It was 2010 and Jesse Rinehart’s first day as the head of his own lab and a faculty member at Yale’s West Campus when an email requested his presence at a meeting to discuss a new recruit—a scientist whom the Systems Biology Institute hoped to lure to West Campus to start a lab. As soon as Rinehart saw what Farren Isaacs, M.S., Ph.D., was working on, he became very excited. “I was like, ‘Oh, my god, I hope this guy comes,’ ” Rinehart says. Isaacs’ current work, he saw, held the solution to a problem that he faced in his own research.
Rinehart, M.S. ’99, Ph.D., ’04, associate professor of cellular and molecular physiology, studies how phosphorylation—the addition of a phosphate chemical group to proteins and other molecules—changes the way cells behave. Phosphorylation is critical for many cellular functions—along with dephosphorylation, it can turn enzymes and receptors “on” and “off”—and in humans there are an estimated 230,000 phosphorylation sites. Phosphorylation is the cell’s most common tool for regulating protein function and passing on signals. It may sound like the nitty-gritty, but it affects human health in a big way: phosphorylation defects can cause high blood pressure, the spread of cancer, and other medical problems.
At the time he started his job on West Campus, Rinehart had developed a bacterial system for adding phosphate groups to proteins to study their effects. But his system didn’t work very well in the bacteria that he was using. “We had invented this amazing manufacturing process that was going to change the world, but we didn’t have the right factory,” Rinehart says. “We were forced to make this in our garage with crappy materials and things that barely worked.” In Isaacs’ unpublished work, Rinehart saw that he had created a bacterial strain that would be the perfect manufacturing plant. “All we had to do was install our technology into this factory, and all of our problems would be solved,” Rinehart says.
That’s essentially what happened. As soon as Isaacs, now an associate professor of molecular, cellular, and developmental biology, accepted Yale’s offer and moved into the lab next door to Rinehart’s, they set to work building Rinehart’s phosphorylated proteins in Isaacs’ bacteria. “Not only did we have exciting, very complementary research programs, but we started at the same time, we were right next door to each other, we were similar in mind and spirit and scientific method—it was just a really, really good match,” Rinehart says. Now they are using their bacterial factories to develop treatments for glioblastoma. Specifically, they are making large amounts of a protein that becomes active when phosphorylated and causes brain tumors to spread. The researchers can then screen for drugs that would inhibit that protein and stop tumors in their tracks.
Changing the genetic code
Rinehart has sought in his research to understand what happens when a protein gets phosphorylated. “How does it change the properties of the proteins? Does it make them more active? Does it turn them off like a switch?” Of particular interest to him is phosphorylation of the amino acid serine, one of the 20 amino acids that make up proteins. Serine phosphorylation is the most frequently used signal in the cellular communications that control most physiological processes. The simplest way to study phosphorylation’s effects on protein function would be to make two versions of the protein, one phosphorylated, one not, and compare their activities. But there was no easy way to alter a protein’s phosphorylation status.
Given what we know about the genetic code, however, it was fairly easy to alter a protein’s amino acid sequence: genes, made of DNA, encode proteins, and, on a smaller scale, three-letter stretches of DNA called codons code for amino acids. To find out how one amino acid affects a protein, scientists can add or delete its codon. Rinehart used this approach to figure out a way to study phosphorylation in bacteria. Only to do it, he had to expand the genetic code to include a new amino acid that was already phosphorylated, plus its corresponding codon. With an expanded genetic code that included phosphorylated serine, or phosphoserine, Rinehart reasoned, he could program a phosphate group into a protein by adding its codon into that protein’s gene.
Putting this approach into practice was not without its hurdles. All the DNA codons already encoded other things—amino acids or stop signals, which tell the cell’s protein production machinery to release the finished protein. Luckily for Rinehart, the genetic code is redundant: more than one codon represents each amino acid, and the same goes for the stop signal. Rinehart used a stop codon called the amber stop codon to encode phosphoserine. The bacterial cell would still have other stop codons that could signal “stop.”
Rinehart also had difficulties at the protein production level. To make a protein, a gene—essentially a series of codons—is copied into a messenger RNA. The messenger RNA, also a collection of codons, travels to the cell’s protein production machine, where transfer RNA matches the codons and amino acids in a process called translation.
To make sure that the amber stop codon would encode phosphoserine, Rinehart needed transfer RNAs that were attached to phosphoserine and would recognize the codon. While working on his doctoral dissertation, he worked closely with a team that discovered a protein that attaches phosphoserine to transfer RNAs, and by 2011, he had developed a translation system that inserted phosphoserine wherever the amber stop codon appeared. Sounds great, right? But exciting as it was, Rinehart says, there was also “a major, major problem.”
The bacterial cell had competing systems that translated the amber stop codon in different ways. The bacterial cell recognized it as a signal to stop translating and release the finished protein; Rinehart’s transfer RNAs recognized it as a signal to insert phosphoserine. That competition rendered the technology weak, Rinehart says. “We could make the proteins we wanted, but there were very low levels; it was almost trace amounts.”
Rinehart was attacking that problem when Isaacs arrived with a bacterium he had created that solved the problem. In his bacterial strain, Isaacs had replaced the amber stop codon with another stop codon throughout the entire genome and deleted the factor that recognized it as meaning “stop.” The amber stop codon took on a new meaning—not “stop,” but “insert phosphoserine.”
“We could easily take our genetically recoded organisms and his phosphoserine system and bring them together, and start to produce custom-designed phosphorylated amino acids, phosphorylated proteins,” Isaacs says. As they reported in a 2013 paper published in Science, the system worked well: in their bacteria, wherever the amber stop codon occurred in RNA, the cell translated it as phosphoserine in the resulting protein. This system would later allow them to study how phosphorylation of a specific protein causes brain cancer to spread, and to look for drugs that, by blocking phosphorylation, may be able to stop the cancer’s progress.
Rinehart and Isaacs decided to study a kinase, a protein that, when active causes brain tumors to spread. And it becomes active, they found, when it is phosphorylated. Their approach appears counterintuitive. Rather than inhibiting phosphorylation, they first used their bacterial factories to generate large amounts of the phosphorylated active kinase. Then they screened for drugs that would inhibit not only that kinase, but also, they hoped, cancer’s spread. They’ve found some candidate treatments that prevent the cancers from migrating in a tissue-culture dish. Now they are testing these candidate drugs in mice implanted with human brain tumors. They hope that in the future, such a compound could prolong human lives.
“It’s a great collaboration,” Isaacs says. “We have developed a great environment to do this work that really lies at the interface of multiple disciplines, and I think that’s really allowed us to sort of do things and achieve things in our science that independently we wouldn’t have been able to do. And that’s precisely what science is about and what collaboration in science is about.”