mapping disease
by Marla Vacek

rom cystic fibrosis to space rats, proteomics is allowing researchers to uncover the secrets of disease.

Genome, transcriptome, and proteome are all fancy terms used by scientists to describe the separate populations of molecules that contribute to our beings, determining who we are. What color eyes we have, how fast we can run, if we are going to have diabetes — it's all in our genes. With the completion of the human genome project came the hope that the molecular links to what makes us tick would emerge from the three billion As, Ts, Cs, and Gs that constitute our DNA. With this promise still unfulfilled, many scientists are turning to proteomics to study the products of our genes — proteins — and how they change structure, interact with each other, and give rise to disease.

According to Lee Graves, associate professor of pharmacology, proteomics is where the action is. "Proteins do everything," Graves says. "They catalyze the metabolic functions of the cell, they break your glucose down to give you energy, they transmit signals from the outside of the cell to the inside of the cell." While DNA is a static store of information, proteins are constantly changing, undergoing modifications, being depleted and degraded, acting differently in different tissues. Even though this complexity makes proteomics more challenging to study than genomics, it also confers the potential to greatly increase the understanding of human disease. Proteomics can be used to discover proteins that are associated with diseases and determine which of these can serve as novel targets for drug development or as biological markers of human disorders.

.: Lee Graves: "Proteomics provides a net to filter thousands of proteins to find just a handful that might make useful candidates for drug targets or biomarkers. There is a fair amount of fishing in science. You just have to have the right net. Photo by Steve Exum; click to enlarge. :.

roteomics enables scientists to study how proteins function in healthy cells as well as what goes wrong when disease strikes. Cystic fibrosis (CF), a common hereditary disease affecting approximately 30,000 children and adults in the United States, is just one of the many disorders that can potentially benefit from proteomics research. Even the identification of the cystic fibrosis gene over a dozen years ago has not led to an effective treatment. Although CF is caused by a defect in just one protein, known as cystic fibrosis transmembrane regulator (CFTR), it creates a variety of symptoms including faulty digestion due to a deficiency of pancreatic enzymes, difficulty in breathing due to mucus accumulation in airways, and excessive loss of salt in the sweat — all caused by one faulty protein.

"To try to understand why a protein works differently in a sweat duct than in an airway, we need to know all the partners, all the other proteins," says Richard Boucher, director of the Cystic Fibrosis Center. According to Boucher, CF is caused not only by the absence of the CF protein's function, but also by the absence of crucial interactions with other proteins in the cell. "There are ways of looking at those tissues and essentially identifying and cataloguing all the proteins in the tissues and then asking which ones are involved in CFTR," he says. Boucher is using proteomics to identify the various parts and determine how they interact with the CF protein to form a functional cell. "Once we know how the system is wired together — that is, what wires or connections are missing because of the missing CFTR protein — then we can reconnect the system or jumpstart it with drugs," Boucher says.

Muscle atrophy is another disorder that proteomics can help us understand. Diabetes, aging, and dystrophies such as muscular dystrophy all result in muscle wasting. Scientists at NASA are working with Graves to study the muscle atrophy experienced by astronauts after extended missions in space. "We are collaborating with a NASA group that has an animal model system to mimic weightlessness, and what we are trying to do is to apply modern methods of proteomics and mass spectrometry to analyze the changes," Graves says. One of the well-observed responses to atrophy is a change in protein expression. By comparing protein levels in normal and atrophic muscle, scientists can profile these differences and determine which proteins are involved in muscle wasting. Tom Hilder, a graduate student in pharmacology, and Jun Han, a postdoctoral fellow, are both working in Graves' lab to identify the important proteins so that therapies can be developed to hinder atrophy.

n addition to the discovery of novel drug targets, proteomics can also be used to identify biomarkers, which are specific profiles that indicate the severity of disease. "For cystic fibrosis, we really need to know how bad the infection and destruction is in the lung at any given time," Boucher says. The current methods to assess the severity of disease — symptoms, chest X rays, and blood tests — are not very helpful, especially in children. Finding markers for lung inflammation and infection would help researchers decide when to initiate therapy and would help in clinical trials of new therapies. Margaret Leigh, professor of pediatrics, is collaborating with Boucher to locate these biomarkers by collecting one to two hundred serum samples from CF patients and then comparing the data with those from normal subjects. Using large 2-D gels and bioinformatics, Boucher can identify which of the 12,000 serum proteins vary between normal subjects and people with CF and of these, which ones increase or decrease with the severity of lung disease.

These biomarkers can be a great tool for pediatricians treating CF babies, who are born with normal lungs at birth and do not get their first infection until somewhere in the first few years of life. Pediatricians can use biomarkers to detect when that first infection occurs and initiate treatment with antibiotics to irradicate the infection before it begins to damage the lungs. The same biomarkers that track the severity of infection and inflammation of the lung can then be used to determine if a new drug ameliorates the patient's condition. According to Boucher, this approach has already produced results with other disorders. "I think that there have been great successes with biomarkers and using proteomics approaches, predominately in cancer."

Graves is working with other scientists to apply proteomics to cancer research. Carolina's Lineberger Comprehensive Cancer Center has breast, prostate, and colorectal cancer projects in the works. "We can profile, or look at a patient's sample, and say that the expression of this protein correlates with metastatic breast cancer or prostate cancer," Graves says. "If we screen out these different patients, we can find something that is now expressed more highly in people that are showing advanced cancer versus those with early cancer or no cancer at all. As a pharmacologist or a biochemist, we can start to design drugs to attack the problem at the molecular level."

Although proteomics entails fishing through thousands of proteins to find just a handful of useful candidates for drug targets or biomarkers, Graves doesn't mind a bit. "There is a fair amount of fishing in science," he says. "You just have to have the right net to make sure you catch something at the end of the day."

Marla Vacek is a Ph.D. candidate in genetics and molecular biology at Carolina.