Feature Article


The Milano mutation: A rare protein mutation offers new hope for heart disease patients

DOE/Lawrence Berkeley National Laboratory

A handful of villagers from this Italian town, Limone sul Garda, possess an extremely rare protein mutation that protects them from atherosclerosis. (photo: DeCou Archive, UCSC)

Researchers at the U.S. Department of Energy's Lawrence Berkeley National Laboratory have discovered the mechanism by which an extremely rare protein mutation shields people from cardiovascular disease. The discovery could lead to more potent drug therapies that target both cholesterol deposition and prevent future accumulation. Results were reported in the February 12, 2002, issue of the journal Biochemistry.

May 28, 2002--Lawrence Berkeley National Laboratory researchers have discovered the mechanism by which an extremely rare protein mutation shields people from cardiovascular disease. The mutation enables the protein to curb oxidation, a harmful process in which molecules with unpaired electrons, also called free radicals, scavenge electrons from healthy tissue. It's believed to play a role in such diverse diseases as Alzheimer's, osteoporosis, and a form of heart disease known as atherosclerosis.

In atherosclerosis, free radicals grab electrons from lipids that line artery walls, sparking an inflammatory response that paves the way for cholesterol deposition. The mutated protein, however, boasts an antioxidant in the form of a sulfur-based residue that mops up unpaired electrons and prevents them from triggering arterial inflammation, according to John K. Bielicki of Berkeley Lab's Life Sciences Division.

Bielicki's research solves a paradox that has puzzled the medical world since 1980, when a middle-aged Italian man was referred to Milan's Lipid Center with high blood triglyceride levels, a risk factor for heart disease. Further testing revealed the patient also possessed very low levels of high-density lipoprotein (HDL), a good cholesterol that exports excess cholesterol from coronary arteries. This process prevents plaque buildup that impedes blood flow and contributes to heart attacks.

Patients with low levels of HDL are susceptible to heart disease, yet the Italian exhibited no signs of pathology. This unlikely combination intrigued scientists, who determined that the patient and a few dozen people from his region possess a mutated form of apolipoprotein A-I protein.

This important protein, known as apoA-I, both manufactures HDL particles and is responsible for their structure. In the mutated form, dubbed apoA-I Milano because of its origin, one of the protein's amino acids is replaced with an amino acid cysteine that has a sulfhydryl group. Somehow, this tiny change enables a handful of Italians to possess low HDL levels and remain free of cardiovascular disease. But how?

In pursuit of the answer, most researchers have focused on the most common form of the protein. About 70 percent of proteins with the Milano mutation come in pairs: one protein attaches to another to form a dimeric complex. The key to this pairing is a disulfide bridge in which the sulfhydryl group from one protein links with the sulfhydryl from another. This pairing restricts HDL size and growth and has been attributed to the HDL deficiency observed in people who have the mutation.

But 30 percent of proteins with the Milano mutation don't form dimeric complexes. They remain unattached as monomeric complexes. In this solo configuration, the sulfhydryl isn't occupied in a disulfide bond. It's free, which enables it to partake in other reactions, says Bielicki. And one of these reactions is quenching ions with unpaired electrons. In other words, the free sulfhydryl form of the Milano mutation is a powerful antioxidant, and Bielicki had a hunch it played a role in the mutation's ability to fight cardiovascular disease.

In a laboratory setting, he compared the mutated protein with the normal apoA-I protein, and determined that only the monomeric form of the mutation protects lipids from oxidation. This confirmed Bielicki's hypothesis. In most people, free radicals can go unchecked as they grab electrons from lipids that line arterial walls. But for the less than 50 people lucky enough to possess the Milano mutation, the monomeric form, with its free sulfhydryl, mops up free radicals' unpaired electrons. This satisfies free radicals' need to scavenge electrons from arterial lipids and prevents a series of reactions that lead to cholesterol deposition.

"We identified a new activity associated with the Milano protein that suggests how it protects against heart disease," Bielicki says. "Next, we can use this knowledge to develop better therapies."

Simply stated, Bielicki believes a mutation found in a handful of Italians could add a powerful component to today's peptide-based cardiovascular disease therapies. Conventional apoA-I protein therapies remove cholesterol from arteries using HDL. Next-generation therapies, however, could couple this process with the antioxidant mechanism found in the mutation, creating a one-two punch that both cleans out cholesterol and prevents oxidation that leads to future rounds of deposition-"a long-term solution," says Bielicki.

So far, he has isolated the structural domain from the mutation that contains the functional cysteine residue. The next step is to include this cysteine in a therapy that homes in on heart disease. Fortunately, apoA-I already possesses this crucial targeting mechanism, providing a pathway on which researchers can model a pharmaceutical. It works like this: when an artery wall suffers oxidative damage and cholesterol deposition, its cells trigger the upregulation of a receptor called ABCA1. This receptor exports cholesterol from the cell. The apoA-I protein is specifically designed to sense this upregulation and attach itself to receptor sites.

This binding process signals the protein to manufacture the HDL that whisks cholesterol out of the arteries. The mutated protein also targets this receptor, which means its antioxidant powers concentrate where oxidation and cholesterol deposition occur. This ensures its ability to guard against the earliest stages of atherosclerosis.

The trick is to develop a simple pharmaceutical peptide that targets the upregulation of the ABCA1 receptor, exports cholesterol like the conventional protein, and fights arterial wall oxidation like the Milano mutation. It would work best where it's needed most, says Bielicki. And to underscore the effectiveness of such a therapy, he adds that more than 20 years after the discovery of the Milano mutation, its carriers remain free of cardiovascular disease.

"It has stood the test of time, promising new therapies to combat the nation's leading cause of death," Bielicki says.--by Dan Krotz

Media Contact: Dan Krotz, LBNL (510) 486-4019, dakrotz@lbl.gov
Technical contact: John Bielicki, LBNL Life Sciences Dept., (510) 495-2208, jkbielicki@lbl.gov


Related Web Links

"Apolipoprotein A-IMilano and Apolipoprotein A-Iparis exhibit an antioxidant activity distinct from that of wild-type Apolipoprotein A-I," John K. Bielicki and Michael N. Oda, Biochemistry, 2002, 41 (6), pp. 2089 -2096. [Full article: HTML or PDF 144KB]

"Genes Can Cause Coronary Artery Disease," Stephanie Trelogan, Genetic Health, September 12, 2000.

"Effects of apolipoprotein and low density lipoprotein receptor gene polymorphisms on lipid metabolism, and the lipid risk factors of coronary artery disease," Taina Korhonen, Department of Internal Medicine, Finland

Funding: The research was conducted at Lawrence Berkeley National Laboratory's Genome Sciences Department, which is supported by DOE Office of Science's Biological and Environmental Research division as well as grants from the National Institutes of Health and an American Heart Association Western Affiliate Postdoctoral Fellowship.

Lawrence Berkeley National Laboratory is a DOE Office of Science national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.

Author: Dan Krotz is a science writer in the Communications Department at Lawrence Berkeley National Laboratory. He covers the broad range of science at the Lab. Before joining LBNL, he covered science and technology for several news media organizations. For more science news, see Berkeley Lab's Science Beat.