In Brief:
Small fluctuations in protein levels within cells can be the difference between health and disease or a drug molecule working or not, but it can be hard to detect these changes.
Scientists developed a new high-throughput tool to look at tiny changes in cellular protein levels across many conditions at once, using only a handful of cells to generate a robust signal that intensifies as a particular protein is degraded.
Researchers hope the tool will accelerate drug discovery by making it easier and faster to screen for new drugs that work by inducing degradation of disease-causing proteins in diseases like cancer.
Imagine a brain teaser where the challenge is to spot the difference between two nearly identical sandcastles. The only difference? The removal of three grains of sand. Scientists face a similarly exasperating challenge when it comes to looking at changes in protein levels within our cells, where small fluctuations can mean the difference between health and disease, or a drug working and not working. A new study in Cell, led by Steven Banik , assistant professor of chemistry in the School of Humanities and Sciences and institute scholar at Sarafan ChEM-H , has provided a solution to this problem, offering a new tool to amplify and visualize these tiny changes.
"Our ideas about what's important in biology are often defined by the tools that we have to look at it," said Banik. "There's a lot of biology happening inside a black box that we can't see. If we can amplify signals that we haven't been able to see before, we can discover new biology or new molecules that might have therapeutic benefit."
Proteins are the molecular machines inside cells that perform all the duties necessary for life. Layers of regulatory machinery govern how much of a certain protein is produced in a cell at any given time, and different kinds of cells contain distinct repertoires of proteins that coordinate what they do – a cardiac cell contains the protein machinery to produce a heartbeat, while a brain cell is equipped with proteins that allow it to fire.
Small decreases in the levels of these proteins can have major consequences on cellular behavior. Being able to study these small changes is also important when developing medicines; using a drug to degrade a rogue malfunctioning protein can help treat diseases like cancer. However, few techniques are sensitive enough to detect these changes for proteins that are not very abundant to begin with, and it can be difficult to study more than one protein or drug molecule at a time.
Turning grains of sand into boulders
Traditional techniques for measuring protein abundance often rely on extracting proteins from many cells and then concentrating the one you want to study in order to visualize it. This is like panning for gold on a beach until you find enough gold flecks to make a pile that you can see without a magnifying glass. You probably would need a dump truck of sand to get enough gold.
To accelerate drug discovery by more quickly zeroing in on the most promising drug candidates, Banik and team sought to devise a more sensitive method that would allow them to pan for gold with small volumes of sand, or in this case, small numbers of cells in a high-throughput screen.
"If you have many grains of sand dispersed all over a table, and you take one grain away, it would be very hard to see that," explained Banik. "But if we had a way to make the grain of sand look like a boulder and then took it off the table, that would be noticeable. And that's what we've done – contributed one of the first methods that uses signal amplification to allow us to see big signals resulting from small protein abundance changes."
The power of the new method lies in its ability to produce boulder-sized signals just as robustly at sand-bucket volumes as it does at dump-truck scale. This enables scientists to measure these changes more efficiently and sensitively than was previously possible, as well as test hundreds of proteins and drugs in parallel. Banik and team worked closely with co-author David Solow-Cordero in the high-throughput screening (HTS) center at the Nucleus , a facility that provides Stanford researchers with access to top-of-the-line instrumentation and expert HTS technical guidance.
"The Nucleus HTS facility is fully equipped with everything automated, things that would have been impossible to do by hand," said postdoctoral scholar and first-author Melissa Gray. "And Dr. David Solow-Cordero was instrumental in helping us understand what we needed to get this to work in high throughput."
Cellular hocus pocus
In the end, developing their new tool required both technical expertise and a bit of cellular engineering magic.
"When people talk about a magic trick, they say that making something disappear is the easiest part," said Banik. "But making something appear is harder. The same applies when you are studying something in biology. To make a signal appear, a lot more has to go right."
Things "going right" relies on programming native cellular machinery to produce a signal that can be read out in the lab, much like flipping a switch to turn on a lightbulb. But the team wanted more than a simple on/off switch. They wanted a lightbulb on a dimmer switch that would allow them to turn up the brightness the more a particular protein is degraded.
To create the first half of the switch, the team fused proteins they wanted to study to a kind of molecular brake that prevents specific regions of the genetic code from being turned on, or lighting up, without degradation activity. The other half of the switch is a protein called a transcription factor that acts as a cellular gas pedal. When more and more of the protein-brake fusion degrades, it allows the gas pedal to propel the cellular machinery forward to make more and more lightbulbs – fluorescent proteins that light up under the microscope. Sand-grain-sized changes in protein concentration thus are amplified into a brighter and brighter light signal that can be easily quantified and visualized.
Part of the promise of this new approach is its tunability. The system can be tweaked to have different-colored lightbulbs turn on independently in the same cell, or even have another output, like an RNA barcode, that can be read out by high-throughput sequencing instead of light. Such a tool could eventually be used to measure or manipulate protein concentrations to better understand how they work together to affect a biological process or drive disease.
"It would be really exciting and fulfilling to see people use this tool to make discoveries that would have taken longer with other methods," said Banik. "And to find interesting drug molecules that do things that we haven't been able to see before because the lens needed to view them didn't exist."
Acknowledgement:
Steven Banik is also a member of Bio-X , the Wu Tsai Human Performance Alliance , and the Wu Tsai Neurosciences Institute . Steven Corsello, assistant professor of medicine in the School of Medicine, is a co-author on the study. Corsello is also a member of Bio-X, the Maternal & Child Health Research Institute , and the Stanford Cancer Institute . Nathanael Gray, the Krishnan-Shah Family Professor and professor of chemical and systems biology in the School of Medicine and Institute Scholar at Sarafan ChEM-H , is a co-author on the paper. Gray is also a member of Bio-X and the Stanford Cancer Institute. Other co-authors on the study not directly mentioned in the text include graduate student trainees in the Chemistry/Biology Interface (CBI) Training Program at Sarafan ChEM-H: Sophia W. Chen, Jennifer A. Co, Qusay Q. Omran, Katelyn L. Randal, and Michelle T. Tang. Other co-authors include Athena Z. Xue, Hlib Razumkov, Jeonghye Yu, Stephanie A. Robinson, and Cara A. Starnbach.
This research was also supported by the Stanford Innovative Medicines Accelerator , the Stanford Cancer Center, Molecular Devices ImageXpress Confocal, the Gordon and Betty Moore Foundation, Stanford's Knight-Hennessy Scholars program, the Stanford Medical Scientist Training Program , the Burroughs Wellcome Fund Careers at the Scientific Interface Award, the Stanford Medical Scholars Research Fellowship Program , a Stanford Undergraduate Summer Research Fellowship in Chemistry, the EDGE Doctoral Fellowship Program, the Damon Runyon Cancer Research Foundation, the NSF Graduate Research Fellowships program, and a Stanford CMAD Fellowship.
