein families have been devised before by the Cravatt lab at TSRI. However, their usefulness as folding probes had been questioned by the scientific community, because the very act of reacting a folding probe with a target folded and functional protein-of-interest stabilizes that state and usually increases the population of the folded and functional fraction, over-representing it. In the new study, however, the researchers used folding probes in combination with cell lysis and ATP depletion, which causes the chaperones in the cell to hold onto the unfolded proteome -- preventing its folding, providing a snapshot of the folded protein-of-interest population, while minimizing overrepresentation of that state by the tagging process.

One of the most important applications of new probes like these will be for the rapid, "high-throughput" screening of very large drug compound libraries to identify drug candidates that prevent protein misfolding by improving the quality of cellular folding. "Using these probes to quantify the concentration of a functional, folded protein-of-interest, we can screen for compounds that boost this concentration, for example," said Zhang, who along with Kelly conceived and designed the study.

In this study, the researchers cleared another hurdle for the use of probes in high-throughput screens with the design of a probe whose fluorescent beacon isn't lit all the time, but only turns on when it reacts with the folded protein-of-interest. "That fluorescent signal quickly shows you the concentration of the folded, functional protein that was in the cell at the time of lysis," Zhang said. "There is no need for the time-consuming removal of fluorescence probes that aren't bound to targets or separation of the probe-protein-of-interest conjugate."

Drugs that reduce the misfolding of specific proteins by altering cell biology of protein homeostasis might one day be used to prevent or delay age-related neurodegenerative diseases, such as Alzheimer's and Parkinson's, and to treat inherited enzyme-deficiency disorders. Zhang, Kelly and their colleagues suspect that anti-misfolding drugs could have even broader applications, given the surprisingly large populations of misfolded proteins in cells and all the ways in which those misfolded proteins could be causing harm.


Story Source:

The above story is based on materials provided by The Scripps Research Institute. Note: Materials may be edited for content and length.


Journal Reference:

  1. Y. Liu, Y. L. Tan, X. Zhang, G. Bhabha, D. C. Ekiert, J. C. Genereux, Y. Cho, Y. Kipnis, S. Bjelic, D. Baker, J. W. Kelly. Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts. Proceedings of the National Academy of Sciences, 2014; DOI: 10.1073/pnas.1323268111

Cite This Page:

The Scripps Research Institute. "New probes quantify folded and misfolded protein levels in cells." ScienceDaily. ScienceDaily, 4 March 2014. <www.sciencedaily.com/releases/2014/03/140304071158.htm>.
The Scripps Research Institute. (2014, March 4). New probes quantify folded and misfolded protein levels in cells. ScienceDaily. Retrieved October 21, 2014 from www.sciencedaily.com/releases/2014/03/140304071158.htm
The Scripps Research Institute. "New probes quantify folded and misfolded protein levels in cells." ScienceDaily. www.sciencedaily.com/releases/2014/03/140304071158.htm (accessed October 21, 2014).

Share This



More Plants & Animals News

Tuesday, October 21, 2014

Featured Research

from universities, journals, and other organizations


Featured Videos

from AP, Reuters, AFP, and other news services

'Cadaver Dog' Sniffs out Human Remains

'Cadaver Dog' Sniffs out Human Remains

AP (Oct. 21, 2014) Where's a body buried? Buster's nose can often tell you. He's a cadaver dog, specially trained to find human remains and increasingly being used by law enforcement and accepted in courts. These dogs are helping solve even decades-old mysteries. (Oct. 21) Video provided by AP
Powered by NewsLook.com
White Lion Cubs Born in Belgrade Zoo

White Lion Cubs Born in Belgrade Zoo

AFP (Oct. 20, 2014) Two white lion cubs, an extremely rare subspecies of the African lion, were recently born at Belgrade Zoo. They are being bottle fed by zoo keepers after they were rejected by their mother after birth. Duration: 00:42 Video provided by AFP
Powered by NewsLook.com
Traditional Farming Methods Gaining Ground in Mali

Traditional Farming Methods Gaining Ground in Mali

AFP (Oct. 20, 2014) He is leading a one man agricultural revolution in Mali - Oumar Diatabe uses traditional farming methods to get the most out of his land and is teaching others across the country how to do the same. Duration: 01:44 Video provided by AFP
Powered by NewsLook.com
Goliath Spider Will Give You Nightmares

Goliath Spider Will Give You Nightmares

Buzz60 (Oct. 20, 2014) An entomologist stumbled upon a South American Goliath Birdeater. With a name like that, you know it's a terrifying creepy crawler. Sean Dowling (@SeanDowlingTV) has the details. Video provided by Buzz60
Powered by NewsLook.com

Search ScienceDaily

Number of stories in archives: 140,361

Find with keyword(s):
Enter a keyword or phrase to search ScienceDaily for related topics and research stories.

Save/Print:
Share:

Breaking News:

Strange & Offbeat Stories


Plants & Animals

Earth & Climate

Fossils & Ruins

In Other News

... from NewsDaily.com

Science News

Health News

Environment News

Technology News



Save/Print:
Share:

Free Subscriptions


Get the latest science news with ScienceDaily's free email newsletters, updated daily and weekly. Or view hourly updated newsfeeds in your RSS reader:

Get Social & Mobile


Keep up to date with the latest news from ScienceDaily via social networks and mobile apps:

Have Feedback?


Tell us what you think of ScienceDaily -- we welcome both positive and negative comments. Have any problems using the site? Questions?
Mobile: iPhone Android Web
Follow: Facebook Twitter Google+
Subscribe: RSS Feeds Email Newsletters
Latest Headlines Health & Medicine Mind & Brain Space & Time Matter & Energy Computers & Math Plants & Animals Earth & Climate Fossils & Ruins