Senile neuritic plaques in the brain are a telling hallmark of Alzheimer's disease (AD). Their origin is just about as complex as the Biblical Book of Genesis family tree. In the beginning, the trunk of the plaques' family tree is a lengthy parent protein called the amyloid precursor protein, APP for short. In fact, APP isn't all that short. It runs from 681 to 782 amino acids long and resides in every cell of the human body.

APP begets the family of molecules that in turn beget those AD hallmark plaques. These come in two sizes, both sheared off from APP. One size, the Beta-amyloid, is 42 amino acids long, carved out of APP and highly toxic to the neurons infested by plaque. Amyloid-beta 40 is somewhat milder.

The generation of A-beta peptides from APP is carried out by an enzyme called gamma-secretase that is composed of a complex of proteins with a presenilin at its core; this enzyme also cleaves a key regulator of cell and organ development called Notch. Therefore, the details of how that secretase works and how to inhibit its action to prevent generation of toxic APP fragments as a way to treat Alzheimer's disease without affecting other important functions is of key importance to medicine. A study titled "A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analyses of Notch 1 and APP proteolysis" published in the Proceedings of the National Academy of Sciences (PNAS), released online Oct 24, 2003, with print publication later this month, sheds new light on the functional arrangement of the enzyme that may help design new drugs to selectively block some, but not all, of its cleaving activities.

The article's senior author is Raphael Kopan, professor of medicine, molecular biology and pharmacology at Washington University in St. Louis.

"I think the most important take-home message of the paper, Kopan told BioWorld Today, "is that the enzyme largely responsible for the development of Alzheimer's disease may work in a different way than previously thought. The more we understand the way this enzyme works, he continued, "the easier it will be to design better and more intelligent approaches to tweak the molecule to do what we want.

The curbing of neuronal plaques makes inhibition of gamma-secretase activity a main objective for new Alzheimer's drugs. Kopan and his co-authors previously found that the secretase enzyme also is required for another protein, Notch by name, to function.

"Notch helps generate many cell types in the body, he observed, "and our team found that inhibitors of gamma-secretase dangerously diminish production of key immune cells.

Next Therapeutics Will Stop Pro-Plaque Secretase

"Ideally, Kopan went on, "the next generation of drugs will be able to prevent gamma-secretase from triggering plaque production without interfering with the enzyme's role in Notch signaling. Our latest finding, which suggests that gamma-secretase may contain multiples of one subunit, is a step in that direction.

To confirm that the enzyme cleaves both APP and Notch, Kopan's team first examined whether the two compete with each other for the enzyme's attention in cultured cells. In their experiments, Notch cleavage was significantly stunted by fragments of APP cleavage debris, upon which gamma-secretase acts.

"The opposite also was true, Kopan pointed out. "In the presence of Notch bits and pieces, he explained, "there was significantly less production of A-beta 40, one product of APP cleavage. We also ranked each of seven different gamma-secretase inhibitors in order of its ability to interfere with cleavage of Notch or APP.

Kopan said enzymes can have either one site where they interact with molecules or separate cutting and binding sites. The PNAS paper suggests that gamma-secretase belongs to a class of enzymes that have "binding sites where molecules can latch on to the enzyme without competing with each other and without becoming subject to cleavage.

"The active site is like a mouth, Kopan allegorized. "It chews whatever it touches but can only chew one thing at a time. The other site, he went on, "is like a hand. It's used for holding and doesn't interfere with the ability of the molecule to chew another object.

Gamma-secretase is a large, complex enzyme composed of four proteins. At its core is the presenilin molecule. "We found, Kopan recounted, "that antibodies designed to find a tag on one presenilin molecule also could latch onto a different presenilin with a different tag. This implies, he noted, "that the two molecules are located close to each other. We confirmed this close proximity by creating an irreversible chemical bond between the two molecules. For this we used a short inhibitor molecule designed by Merck & Co. Inc.

The researchers then examined the effects of presenilin mutation found in people who develop the early, genetically linked form of Alzheimer's disease. They reintroduced mutated presenilin proteins from such patients to cultured cells missing both presenilin molecules. The mutant proteins failed to completely restore gamma-secretase activity, but the cells still produced A-beta 42, the product of APP cleavage that forms the neuronal plaques in the brain.

"Notch is a short-range communication channel, Kopan said. "That means cells in contact with each other activate Notch on the surface of each other. And the cell that manages to activate more Notch on each neighbor is able to prevent that neighbor from choosing the same development outcome as itself.

Notch's All-Body Numbers Game

"In development and in the adult," he added, "when your body produces new skin cells, for instance, or new blood cells, or replaces any organ as it does over time, the way the body goes about it is to make more cells than it initially needed, and choosing the right population numbers for the right task.

"In our bodies, immune system T lymphocytes require Notch in order to properly develop. In the pancreas, Notch is required to allow the proper allocation of cellular fates to occur. The same is true in our intestine and our skin, and many such cellular decisions depend on the proper activation of Notch signals. That's why we were interested in the Notch molecule.

"To me, collectively, the reason why this is an exciting paper from my very nonobjective point of view is that if one wants to improve approaches for therapies, the more one understands how the enzyme is put together and actually works, the better those very smart chemists will be able to come up with a strategy to eliminate the unwanted cuts while preserving those you want.

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