From the Oct. 16, 2006, Princeton Weekly Bulletin
Sometimes what appears to be a problem at first glance could lead to a solution if a scientist knows how to look at it in the right way.
That’s what happened to Michael Hecht and his team of Princeton researchers while attempting to put together a set of novel proteins. The team has achieved a reputation in the scientific community for successfully custom-designing proteins that could form the basis for new developments in medicine and biotechnology.
This time, however, their efforts yielded an annoying tangle of stringy fibers rather than the neat helical shapes that Hecht’s group found in earlier attempts to design novel proteins. But upon further examination, the team realized that these minuscule fibers could provide answers to some larger questions regarding how proteins fold and even begin to shed light on how to treat illnesses such as Alzheimer’s disease.
Hecht’s team of undergraduates, graduate students and postdoctoral fellows designs and builds novel protein molecules “from scratch” by assembling chains of amino acids, the 20 compounds that are among the building blocks of life. Long chains of amino acids, if the team assembles them in the right way, form proteins — some that exist in nature, others that do not.
“For the past 15 years, we’ve been focusing on novel proteins that do not exist in nature,” said Hecht, a professor of chemistry. “We’re not making useful proteins yet, but we are laying the foundation for the future. The ability to make new proteins that fold into the desired structures may one day have uses in biotechnology and medicine.”
Chains of amino acids typically form two basic shapes: helical spirals or extended strands. When Hecht’s team designed proteins from the spiral structures, they tended to fold up nicely into complex but well-organized shapes reminiscent of the naturally-occurring proteins found in cells. In contrast, proteins designed from the strands have been less well-behaved, tending to misfold in ways that cause them to aggregate into tangled fibers instead of compact shapes.
“We could string them together, but they refused to fold properly,” Hecht said. “All we got were longer and longer fibers. It was a bit frustrating for the protein designers, because it meant we couldn’t take advantage of all the architectures that natural proteins use.”
When the team first saw that these proteins were misbehaving about eight years ago, Hecht called upon the skills of Jennifer Patterson, then an undergraduate in his lab, who had previous experience using an electron microscope. She used one of the powerful magnification devices elsewhere on campus to take pictures of the strand-based chains. When he inspected the chains up close, Hecht had a flash of recognition.
“I remembered a lecture I attended a few years previous on Alzheimer’s disease,” Hecht said. “One of the slides had featured images of the tangled filaments that form in the brain of Alzheimer’s patients, and I realized that the structures Jennifer saw in her electron microscope images bore an uncanny resemblance to those observed in Alzheimer’s patients.”
Bigger medical questions
Alzheimer’s disease is noted for the insoluble plaque that builds up around the neural cells of victims. The plaque is formed of a protein called A-beta, which aggregates into an abnormal fiber-like structure called an amyloid that, in sufficient quantities, can cause affected regions of the brain to atrophy.
“Amyloids are themselves misfolded proteins, and our lab has extensive experience with protein folding,” Hecht said. “The logical questions were: What is it about the A-beta chain of amino acids that causes it to misfold into these stringy fibers, and why does it accumulate to such harmful effect?”
Several other neural disorders, most notably Huntington’s and Parkinson’s diseases in humans as well as “mad cow” disease and scrapie in animals, are also associated with misfolded proteins. So while team members may not have solved their ongoing problem with the design of extended strands, they now had the opportunity to explore a connection with bigger medical questions. What they needed was a way to stop the A-beta protein from aggregating into fibers.
“Since we didn’t have a clear structural model for how A-beta forms the fibers, we couldn’t custom design an inhibitor of the fiber formation process,” Hecht said. “Instead, we devised a method that would allow us to screen through large collections of arbitrarily chosen molecules that might do what we wanted. The screen would allow us to find a rare needle in a large haystack.”
To make the screen user friendly, they wanted something that would light up in the presence of a compound that inhibited A-beta aggregation. The light came from a special protein taken from fluorescent sea creatures. Green fluorescent protein, or GFP, is used as a marker for biological and chemical processes in labs worldwide, and the team found a way to link the fluorescence of GFP to the presence of a molecule that inhibited A-beta aggregation.
“We had an effective screen and were ready to search through a large collection of drug-like compounds for those rare ‘winners’ that might inhibit A-beta aggregation,” said Hecht. “The problem, however, was that we did not have our own large collection of drug-like compounds. Although our lab had expertise in making proteins, we didn’t have the ability to synthesize the small molecules that might be useful as future drugs.”
So Hecht began talking to other scientists who synthesized small molecules. Although many research groups custom-build molecules for different purposes, finding the right collaborator took a few years. “Eventually we found Young-Tae Chang at New York University, who is making libraries of drug-like molecules, and initiated a research partnership with him.”
By applying their new fluorescence-based screen to Chang’s collection of drug-like molecules, the scientists were able to discover several ‘hits’ — compounds that inhibit the aggregation of A-beta into fibers.
Hecht and his team are excited by these initial results, but they caution that the process will not lead to an anti-Alzheimer’s drug anytime soon. Nonetheless, they are optimistic that the approach may eventually identify a compound that can form the basis for a future therapeutic for treating Alzheimer’s disease.
Hecht’s team published a paper on their screen in the Aug. 18 edition of the journal ACS Chemical Biology, published by the American Chemical Society. This research was led by Woojin Kim, one of the team’s graduate students, who is first author on the paper.
Kim is also spearheading efforts to determine what aspects of A-beta promote aggregation, and the team has written several other papers on their findings. In addition, Hecht said the team plans to explore how well their selected compounds halt the accumulation of A-beta fibers in transgenic fruit flies.
While the team still has its original problem, it could be on the right track to a solution for another.
“Though we’re still having problems making strand-rich proteins that fold properly, these difficulties have led us to examine the problems that nature sometimes has with protein folding,” he said. “Those investigations led us to redirect our research to address the molecular causes of Alzheimer’s, a disease that is becoming increasingly prevalent as medical advances cure other diseases and enable more people to live longer.”
