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    California Pacific Currents 2004

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    Capturing Cell Magic in Slow Motion: Vishu Lingappa’s Search for Radically New Therapies

    Just as most people relish a well-told story, scientists and physicians crave an elegant explanation of how things work. Biologists derive great satisfaction in the DNA-makes-RNA-makes-protein mantra of molecular biology. Clinicians welcome a therapy with a proven mechanism of action and data from double blind clinical trials. The facts are intellectually satisfying and useful in day-to-day research and practice.

    But facts change. On closer inspection, black and white becomes a speckled grid of grays. New insights often reveal a complexity that, if not explored fully, can limit progress in basic science and medicine.

    “We really don’t understand how many things work,” says Dr. Vishwanath (Vishu) Lingappa, MD, PhD. “Medical folks tend to be very skittish when you talk about uncertainty. Yet the truth is that many diseases are not satisfactorily treatable because we fundamentally still don’t understand the underlying biology.” In his new role as Senior Scientist at the California Pacific Medical Center Research Institute, Dr. Lingappa intends to investigate this realm of uncertainty. Based on his 20 years as a teacher, physician, and researcher—including close collaborations with two Nobel Prize winners—and on his recent groundbreaking work with proteins and viruses, he has now described several previously hidden cell mechanisms and is positioning himself to translate these insights into new tools to diagnose and treat disease.

    Cell Biology in Slow Motion
    In one of Dr. Lingappa’s most profound recent discoveries, he and his colleagues have identified new pathways in a key phase in the life cycle of viruses. Viruses are infectious agents against which there are very few effective drugs. Each virus must house its genes in a simple protective shell—called a capsid—before it leaves the human host cell to infect other cells. This capsid “spacesuit” is made of proteins.

    “The textbooks still say capsids are formed via self-assembly,” says Dr. Lingappa. “In other words, the proteins are made and then—poof—they assemble themselves into the capsid. Well, when we used our system to slow down capsid formation in the test tube, we saw that it’s not self-assembly at all. There are energy-dependent steps and steps dependent on host proteins, all playing a role. These constitute brand new biochemical pathways that appear to occur in all viruses but with distinctive variations in each viral family. We believe they will prove to be new targets for antiviral therapy.”

    Dr. Lingappa and his collaborators have outlined about a half dozen discrete steps that are necessary to form a capsid, and the team has demonstrated such pathways in three unrelated families of viruses. Every step, Dr. Lingappa says, is a new target for therapy.

    “The history of pharmacology tells us that anytime you have a new pathway, you can find drugs that block that pathway,” he says. “This is a program with concrete clinical applications where the need is dire.” According to Dr. Lingappa, virologists are still somewhat taken aback by this new information. However, he sees a potential sea change in our thinking about viruses and, citing the current paucity of anti-viral drugs, he goes on to predict that his group’s work may have applications for an array of viral diseases, including those caused by the human immunodeficiency virus (HIV), hepatitis B and C, and Ebola, not to mention the common cold. To help convert his insights into new drugs, he has formed Prosetta Corporation, an early-stage biotechnology company.

    A Fast Elevator
    Dr. Lingappa was born in Lafayette, Indiana, and grew up in Worcester, Massachusetts. Both his parents were microbiologists, making a science career seem perfectly reasonable. But when his family took a whole year off to travel the world, it exposed him to the human condition and made medicine seem an even more useful
    calling. In 1975, he headed for medical school.

    The mysteries of the cell, however, still beckoned. In fact, the special tricks of the test tube and the habits of thought that later allowed Dr. Lingappa to slow down the viral capsid assembly were learned beginning on his very first day at Weill Medical College of Cornell University in New York City. On that day, he skipped class, ran across the street to Rockefeller University, snuck past a guard, and dropped in unannounced on a relatively unknown assistant professor named Günter Blobel. The two hit it off. The out-of-breath scholar had found a mentor and in a matter of months had started on a PhD track. (In fact, all three Lingappa siblings eventually became MD-PhDs.) He soon found himself reading the then unpublished manuscript introducing the “signal hypothesis,” a piece of breakthrough thinking for which Blobel won
    the 1999 Nobel Prize.

    “It was exhilarating,” recalls Dr. Lingappa. “I felt as if I had walked into an elevator and someone had pushed the button to the top floor. There I was, a first-year medical student, exposed to this new view of cells and using a system that would slow it all down in a test tube.”

    A brief aside about the signal hypothesis itself is in order here. Not only did it shape Dr. Lingappa’s early scientific interests, but it also supports his still-evolving vision that gene expression must be conceptualized as a multi-dimensional process. It involves protein shape (the traditional three dimensions, determined by how the linear chain of amino acids folds itself into a very specific configuration); protein time (the concept that proteins require time and intermediate steps to fold and combine into functional units, as documented in his slow-motion viral capsid studies); and protein place (the notion that the same protein in different locations can carry out different functions).

    The signal hypothesis solved a long-standing riddle about protein place. Specifically, it explained how the billions of proteins crowded inside each cell are transported from where they are manufactured to the exact location where they are supposed to be—for example, to one of the cell’s many specialized subcompartments, or to the cell’s exterior membrane, or perhaps secreted outside the cell. Each protein, it turns out, contains a special “zip code” section that governs its transport across or into various fatty membranes to the intended location. This sophisticated address tag system is found in animals, plants, and yeast. It is one of those ultra-elegant biological systems that can make grown scientists weep.

    The technical breakthrough that paved the way for the signal hypothesis involved the development of a cell-free system that creates (slowly) in test tubes what normally occurs in cells with blinding speed and is shrouded by the complex and crowded in vivo environment. “It’s like watching a slow-motion video of a master magician who just pulled a rabbit out of a hat,” says Dr. Lingappa. “We can use this system to detect all manner of events previously not appreciated in biology, some of which have important implications for medicine.”

    New Twists in Protein Folding
    For the past two decades in his research at the University of California, San Francisco, Dr. Lingappa has continued refining and applying this special approach to cell analysis. In addition to his ongoing efforts to characterize capsid assembly for HIV and hepatitis B, he has turned his slow-motion camera to the biosynthesis and folding of proteins involved in cardiovascular and neurodegenerative diseases.

    In the late 1980s and early 1990s, Dr. Lingappa collaborated with Dr. Stanley Prusiner on seminal experiments leading to the discovery of prions—a discovery for which Dr. Prusiner was awarded the 1997 Nobel Prize. Normal prions are proteins found in the brain and the immune system. Dr. Prusiner’s group showed that prions sometimes fold into a rare shape; that is, the linear chain of amino acids that bend and twist into the “normal” shape finds itself twisted into an odd position. With prions, this unusual folding position causes problems and even starts a “chain reaction” of prion formation that is, essentially, infectious. The most notorious diseases involving infectious prions are mad cow disease, scrapie in sheep, and kuru and new variant Creutzfeld-Jacob disease in humans. While Dr. Lingappa’s previous work contributed to an understanding of why prions kill neurons, his recent studies suggest that the principles of protein folding involved in prion biology are actually more general than commonly appreciated.

    In fact, for Dr. Lingappa, protein folding is about much more than prions. Instead, it has become a natural extension of his continuing study of the unconventional dimensions of gene expression. Until recently, most scientists and clinicians still considered the final shape of any protein as predetermined by the linear sequence of 20 amino acid building blocks and, in turn, the linear sequences of four nucleic acids spelled out on the gene. The final shape of the protein, it was commonly thought, was predetermined in the gene sequence in the same way that a building’s final shape was inherent in its architect-drawn blueprint. As any building contractor can tell you, though, there is a maze of building codes, nosy neighbors, excavation surprises, and homeowner whimsies that alter the final look of a home on its journey from the drawing board. Only in recent years have molecular biologists started to notice a similar gamut of local cell rules and special environmental circumstances that seem capable of altering the final shape of a protein. Early prion researchers were among the first to show that a string of amino acids could fold up in more than one way and become infectious.

    Now Dr. Lingappa and his new colleagues at California Pacific are putting forward the startling idea that “alternative protein folding” is actually common—albeit hard to detect—in many cells. They call this proposal (that proteins have many possible folded states) the bioconformatic hypothesis.

    “The prion is not the only protein that has different folding forms,” he says. “It’s just that the properties of prions are so dramatic. An infectious protein? It hits you over the head. Also, these protein-folding changes were noticed in neurodegenerative disorders first because you don’t have to knock out that many neurons before you see that something is wrong with a person. Whereas, even if you lost over 50% of the function of organs such as your pancreas, kidneys, or liver, you’re still doing fine. But now we’ve gone back and looked at proteins involved in diabetes, cancer, obesity, and osteoporosis and we’ve seen that many of the same principles apply.”

    According to Dr. Lingappa, many cells have the ability to fold up specific proteins in diverse shapes and therefore access diverse functions depending on circumstances. Once again, his group has used their slow-motion technique to search for the alternatively folded proteins as well as the cell enzymes and factors that regulate protein folding. The results are preliminary but provocative. He speculates, for example, that some alternative protein forms may be useful in an organism’s early development or under certain cell conditions like stress. As an adult, however, these vestigial protein shapes are not needed and may in fact take on harmful roles, as they apparently do in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and perhaps even amyotrophic lateral sclerosis (ALS).

    An Exciting New Era at California Pacific
    With the recent move to the Research Institute, Dr. Lingappa has cut back on his teaching and clinical practice and now devotes even more time to research on viral capsids and alternative protein folding. He says that the leadership of Scientific Director Dr. Warren Browner—who was his resident during his internship year at UCSF back in 1980—was a major factor in his decision to join the California Pacific team. “I’ve known Warren for decades and he’s someone I’ve always admired, respected, and trusted,” says Dr. Lingappa.

    Already, he is thriving in the new environment and sees potential for many collaborations with his new colleagues. “We believe the concepts of protein bioconformatics are very relevant in ALS, for example,” he says, “and California Pacific has a wonderful ALS research and clinical community.”

    In fact, by pursuing his broader-than-usual definition of protein structure and function, Dr. Lingappa is building a paradigm of cell biology that spans medical specialties. The potential clinical applications include neurodegenerative diseases, cardiovascular and pulmonary disease, viral infections, diabetes, and cancer. He is satisfied, for now, to explore these disease-specific areas one by one before asking the scientific community to accept any grand new rule that ties together the disparate data into an elegant theory of protein bioconformatics.

    In the meantime, Dr. Lingappa will do experiments and collect data. And, as he has always counseled medical students in his physiology classes and in his unusually meditative textbook Physiological Medicine (McGraw-Hill, 2000), he will listen to his patients. “Instead of running away from uncertainty, one can embrace it and use it to great therapeutic advantage in clinical practice,” he says. “Every patient with a chronic disease is an expert of sorts because they live with the disease 24/7. Patient-specific symptoms teach us something important about disease heterogeneity—how it can manifest differently in different individuals. The uncertainty caused by our limits of medical knowledge doesn’t have to be scary or demoralizing to either the patient or the clinician. It should make the clinician more cautious—remember ‘first, do no harm’—humble, and attentive to the patient’s views and experience. It should empower patients to note and share their experiences, leading to a much more collaborative and engaged relationship with their doctors, in which each contributes to understanding that individual’s medical jigsaw puzzle, in order to treat the disease in that patient more effectively.”

    In the years to come, Dr. Lingappa will continue wrestling with uncertainty and writing his story of how things work deep within cells. Someday soon—or perhaps two decades from now—his descriptions may strike a new chord of scientific understanding among his fellow researchers and clinicians and bring fresh hope to patients with chronic diseases.