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.
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.
Contact: Jason Bardi
jasonbscripps
858-784-9254
Scripps Research Institute
scripps
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