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Controlled Cell Death
Tiny proteins shed light on apoptosis, targeted drug delivery, and human disease triggers
Jeanne Hardy and a student examining images of cells on a screen.

With the Institute for Applied Life Sciences and new state-of-the-art equipment, Hardy envisions the campus serving as a "nucleating point" to understanding the interplay between structure and drug delivery. 

Apoptosis, or programmed cell death, has been tied in recent years to cancer and neurodegenerative disorders such as Alzheimer’s disease. UMass Amherst biological chemist Jeanne Hardy, above, and her research group are studying caspases—the proteins that control apoptosis—and making huge strides towards cures for these damaging diseases.

Hardy’s research is part of a coordinated effort through the campus’s new Institute for Applied Life Sciences (IALS), funded largely with $95 million from the Massachusetts Life Sciences Center, which is focused on taking fundamental research in the life sciences and moving it more quickly to application. In her research, Hardy focuses on tiny proteins—small elements of the larger human system.

“We are working on the proteins that come from humans so the discoveries we make are directly relevant to human disease,” says Hardy.

Cell death, Hardy explains, can be good or bad depending on the context. In cases of cancer, cell death is a necessary part of the solution—the body senses that the cells are damaged and triggers apoptosis. In cases of Alzheimer’s and neurodegenerative disorders, however, the opposite is true. These diseases trigger unwanted apoptosis, killing necessary cells instead. Much remains unknown about the mechanisms for cell death, which is why Hardy and the team are studying caspases. By closely examining the structures of these proteins, Hardy is investigating how modulating their role in apoptosis could impact human health.

Reagents allow the separation of desired human proteases from other cell proteins.
Caspases are proteases, or enzymes that conduct proteolysis, meaning they “bite” other proteins in half.  There are 14 members in the caspase family, each with its own number. In a project funded by the National Institutes of Health and the UMass Amherst Armstrong Fund for Science, Hardy is studying the connection between caspase-6 and Alzheimer’s disease. She explains that if caspase-6 is activated in neuron cells, it triggers a degenerative process that leads to the neurofibrillary tangles known for disrupting brain functioning in Alzheimer’s patients. Hardy is working to develop a small, drug-like molecule that will specifically inhibit caspase-6 to treat Alzheimer’s. To accomplish this, Hardy is using x-ray crystallography—a procedure that beams-rays through lab-grown crystals made up of trillions of copies of the protein—to examine the structure of caspase-6. By observing the ways the x-rays are diffracted, Hardy can see exactly how the protein is structured.

“You can see atom by atom what that protein looks like, which is really useful if you want to make a drug because you know precisely what your target looks like,” says Hardy.

Hardy explains that the therapeutic “bullet” that will block caspase-6 must be strategically designed to not block caspase-3 (and other caspases), or it will prevent the body from killing off abnormal cells. The fact that various caspases play various roles actually allows Hardy’s team to harness caspase function for a number of different outcomes. When caspase-3 is activated, it triggers apoptosis and surrounding cells commit suicide. Hardy and the team are working with chemists Vincent Rotello and Sankaran “Thai” Thayumanavan on methods to utilize the trigger by delivering caspase-3 into cancer cells. In these applications, caspase-3 can act as a drug that kills cancer cells without collateral damage to healthy cells. Thayumanavan and his team have already developed a nanogel capable of delivering therapeutics to targeted cells, and the Rotello group has developed nanoparticles that can transport caspases and other proteins. Because cancer only proliferates when cancer cells evolve to evade apoptosis, the delivery of caspase-3 could be a novel cure.

Samples of purified proteases and protease substrates are stored on ice to slow their self-destruction.
To watch which caspases are active and where, Hardy and the team worked with stem cell biologist Kimberly Tremblay and biologist Gerald Downes to develop a reporter—a tool used by researchers to detect caspase activity. The reporter has come to be in high demand. To create the reporter, the team used green fluorescent protein, yet also attached a 27-amino acid peptide “tail” which prevents the protein’s signature green color. If an active caspase is present, it will cleave the tail and the reporter will turn green. This allows real-time monitoring of caspase action in living organisms.

Inspired by their work to find new ways to inhibit caspases, one of Hardy’s graduate students, Muslum Yildiz, has shown that he can alter the structure of dengue virus protease—the virus responsible for ‘bone-break fever.’ The deadly virus is a growing concern in tropical regions, and has recently spread through mosquitos to North America. Yildiz has shown how inhibitors that lock the protease into a new conformation prevent enzyme activity and could prevent infection.

The Council for International Exchange of Scholars recently awarded Hardy a Fulbright Scholarship to further her work on Alzheimer’s research with collaborators in Tokyo and Paris. In her research abroad, Hardy will incorporate the latest developments in nuclear magnetic resonance (NMR) spectroscopy, a technique critically important to her research.

Above is a gel that allows assessment of the size and purity of the proteases under investigation. The marker in the central lane contains protein of known size for comparison.
Hardy will return to new NMR spectrometers purchased as part of the capital expenditure for the new IALS research facility. Hardy’s research will be central to two of the Institute’s new centers, Models to Medicine and Bioactive Delivery Bridge. Researchers in the Models to Medicine center are translating fundamental discoveries into the identification and validation of new therapeutic targets, while those in Bioactive Delivery Bridge are focusing on the discovery and application of innovative drug delivery vehicles for those targets.

Hardy says she is excited to return to this game-changing equipment and eager to use a one-of-a-kind high-throughput circular dichroism spectrometer to advance her research.

“There are a lot of ways that this investment will change the kinds of experiments we do and the speed with which we can do them. I foresee it being a nucleating point for other organizations to come here…to see us as the ‘go-to place for understanding the interplay between structure and drug delivery,’” Hardy says.

Amanda Drane '12