Scientists use light to control the logic networks of a cell
New technique illuminates role of previously inaccessible proteins involved in health and disease
Proteins are the workhorse molecules of life. Among their many jobs, they carry oxygen, build tissue, copy DNA for the next generation, and coordinate events within and between cells. Now scientists at the University of North Carolina at Chapel Hill have developed a method to control proteins inside live cells with the flick of a switch, giving researchers an unprecedented tool for pinpointing the causes of disease using the simplest of tools: light.
The work, led by Klaus Hahn and Nikolay Dokholyan and spearheaded by Onur Dagliyan, a graduate student in their labs, builds on the breakthrough technology known as optogenetics. The technique, developed in the early 2000s, allowed scientists, for the first time, to use light to activate and deactivate proteins that could turn brain cells on and off, refining ideas of what individual brain circuits do and how they relate to different aspects of behavior and personality.
But the technique has had its limitations. Only a few proteins could be controlled by light; they were put in parts of a cell where they normally didn’t exist; and they had been heavily engineered, losing much of their original ability to detect and respond to their environment.
In their new work, published recently in Science, Hahn, Dokholyan and Dagliyan expand optogenetics to control a wide range of proteins without changing their function, allowing a light-controllable protein to carry out its everyday chores. The proteins can be turned on almost anywhere in the cell, enabling the researchers to see how proteins do very different jobs depending on where they are turned on and off.
“We can take the whole, intact protein, just the way nature made it, and stick this little knob on it that allows us to turn it on and off with light,” said Hahn, Thurman Distinguished Professor of Pharmacology and a UNC Lineberger Comprehensive Cancer Center member. “It’s like a switch.”
The switch that Hahn, Dokholyan and colleagues developed is versatile and fast – they can toggle a protein on or off as fast as they can toggle their light. By changing the intensity of light, they can also control how much of the protein is activated or inactivated. And by controlling the timing of irradiation, they can control exactly how long proteins are activated at different points in the cell.
“A lot of aspects of cell behavior depend on transient, fast changes in protein activity,” said Hahn. “But those changes have to happen in exact locations. The same protein can cause a cell to do different things if it’s active in different places, building flexible logic networks in different parts of the cell, depending on what it is responding to.”
To make their breakthrough, Hahn and Dagliyan used a computational approach to identify which parts of a protein could be modified without changing the protein’s normal operation, and showed that loops of protein structure commonly found on protein surfaces can be readily modified with different ‘knobs’ to control proteins with light, or even to respond to drugs.
Imagine sticking a video camera on a bus; put it on the gas pedal and it will obstruct its function, so the bus will not drive properly. But put it on the hood, and the bus will continue to drive just fine. The new computational approach pointed the researchers toward each protein’s hood.
Because the tools keep the natural protein function intact, the new technique allows scientists to study proteins in living systems, where proteins normally live and work in all their natural complexity. This ability to manipulate proteins in living systems also provides an opportunity to study a wide range of diseases, which often arise from the malfunctioning of a single protein.
“In order to understand what’s happening you need to see the parts moving around,” said Hahn. “It’s that dynamic behavior that you need to know to understand what’s going on.”
The University of North Carolina at Chapel Hill (also known as UNC, UNC-Chapel Hill, Chapel Hill, North Carolina, or simply Carolina) is a coeducational public research university located in Chapel Hill, North Carolina, United States.
It is the second largest university in North Carolina. North Carolina has been consistently ranked among the highest ranked universities in the United States and is one of the original eight Public Ivy schools that provide an Ivy League experience for a public schooling price. After being chartered in 1789, the university first began enrolling students in 1795, which allows it to be one of three schools to claim the title of the oldest public university in the United States.
The first public institution of higher education in North Carolina, the school opened its doors to students on February 12, 1795. The university offers degrees in over 70 courses of study through fourteen colleges and the College of Arts and Sciences. All undergraduates receive a liberal arts education and have the option to pursue a major within the professional schools of the university or within the College of Arts and Sciences from the time they obtain junior status. Under the leadership of President Kemp Plummer Battle, in 1877 North Carolina became coeducational, while desegregation ended when African-American graduate students were admitted under Chancellor Robert Burton House in 1951. In 1952, North Carolina opened its own hospital, UNC Health Care, for research and treatment, and has since specialized in cancer care. The school’s students, alumni, and sports teams are known as “Tar Heels”.
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Researchers from North Carolina State University, the University of North Carolina at Chapel Hill and First Affiliated Hospital of Zhengzhou University have developed a synthetic version of a cardiac stem cell. These synthetic stem cells offer therapeutic benefits comparable to those from natural stem cells and could reduce some of the risks associated with stem cell therapies. Additionally, these cells have better preservation stability and the technology is generalizable to other types of stem cells.
Stem cell therapies work by promoting endogenous repair; that is, they aid damaged tissue in repairing itself by secreting “paracrine factors,” including proteins and genetic materials. While stem cell therapies can be effective, they are also associated with some risks of both tumor growth and immune rejection. Also, the cells themselves are very fragile, requiring careful storage and a multi-step process of typing and characterization before they can be used.
Ke Cheng, associate professor of molecular biomedical sciences at NC State, associate professor in the joint biomedical engineering program at NC State and UNC, and adjunct associate professor at the UNC Eshelman School of Pharmacy, led a team in developing the synthetic version of a cardiac stem cell that could be used in off-the-shelf applications.
Cheng and his colleagues fabricated a cell-mimicking microparticle (CMMP) from poly (lactic-co-glycolic acid) or PLGA, a biodegradable and biocompatible polymer. The researchers then harvested growth factor proteins from cultured human cardiac stem cells and added them to the PLGA. Finally, they coated the particle with cardiac stem cell membrane.
“We took the cargo and the shell of the stem cell and packaged it into a biodegradable particle,” Cheng says.
When tested in vitro, both the CMMP and cardiac stem cell promoted the growth of cardiac muscle cells. They also tested the CMMP in a mouse model with myocardial infarction, and found that its ability to bind to cardiac tissue and promote growth after a heart attack was comparable to that of cardiac stem cells. Due to its structure, CMMP cannot replicate – reducing the risk of tumor formation.
“The synthetic cells operate much the same way a deactivated vaccine works,” Cheng says. “Their membranes allow them to bypass the immune response, bind to cardiac tissue, release the growth factors and generate repair, but they cannot amplify by themselves. So you get the benefits of stem cell therapy without risks.”
The synthetic stem cells are much more durable than human stem cells, and can tolerate harsh freezing and thawing. They also don’t have to be derived from the patient’s own cells. And the manufacturing process can be used with any type of stem cell.
“We are hoping that this may be a first step toward a truly off-the-shelf stem cell product that would enable people to receive beneficial stem cell therapies when they’re needed, without costly delays,” Cheng says.
An interdisciplinary team of researchers has developed a smart patch designed to monitor a patient’s blood and release blood-thinning drugs as needed to prevent the occurrence of dangerous blood clots – a condition known as thrombosis.
In an animal model, the patch was shown to be more effective at preventing thrombosis than traditional methods of drug delivery. The work was done by researchers at North Carolina State University and the University of North Carolina at Chapel Hill.
Thrombosis occurs when blood clots disrupt the normal flow of blood in the body, which can cause severe health problems such as pulmonary embolism, heart attack or stroke. Current treatments often rely on the use of blood thinners, such as Heparin, which require patients to test their blood on a regular basis in order to ensure proper dosages. Too large a dose can cause problems such as spontaneous hemorrhaging, while doses that are too small may not be able to prevent a relapse of thrombosis.
“Our goal was to generate a patch that can monitor a patient’s blood and release additional drugs when necessary; effectively, a self-regulating system,” says Zhen Gu, co-corresponding author on a paper describing the work. Gu is an associate professor in the joint biomedical engineering program at NC State and UNC.
“Two years ago, I spoke with Zhen Gu about the significant clinical need for precise delivery of blood thinners,” says Caterina Gallippi, a co-corresponding author and associate professor in the joint biomedical engineering program. “We, together with Professor Yong Zhu in the mechanical engineering department at NC State, assembled a research team and invented this patch.”
The patch incorporates microneedles made of a polymer that consists of hyaluronic acid (HA) and the drug Heparin. The polymer has been modified to be responsive to thrombin, an enzyme that initiates clotting in the blood.
When elevated levels of thrombin enzymes in the bloodstream come into contact with the microneedle, the enzymes break the specific amino acid chains that bind the Heparin to the HA, releasing the Heparin into the blood stream.
“The more thrombin there is in the bloodstream, the more Heparin is needed to reduce clotting,” says Yuqi Zhang, a Ph.D. student in Gu’s lab and co-lead author of the paper. “So we created a disposable patch in which the more thrombin there is in the blood stream, the more Heparin is released.”
“We will further enhance the loading amount of drug in the patch. The amount of Heparin in a patch can be tailored to a patient’s specific needs and replaced daily, or less often, as needed,” says Jicheng Yu, a Ph.D. student in Gu’s lab and the other co-lead author of the paper. “But the amount of Heparin being released into the patient at any given moment will be determined by the thrombin levels in the patient’s blood.”
The research team tested the HA-Heparin smart patch in a mouse model. In the experiments, subjects were injected with large doses of thrombin, which would result in fatal blood clotting of the lungs if left untreated.
In the first experiment, mice were either left untreated, given a shot of Heparin, or given the HA-Heparin smart patch. The mice were injected with thrombin 10 minutes later. Fifteen minutes after the thrombin injection, only the mice who received no treatment died.
In the second experiment, the thrombin was injected six hours after treatment. Fifteen minutes after the thrombin injection, all of the mice with the HA-Heparin smart patch were fine, but around 80 percent of the mice that received the Heparin shot had died.
“We’re excited about the possibility of using a closed-loop, self-regulating smart patch to help treat a condition that affects thousands of people every year, while hopefully also driving down treatment costs,” Gu says. “This paper represents a good first step, and we’re now looking for funding to perform additional preclinical testing.”
Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year.
Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology.
Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.
Dengue virus (DENV) is transmitted by mosquitoes and is endemic in over 120 countries, causing over 350 million infections yearly. Most infections are clinically unapparent, but under specific conditions, dengue can cause severe and lethal disease. DENV has 4 distinct serotypes and secondary DENV infections are associated with hemorrhagic fever and dengue shock syndrome. This enhancement of infection complicates vaccine development and makes it necessary to induce protective immunity against all 4 serotypes. Since whole virus vaccine candidates struggle to induce protective immunity, we are developing a nanoparticle display vaccine approach. We have expressed, purified and characterized a soluble recombinant E-protein (sRecE). Regardless of nanoparticle shape or size, particulation of sRecE enhances DENV specific IgG titers and induces a robust, long lasting neutralizing antibody response and by adsorbing sRecE to the nanoparticles, we prolong the exposure of sRecE to the immune system.
Nanoparticle display shows great promise in dengue vaccine development and possibly other mosquito-borne viruses like zika virus.