© BRIAN COATS FOR UT SOUTHWESTERN MEDICAL CENTERBruce Beutler published his first scientific paper at the age of 16. As an apprentice in his father’s lab at the City of Hope National Medical Center in Duarte, California, Beutler learned how to purify proteins and assay their activity—work that led to a pair of publications on the enzyme glutathione peroxidase, including that first one in the Annals of Human Genetics in 1974.
“I don’t think there were any other students in my high school who were spending their afternoons and weekends that way,” laughs Beutler. “But in those days I was very ambitious.” Graduating from the University of California, San Diego, when he was 18, Beutler went on to obtain a medical degree from the University of Chicago. “That was something my father advised me to do. He said, ‘If you go to medical school, you’ll learn all about the workings of one specific organism.’” This knowledge, Ernest Beutler told his son, “will be extremely useful to you, no matter what you decide to do in biology.”
“He also said, ‘If you don’t do well, you’ll have something to fall back on. You can always see patients.’” But for the young Beutler, that possibility seemed remote. “It never occurred to me that I might fail. Such thinking seemed counterproductive. If you allow for the possibility of failure, I think you’re less likely to succeed.”
That indefatigable zeal kept Beutler on track as he labored to unravel the mechanisms by which mammals detect and eliminate infection—work that would earn him a share of the 2011 Nobel Prize in Physiology or Medicine. Here he talks about admiring a mutation, constructing a catwalk, and making his family proud.
“I think it’s foolish for people to believe that all the easy things have been discovered.”
Beutler breaks out
The call of the lab. After completing his internship and residency at The University of Texas (UT) Southwestern Medical Center, Beutler “missed having the opportunity to make real discoveries.” So in 1983, he became a postdoc in the laboratory of Anthony Cerami at Rockefeller University. Cerami was studying cachexia, a form of wasting that often accompanies cancer or serious infections. In the latter case, the presence of a microbial molecule—such as the lipopolysaccharide (LPS) that coats the outer membrane of many bacteria—was thought to trigger the release of some yet-to-be-discovered factor that prevents host tissues from storing energy. “I was chomping at the bit to purify proteins again, so I energetically set about finding the molecule responsible.”
Double duty. The protein Beutler wound up isolating from mice, dubbed cachectin, shut down fat cells’ ability to take up triglycerides. But that wasn’t all it could do. “Cachectin turned out to be the mouse ortholog of human tumor necrosis factor [TNF], which had itself been recently isolated,” he says. So cachectin could also kill tumor cells. “For the first day or so I was actually disappointed, because some of the novelty was compromised. But then I realized, we now knew a great deal about the activities of TNF that no one else knew”—including the fact that it was an extremely toxic protein. “Those who had been working with TNF had been treating it as innocuous, and as a potential chemotherapy agent.” But Beutler found that mice injected with cachectin, now known as TNF, would die of shock within hours—and that blocking this protein with antibodies protected them from this form of toxic shock. The papers outlining these discoveries—which included a handful in Science and Nature throughout 1985 and 1986—“changed the whole thinking on TNF.”
Thwarting TNF. Beutler continued to pursue TNF when he left Rockefeller to start his own lab at UT Southwestern in 1986. His first order of business was devising a way to block the molecule’s actions. The plan was to sop up circulating TNF using its receptor as the sponge. The receptor had been cloned in another laboratory, and when the paper came out, Beutler says, “we more or less pounced on it.” He and his trainees, graduate student David Crawford and postdoc Karsten Peppel, attached the TNF-binding domain of the receptor to a hunk of antibody molecule, generating a large fusion protein that would be difficult for the body to eliminate. “It worked like a charm,” says Beutler. “The hybrid had a long half-life in vivo and was effective at neutralizing TNF.” That chimeric receptor, described in the Journal of Experimental Medicine in 1991, is now used to treat autoimmune disorders that stem from an inappropriate generation of TNF, such as rheumatoid arthritis and psoriasis.
Swimming upstream. Why TNF is produced in autoimmunity, Beutler says, “nobody really knows. That’s one of the big mysteries.” Could it be that the presence of a microbial component like LPS triggers an inflammatory response that then goes awry? “I thought if we could find the LPS receptor, we’d have a handle on the problem—and we’d learn how the body ‘knows’ it’s infected. It was that work that led to the Nobel Prize.” But it didn’t lead there quickly. “First I tried biochemistry. It was known that there were mice that were resistant to LPS”—animals that did not mount an immune response when exposed to this bacterial product. “It was speculated that these animals must have something wrong with their LPS receptor. So I tried to look for the receptor by comparing the membrane proteins of those mice to a control strain that was sensitive to LPS. But that got me absolutely nowhere.” Three years and several different approaches later, Beutler was no closer.
“Then I decided to try positional cloning”—an approach that narrows down the location of a gene by determining which DNA markers tend to be inherited along with a given trait. At the time, the gene was “believed to be within an area that constituted about an eighth of chromosome 4.” With their first round of matings—which produced about 500 mice—they whittled that down to one-sixteenth of chromosome 4. “So we didn’t make a huge amount of progress.” Another series of matings—and another 2,000 mice—brought it down to about 2.6 million base pairs. “That was still an absolutely enormous area. But we couldn’t seem to reduce it any more than that.” So they started sequencing. “Our days consisted of setting up mini preps in the afternoon and then looking at ladders of bases on X-ray films the next morning. My assistant Betsy Layton would read the sequence to me and I would type. Then we would exchange duties. For 3 years, that was how it went.”
Red herrings & the Big Kahuna. Occasionally, those sequences would yield something that looked promising. “We might find a gene that appeared to exist in many different splice forms and think, ‘Could this be the basis of receptor diversity in the innate immune system, and maybe it’s the splice form that codes for the LPS receptor that is defective in these mice?’” Then they would sequence the corresponding cDNAs from both resistant and sensitive mice and find no difference. “And our hopes would be dashed. It wasn’t until we’d covered more than 90 percent of the region that we found what we were looking for”—thanks to the rise of genomics.
About midway through the project, geneticists had begun to catalog expressed sequence tags (ESTs)—short fragments of cDNAs that correspond to active genes. Every afternoon, Beutler’s crew would search the EST databases to see if any of their chromosome 4 sequences matched those of an expressed gene. “One night I was home, going over the results, and I saw a very strong hit.” One of the team’s sequences matched a gene that encoded a receptor called TLR4. But was this really the gene? TLR4 appeared to have some homology with another receptor involved in inflammation—one that binds IL-1—as well as with a protein called Toll that Jules Hoffmann had linked to immunity in Drosophila. “That encouraged me,” says Beutler. “But perhaps not as much as the fact that we had covered 90 percent of the area and we were running out of DNA. I thought: this must be the gene because we’re almost out of places to look.” It wasn’t until they saw a mutation—a single base-pair change in the gene from the LPS-resistant mice—that they knew they’d found the LPS receptor. “I kept coming back to look at the computer, thinking it might just be a mistake or that it was my imagination,” says Beutler. Postdoc Alexander Poltorak, the lead author on the resulting 1998 Science paper, had a similar reaction. “Both of us would come back to admire the mutation two or three times a day.”
The screen team. “Since then,” Beutler says, “I have been absolutely in love with genetics!” Now back at UT Southwestern, after spending 11 years at the Scripps Research Institute, Beutler continues to make mutant mice and confer with colleagues to devise new screens for identifying intriguing phenotypes, especially those that pertain to an ability to respond to infection. “It’s really wonderful to know that when you come to the lab in the morning, it’s likely that somebody is going to have uncovered a bizarre new phenotype—and that it’s going to be relatively easy to figure out what gene has been hit.” In about a third of the cases, he says, they discover something exciting. “This is a very effective way to find the unexpected, to see things that have maybe never been seen before.”
California dreamin’. “I remember having dreams about the [LPS receptor] gene. One time, I was in a hotel in the mountains in San Bernardino and I woke up in middle of the night suddenly convinced that I knew exactly what the gene was. I wrote it down on some paper at the bedside and fell back into a very satisfied sleep. When I woke up in the morning, I found it was absolute gibberish—some nonsensical enzyme that gave no insight at all. Of course, in the dream it made perfect sense.”
Catching the fever. Through all the ups and downs, Beutler stuck with the search for the LPS receptor. “It was pure obsession—a deep, visceral desire to find the gene. It was a kind of addiction. You might compare it to gold mining. People became addicted to that. They caught the fever and couldn’t stop. Because every day might bring the mother lode. I had this one goal and I always felt like success was just around the corner. It was so tantalizing. Although it was stressful and a lot of work, that was the most intense and exciting time of my career.”
If we could do then . . . “If we had to solve the problem of why this particular mouse strain does not detect LPS starting today, we would know the answer in about a week. We would just sequence the whole genome of the resistant strain and compare it to the reference sequence or to the sensitive strain. Massively parallel sequencing has changed the game completely.”
Don’t sweat the big things. “I tell trainees to try not to be deterred by the difficult funding situation. Because it’s always been difficult. And even though it seems like there’s so much more competition now, with advances in technology there really are more things to work on. I think it’s foolish for people to believe that all the easy things have been discovered, or that we’ve reached the end of what we can know. I see science as providing more opportunities than ever before. And I think it will only continue to accelerate.”
Capitol connection. In Stockholm, Beutler addressed a group of parliamentarians that meets with invited investigators to discuss issues of scientific importance. On his return, Beutler happened to mention the meeting to Texas Senator Kay Bailey Hutchison, who then organized a similar session for a handful of senators and Nobelists. Such high-powered powwows might be one way to influence policy. “If scientists remain mute, then we ought not to complain when legislation doesn’t go our way.”
R-E-S-P-E-C-T. “I went through some rough times with my sons in their adolescence. In those days, they thought I was sort of a nerd and they couldn’t understand why I was doing what I was doing. But when I won the Nobel Prize, they were so proud. I think that’s the greatest thing about the Nobel Prize. It’s sort of like a prize for them, too.”
Beutler finds balance
Home sweet home. Beutler worked with a builder to design his dream house in Dallas. “It’s kind of Tuscan, with a Southwestern feel”—and a catwalk that looks down over the living room. “I spend a good deal of time up there just musing.”
For the birds. “I used to be an avid birder. When I was in San Diego, I liked to go to the San Gabriel Mountains or sometimes to the desert or the seashore. But I’m happy to look for birds anywhere. I like being close to nature and seeing things I haven’t seen before.”
Embracing Bach. Beutler fell for Bach at the age of 15. “I went to a performance of the St. Matthew Passion, and I remember getting chills. I’d never heard a great choral masterpiece performed live before. I believe we evolved an ability to recognize the emotional quality of the human voice, and that explains much of our appreciation of music.” He even tried his hand at writing his own fugues. “I wanted to feel what was so special about creating music in the style of Bach. Natural scientists discover things—but Bach invented them. It’s good to strive to be as inventive as one can be—particularly in science.”
• Discovered that tumor necrosis factor (TNF) mediates endotoxic shock, and turned its receptor into a therapeutic agent that eradicates TNF activity.
• Using a classical genetic approach, identified the receptor that recognizes the microbial molecule lipopolysaccharide (LPS). Demonstrated that this LPS receptor, called TLR4, is a key player in innate immunity, as it alerts the body to a broad range of bacterial infections. These efforts were recognized by a 2011 Nobel Prize.
• Launched a program to generate and study hundreds of informative mouse mutants, many of which show defects in immune function. These mice are fostering the discovery of molecules involved in immune signaling.