A clear solution to a protein puzzle

An enzyme’s structure plus oil and water may yield a clue to Alzheimer’s disease.

Several years ago, when researchers discovered intramembrane proteases—a class of hydrophilic enzymes that seemed to work despite being surrounded by cells’ oily membranes—many scientists were perplexed. Some were downright skeptical. After all, oil and water don’t mix.

Ya Ha, Ph.D., assistant professor of pharmacology, and colleagues may have revealed the intramembrane proteases’ recipe for success with their publication in October in the journal Nature of the protein’s structure. In addition to providing a solution to a slippery biological mystery, Ha’s work could shed light on the mechanisms underlying Alzheimer’s disease.

The chewing up of proteins, a process known as proteolysis, is the job of proteases. But protein-splitting reactions require water, not normally found in the greasy interior of cell membranes. In 1997, Nobel laureates Joseph L. Goldstein, Ph.D., and Michael S. Brown, Ph.D., suggested in an article in Cell that a protease involved in regulating cholesterol somehow did its work within the cell membrane. This protease must be “unusual,” they acknowledged, but they proposed that gamma-secretase, the enzyme that cleaves amyloid protein into the toxic fragments seen in the brains of Alzheimer’s patients, might also operate in the same manner.

When he came to the School of Medicine from Harvard University five years ago, structural biologist Ha was convinced that gaining structural information through X-ray crystallography was the key to understanding intramembrane proteases. He started with gamma-secretase. But despite his best efforts, it could not be coaxed into forming crystals, the first step in determining a protein’s molecular structure by X-ray crystallography.

When a family of bacterial enzymes with similar activity known as rhomboid proteins was discovered in 2001, Ha seized on those as an alternative. Over the next four years, Ha worked with postdocs Yongcheng Wang, Ph.D., and Yingjiu Zhang, Ph.D., to obtain the first X-ray data of the rhomboid enzyme molecules and created a visualization of the structure.

At last, they saw how an intramembrane enzyme is built: in the middle of a sea of fat, the rhomboid protease creates a protective bubble to shelter water molecules (whose source is unknown; they may come from a surrounding aqueous solution) in its active site. The enzyme is serpentine, crisscrossing the cell membrane six times. Five of these segments bundle together to create a water-filled chamber within the membrane.

However improbable this enzyme’s mechanics, they are medically important because the enzyme belongs to the family that includes human gamma-secretase. “Compounds that inhibit the production of toxic amyloid peptides are now believed to be one of the most promising approaches to the development of drugs for Alzheimer’s disease,” says Vincent T. Marchesi, M.D., Ph.D., the Anthony N. Brady Professor of Pathology and an expert on both membrane protein structure and Alzheimer’s disease. “Ha’s findings are an important contribution to this effort.”

Ha says that the rhomboid protease is a good model system for intramembrane proteases in general, but he confesses that he still has his eye on gamma-secretase. “I would love to see it,” he says. While the two enzymes are not related by their protein sequence or by evolution, Ha believes that they share common features because they face the same challenge of mixing water with oil. “Once you have a few structures, you’ll see a pattern start to emerge,” Ha explains. “That will give us a better understanding of how inhibitors might work. And then maybe we can design better inhibitors, and maybe those inhibitors can be used as drugs.”

—Pat McCaffrey

Yale scientist finds two genetic anomalies linked to macular degeneration

In late 2004, Josephine J. Hoh, Ph.D., associate professor of epidemiology and of ophthalmology and visual science, used new DNA chip technology to identify a gene for age-related macular degeneration (AMD). She found a single nucleotide polymorphism (SNP)—in this case a variant in the coding region of complement factor H (CFH) gene on chromosome 1—that is linked to an increased risk of developing both wet and dry forms of AMD.

Now, in findings published in the journal Science in October, Hoh has reported finding an independent SNP in a section of chromosome 10 that regulates the HTRA1 gene. The variant appears to increase the expression of the protein HTRA1 in the eyes of homozygotes, and people carrying two copies of the risk allele have a much higher risk of wet AMD.

“We found that patients with the HTRA1 SNP in both chromosomes were 10 times more likely to have wet AMD than those who have it in just one chromosome or no SNP,” said Hoh. “This may point to possible directions for effective treatment of wet AMD.”

AMD causes light-sensitive cells in the retina to break down, resulting in progressive loss of central vision. Of the two forms of AMD, the dry form is more common than the wet form. Wet macular degeneration can lead rapidly to blindness, while the dry form of AMD progresses more slowly.

The discovery marks the first time that a cross-ethnic approach was used to efficiently decipher a complex disease such as AMD. Hoh exploited differences in the progression of AMD in Caucasian and Chinese patients. In Caucasians, the proliferation of abnormal blood vessels is compounded by the development of large waste deposits called drusen. Chinese patients, she said, develop little or no drusen and progress directly to wet AMD.

Using DNA samples from Chinese patients with AMD, Hoh discovered the disease-causing variant in the control section of the HTRA1 gene. She then hypothesized that this same genetic variant causes wet AMD in Caucasians. “Finding a gold mine is very much easier if you have a good map showing where to dig. The discovery in the Hong Kong Chinese samples gave me the map,” she said. The confirmation of her hypothesis that the HTRA1 variant causes wet AMD in Caucasians was done in collaboration with Kang Zhang, M.D., Ph.D., of the University of Utah School of Medicine.

These studies demonstrate that each of these two major genes, CFH and HTRA1, affects the risk for a distinct component of the AMD phenotype: CFH influences the production of drusen associated with dry AMD, whereas HTRA1 influences blood vessel development, the hallmark of the wet disease type. When the two processes are combined, they lead to the composite characteristics seen in some cases of AMD.

“The marker variant we have identified in HTRA1 is very much associated with AMD, but function of the gene that causes wet AMD is unknown. We need to conduct further functional studies in order to understand the biological mechanisms,” said Hoh.

—Robin Orwant

et cetera

Cancer mutations common

Cancer gene mutations are found in about 1 percent of the general population, more frequently than previously thought, according to Yale researchers.

The study published in the December 6 issue of the Journal of the National Cancer Institute looked for the presence and rate of BRCA1 and BRCA2 mutations in women with newly diagnosed ovarian cancer. Those mutations have been linked to elevated lifetime risks for breast, ovarian and other cancers.

The scientists screened for the most common mutations as well as rare or hard-to-distinguish variants. They then calculated the incidence of those variants in the general population and in individuals with family members who have had cancer. The Yale team found that the lifetime risk to age 80 is not the same for all mutations.

“The exact nature of the level of risk for the particular mutations needs to be further explored,” said lead researcher Harvey A. Risch, M.D.,Ph.D., professor of epidemiology. “This study is an important first step in that direction.”

—John Curtis

A gene for nicotine addiction

AThe World Health Organization estimates that more than 1 billion people smoke worldwide, and that smoking causes nearly 5 million premature deaths every year. Growing scientific evidence suggests a genetic link to nicotine addiction.

In findings published in the January issue of Biological Psychiatry, Joel E. Gelernter, M.D., professor of psychiatry, genetics and neurobiology, and colleagues reported on the risk of nicotine dependence in 634 small nuclear families with members who were also dependent on cocaine or opioids. In 507 of the African-American or European-American families, at least two members were nicotine-dependent. The researchers identified one significant genome-wide linkage on chromosome 5 for the African-Americans and a strong linkage on chromosome 7 for the European-Americans. “These data add to the growing evidence for specific locations for genes that influence risk for nicotine dependence,” said Gelernter.

—Amy Chow


			Originally published in Yale Medicine, Spring 2007. Copyright © 2007 Yale University School of Medicine. All rights reserved.