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Why Do Amyloid Diseases Strike Different Tissues?

Date:
April 26, 2005
Source:
Scripps Research Institute
Summary:
Researchers at The Scripps Research Institute are reporting the results of a recent study that addresses why different tissues in the human body vary in their susceptibility to "amyloid" diseases, which include Alzheimer's disease and a cluster of ailments called the familial amyloidoses.

Export of newly synthesized cargo (ball and stick icons) from the endoplasmic reticulum (ER) to the Golgi occurs through cargo selective budding regions (lower electron micrograph image). (Figure prepared by Mike Pique)

Researchers at The Scripps Research Institute are reporting the results of a recent study that addresses why different tissues in the human body vary in their susceptibility to "amyloid" diseases, which include Alzheimer's disease and a cluster of ailments called the familial amyloidoses.

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The familial amyloidoses, on which the researchers focused their study, are caused by various mutations to a human protein called transthyretin (TTR). These mutations render transthyretin unstable and predisposed to misfolding from a normal, safe structure into dangerous, sticky ones that glom together and form microscopic fibrils, which then cluster to form larger amyloid plaques that deposit in peripheral nerves, organs, and sometimes in the central nervous system.

Strangely, some TTR mutations cause the fibrils to target the heart, others cause the fibrils to form in the peripheral nervous system, and still others cause the fibrils to form in the gut or in the brain. In the latest issue of the journal Cell, the Scripps Research team is describing the chemical and biological basis for this tissue selectivity.

It is not only, say the scientists, that certain tissues like the brain are more susceptible to the amyloid plaques because they are specifically targeted by misfolded TTR proteins, but rather because cells that secrete proteins into these tissues are the ones that secrete the bad proteins most efficiently.

"Most of the destabilized TTR variants tend to be secreted within susceptible tissues just as efficiently as normal TTR proteins, even though they are substantially destabilized," says Scripps Research Professor Jeffery W. Kelly, Ph.D., who led the research with Scripps Research Professor William E. Balch, Ph.D. Kelly is the Lita Annenberg Hazen Professor of Chemistry, a member of The Skaggs Institute for Chemical Biology, and Vice President of Academic Affairs at The Scripps Research Institute.

"The ability of the cell to efficiently release misfolded protein provides a striking and unanticipated new view of the operation of cellular secretion pathways," says Balch, who is a professor in Scripps Research's Department of Cell Biology and the Institute for Childhood and Neglected Diseases. "These results suggest that we may be able to correct these diseases by small molecules that target fundamental rules guiding protein folding and secretory pathway function."

Amyloidosis is All in How the Protein Folds

For decades, scientists have known that proteins have the propensity to fold into a particular three-dimensional structure based on the particular sequence of amino acids the body strings together. Scientists have also known that the structure of a protein is essential for the protein's function, and that an unfolded protein may not be functional. In the last few years, they have also become increasingly aware of the danger of protein misfolding and misassembly.

Misfolding can change a protein from something that is useful into something that is prone to misassembly, making it harmful--even toxic. And even as a properly folded protein may be essential for human health, proteins that are misfolded are the cause of many different misfolding diseases, such as Parkinson's, Huntington's, and the amyloid diseases mentioned above.

Familial amyloid polyneuropathy (FAP), for instance, is a collection of more than 80 rare amyloid diseases caused by the misfolding of one mutant transthyretin (TTR) protein, which the liver secretes into the bloodstream to carry thyroid hormone and vitamin A. Normally, TTR circulates in the blood as an active "tetramer" made up of four separate copies, or protein subunits, that interact with each other.

These tetramers, normally composed of identical protein subunits, come from two different genes. When one of the genes has a heritable defect, hybrid tetramers form that are composed of mutant and normal subunits. The inclusion of mutated subunits makes the tetramer less stable and causes the four subunits to more easily dissociate. Once the subunits are free, they misfold and reassemble into the rod-like amyloid fibrils. The process of fibril formation causes the disease FAP by compromising peripheral nerve and muscle tissue, disrupting their function and leading to numbness, muscle weakness, and--in advanced cases--failure of the autonomic nervous system, including the gastrointestinal tract. The current treatment for FAP is a liver transplant, which replaces the mutant gene with a normal copy. However, small molecule therapies developed previously by the Kelly laboratory are now being tested in placebo-controlled human clinical trials.

An analogous disease called familial amyloid cardiomyopathy (FAC), which is caused by deposition of a few variants of TTR in the heart, leads to cardiac dysfunction and ultimately congestive heart failure. About one million African-Americans carry the gene that predisposes them to FAC. Another amyloid disease affecting the heart, Senile Systemic Amyloidosis (SSA), afflicts an estimated 10 to 15 percent of all Americans over the age of 80 and is associated with deposition of wild type TTR.

Similarly, misfolded and misassembled amyloid beta proteins are thought to be a major player in Alzheimer's disease, because they can accumulate into the fibrils and plaques that autopsies reveal in the brains of patients with the disease. These fibrils and plaques and their precursors are implicated in neuronal loss.

Some scientists have tried to confront amyloid diseases in the laboratory by administering drugs designed to inhibit the growth of fibrils from the misfolded state. However, this has often proven ineffective because fibril formation is strongly favored once an initial, misfolded "seed" fibril forms.

A few years ago, Kelly and his colleagues developed a new way to prevent mutant TTR protein from forming amyloid fibrils. Instead of preventing the abnormal, misfolded protein subunits from conglomerating to form plaques, they were able to prevent them from becoming misfolded and abnormal in the first place.

They administered small molecules that bound to the TTR proteins and stabilized them in their natural tetrameric state. This kept the proteins folded in their proper form, making it harder for the TTR subunits to dissociate, inhibiting the formation of fibrils--an approach offering promise for the treatment of TTR amyloidoses.

Protein Export, Mutations, and Quality Control

While work pursuing new therapeutic strategies has continued in the Kelly laboratory, he and his colleagues have also been asking basic questions about the biology of amyloid diseases. Particularly, they are interested in discovering what controls the onset, tissue selectivity and progression of these diseases.

To investigate these issues, Kelly and his colleagues established a collaboration with Balch, who has studied cell export machinery for a number of years.

Exporting proteins is one of the ways that cells in the various tissues in the body maintain specialized functions. Examples of tissue-specific protein secretion abound. Cells in the liver secrete highly abundant serum proteins such as coagulation factors, albumin and TTR. Cells in the skin secrete inflammatory proteins at the site of a cut to ward off infection from bacteria entering through the cut. Cells in the brain secrete proteins that are involved in modulating neurotransmission. And cells in the gut secrete proteins designed to digest proteins.

The export machinery that drives this secretion is located inside the cell on the convoluted membrane surface of the cell organelle known as the endoplasmic reticulum. Here a complicated series of events involving hundreds of different molecular components will gather together proteins the cell is going to export by folding and packaging them in anticipation of shipping.

As in many other areas of biology, the protein export machinery was thought to play a role in insuring that proteins that are problematic--such as those that are prone to misfolding--will not be secreted. These pathways are envisioned to select and degrade these proteins before they are exported.

Scientists have long assumed that this process was somewhat analogous to the quality control checks that might exist on an assembly-line in some generic factory. A person in a white coat examines each package as it goes down the line, compares it to a standard, and if any package is damaged, then discards the damaged product. Quality control in protein secretion was thought to function similarly: any protein not comparing favorably to wild-type stability would not pass quality control and would be degraded.

Scientists have long assumed that thermodynamic stability would determine whether a protein like TTR would be secreted or not. Thermodynamic stability is an indication of a protein's inherent tendency to be in one state or another--folded, unfolded, or misfolded. One way to look at this is if you have a population of proteins that are highly stable thermodynamically, then perhaps 99 out of every hundred will be folded correctly. In a population of less thermodynamically stable proteins, perhaps only half will be properly folded under the same conditions.

Strangely, Kelly, Balch, and their colleagues found that the efficiency of protein secretion is not correlated with the thermodynamic stability of TTR. They did cell-based experiments and looked at the secretion of 32 TTR variants, including 23 that have actually been associated with disease pathologies in patients.

These 23 proteins have amino acid substitutions that make them prone to misfolding, and therefore one might expect that these inherent instabilities might make them more prone to degradation by the cell's quality control mechanism than the normal TTR proteins. However, the mutants were not all degraded by the cell.

"Most of the mutant proteins that cause the disease are secreted with wild-type efficiency," says Kelly. However, he adds, some tissues are less permissive in terms of TTR secretion, making it likely that tissue specific secretion propensity influences the tissue specificity of the TTR amyloidoses.

Thermodynamics, Kinetics, and Both

Asked what it says about quality control if a protein that is unstable is exported just as efficiently as one that is stable in cells that are more permissive, Kelly and Balch answer that quality control may be the wrong concept. You can't think just about the thermodynamics of the protein, they say.

Kelly and his colleagues did find, however, that both the thermodynamic AND the kinetic stabilities of the TTR mutants taken together contribute to the energetics of the fold and predict secretion efficiency.

What is the difference? Whereas thermodynamic stability is a measure of how likely a protein will be folded or misfolded, kinetic stability is a measure of how easy it is for an individual protein to pass from one state into another--or how fast the process occurs. So a kinetically stable protein will take a long time to go from a folded state to a misfolded state.

Mutant TTR proteins get help at folding within the endoplasmic reticulum, where the microenvironment is so crowded that folding correctly is difficult for any protein. There, the body employs what are known as molecular chaperones to help the proteins fold properly.

The chaperones even help TTR mutants to fold correctly. This creates an unfortunate situation because the chaperone-assisted folding will take place rather quickly, in seconds. Once a mutant TTR protein is properly folded, its kinetic or thermodynamic stability is generally high enough that it will stay properly folded long enough to be secreted. This deceptive appearance will prevent it from being selected for degradation.

"Destabilized proteins that adopt a native or near-native fold can get out of the cell just as efficiently as wild type protein," says Luke Wiseman, a graduate student in Scripps Research's Kellogg School of Science and Technology who is one of the lead authors on the Cell paper.

Therein lies the problem. The energy barrier of most of the TTR mutants is not so high that these proteins will never misfold--they just do it very slowly. The mutants will stay folded long enough to be secreted out of the cell. Then, given enough time, their inherent thermodynamic instability will cause some of them to misfold, initiate amyloid deposits in the tissues in which they are secreted, and create disease pathologies.

"Once they are out there, they are destabilized, and they form the aggregates that cause the disease," says Wiseman.

In their studies, the researchers found that different tissues have differing abilities to secrete proteins that are destabilized. This could explain one of the most confounding things about amyloid diseases--certain mutations give rise to tissue-specific amyloid diseases. Familial amyloid polyneuropathy patients have amyloid plaques in their peripheral neurons, for instance, and familial amyloid cardiomyopathy patients have amyloid plaques in their hearts, whereas CNS selective amyloid patients have deposits in their brains.

Differences in tissue-specific folding pathways can explain why the most destabilized TTR mutants are secreted by cells in the brain, but are not allowed out of cells in other tissues. One possible explanation for the low apparent scrutiny exhibited by the brain is that the hormone thyroxine present at high levels in the brain cells may be binding to the protein TTR during secretion, transiently stabilizing mutant forms of the protein and allowing their export.

While their current study focuses on TTR amyloidosis, it has broad implications for protein misfolding diseases in general, say Balch and Kelly. For example, in cystic fibrosis, a chloride channel (CFTR) that is required on the surface of lung cells to make them function properly, is trapped instead in a partially folded state in the endoplasmic reticulum and degraded. By understanding the new rules that couple protein folding kinetics and thermodynamics with export, CFTR and other misfolding diseases may also be susceptible to correction by small molecules.

The article, "The Biological and Chemical Basis for Tissue-Specific Amyloid Disease," is authored by Yoshiki Sekijima, R. Luke Wiseman, Jeanne Matteson, Per Hammarstrom, Sean R. Miller, Anu R. Sawkar, William E. Balch, and Jeffery W. Kelly and appears in the April 8, 2005 issue of the journal Cell.

###

The research was funded by the National Institutes of Health, The Skaggs Institute for Research, and the Lita Annenberg Hazen Foundation, and individuals involved were supported by fellowships sponsored by the Fletcher Jones Foundation and the Norton B. Gilula Graduate Student Fellowships at The Scripps Research Institute.

About The Scripps Research Institute

The Scripps Research Institute, headquartered in La Jolla, California, in 14 buildings on 100 acres overlooking the Pacific Ocean, is one of the world's largest independent, non-profit biomedical research organizations. It stands at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel.


Story Source:

The above story is based on materials provided by Scripps Research Institute. Note: Materials may be edited for content and length.


Cite This Page:

Scripps Research Institute. "Why Do Amyloid Diseases Strike Different Tissues?." ScienceDaily. ScienceDaily, 26 April 2005. <www.sciencedaily.com/releases/2005/04/050425210818.htm>.
Scripps Research Institute. (2005, April 26). Why Do Amyloid Diseases Strike Different Tissues?. ScienceDaily. Retrieved October 25, 2014 from www.sciencedaily.com/releases/2005/04/050425210818.htm
Scripps Research Institute. "Why Do Amyloid Diseases Strike Different Tissues?." ScienceDaily. www.sciencedaily.com/releases/2005/04/050425210818.htm (accessed October 25, 2014).

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