At every moment, the liver makes and pours into the blood thousands of units of a protein that works like a cable car: it alternates in linking itself up with two compounds that are essential to the harmonious working of the organism and carries them through the inside of arteries and veins and then distributes them to the cells in the body. The name of this protein, transthyretin, is actually an abbreviation that announces its function: transporting thyroxin, a hormone produced by the thyroid gland, and retinol, the active form of vitamin A. Associated now with one and then with another of these compounds, transthyretin circulates through the blood throughout almost the whole of life.
With old age, though, transthyretin tends to join up into long strings or fibrils, that build up over decades in the heart. The long strings turn themselves into a kind of wall between the cells, which jeopardizes the working of the cardiac muscle and makes it difficult to pump the blood. One out of every four persons over the age of 80 lives with this problem, called senile systemic amyloidosis.
American chemist Jeffery Kelly, from the Scripps Research Institute, in the United States, put forward ten years ago a theoretical model to explain the appearance of this form of amyloidosis. When they are old, the proteins are taken to be degraded in a sort of recycling plant of the cells called lysosomes – the blocs called monomers, which act as raw material for forming the strings that build up in the cells and afterwards form extracellular deposits called amyloid fibrils, acting like veritable walls between the cells, hindering their workings.
But it may not be quite like that. In a series of studies published recently, researchers from the Federal University of Rio de Janeiro (UFRJ) show a mechanism that explains in a more simple fashion the formation of the fibrils that are typical of senile amyloidosis from the accumulation of transthyretins. A similar process is found in two other characteristic diseases of old age, Parkinson’s disease, which leads to the lack of control over the movements of the body, with trembling, above all in the hands, and Alzheimer’s disease, which brings about the loss of memory; each one of them are caused by the union of a different protein.
In this new model, the transthyretin does not get to be dismantled in the lysosomes, and it remains whole in the blood, without, though, linking up with thyroxin and the active form of vitamin A, the concentration of which diminishes with age. It would then take on a modified form, which would leave more exposed the segments that repel the molecules of water and serve as a point of contact between the two transthyretins. Afterwards, for the same reason, other transthyretin molecules would join up together in succession. The result would be amyloid fibrils, now formed by the accumulation of whole transthyretins, and not by the joining of the units that make them up, the monomers.
“This proposition is more coherent, since it does not presuppose the protein passing through the lysosomes, where it has never been seen”, says biologist Debora Foguel, from UFRJ, who had funding from the State of Rio de Janeiro Research Support Foundation (Faperj) and from the Ministry of Science and Technology (MCT). The researchers from Rio arrived at this new model after experiments in which they submitted transthyretins to extremely high pressures, up to 3,500 times higher than the pressure at sea level, which is one atmosphere. Alternating between high and low pressures altered the form of the protein, which began to form fibrils in less than 30 minutes. Before this, using chemical reactions, fibrils would take up to four days to be constituted. A quicker way of getting amyloid fibrils, which opens up the possibility of using this method in the selection of chemical compounds to be used as medicines.
Indeed, the team coordinated by Debora and by physician Jerson Lima da Silva, from UFRJ’s Nuclear Magnetic Resonance Center, is testing compounds capable of fighting the formation of the fibrils that are characteristic of senile systemic amyloidosis, Parkinson’s disease and Alzheimer’s disease. Among the five kinds of compound analyzed in the last few months, the most promising candidates, which proved to be capable of preventing the formation of the fibrils characteristic of Alzheimer’s and of transthyretin itself are compounds from the family of anilinonaphthalene sulfonate, a yellow liquid used only as a chemical reagent, to map the entanglement of proteins. “But the results are very preliminary”, Debora warns. “Years of research will still be necessary before some medicine is achieved that can be used by human beings.”
Old and young
While the normal or wild form of transthyretin builds up slowly and only causes problems in the hearts of old people, its altered forms – or mutants are more aggressive and attack the kidneys and nerves of another public, the young; this is familial amyloidotic polyneuropathy. Although rare in the majority of countries, with an incidence of one case in 1 million persons a year, it is more frequent in isolated communities in Portugal, Sweden and Japan, and can kill before the age of 30. In Brazil, 30 cases have now been studied, in 24 families, all prompted by the most common mutant form of this protein. When diagnosed, the problem can only be solved with a transplant of the liver, the organ that produces the protein.
Working today in collaboration with Kelly, the team from UFRJ showed that two of the 80 mutant forms of transthyretin now identified the most common of them, known by the abbreviation V30M, frequent in Portugal, and the most aggressive, L55P, which affects mainly the Swedes are very similar to the normal protein and are also reminiscent of an hourglass. The difference is so small that, on its own, it would not be sufficient to explain the aggressiveness of the modified forms or mutants.
In an article published in May in the Journal of Molecular Biology, Debora describes how this small difference arises: small defects in the gene that contains the recipe for this protein, located in chromosome 18, cause the exchange of just one of the 127 units (amino acids) that make up each one of the four blocs of transthyretin.The replacement of only one amino acid a methionine for a valine in V30M, or a proline for a leucine in L55P makes the protein take on a more fragile form than the wild one.
“For being more fragile than the wild protein, the mutant protein is less stable and can more easily take on the form that generates amyloid fibrils”, Debora explains. When they compare the capacity for aggregation of the three forms of the protein V30M, L55P and the wild one, the researchers from Rio found that the wild version of the protein forms amyloid fibrils more slowly than the other two. They also saw that the transthyretin with the most aggressive alteration, L55P, generated the fibrils more quickly than the most common mutant form, the V30M. These results explain why the symptoms manifest themselves earlier, around the age of 20, in people who produce the most aggressive form of the protein, L55P, and later in individuals whose amyloid fibrils are formed from the wild variety.
The clinical importance of the work of the team from Rio becomes clear from the results of another study, published on August 19 in the Proceedings of the National Academy of Sciences. This time, Debora decided to compare the behavior of the amyloid fibrils resulting from the union of the wild transthyretins with the mutant forms, under hydrostatic pressure. After submitting the fibrils to a pressure of 3,000 atmospheres, there came the conclusion: each fibril was dismantled in a different manner. While the fibril made up of the wild transthyretins would break down into smaller unit, those made of the mutant proteins were not completely dismantled, but would just generate shorter fibrils. “The shortened fibrils are very resistant to pressure and apparently more toxic for the cells”, Debora explains.
Once more, the data is compatible with clinical reality. Maria João Saraiva, from Oporto University, in Portugal, had studied victims of familial amyloidotic polyneuropathy arising from the accumulation of V30M proteins and found that the people with the more advanced symptoms of the disease showed fibrils of an intermediate size, and not the longest, as one would expect. These results overthrow an old belief of specialists in proteins. “It was always thought that every kind of amyloid fibril was the same and would behave in the same way”, comments the biologist from UFRJ.
“We showed that it’s not so.” But what would be the behavior of an amyloid fibril made up of a protein other than transthyretin? Debora next tested amyloid fibrils made up of another protein, alpha-synuclein, connected with the development of Parkinson’s disease a disease in which the individual progressively loses the control of his movements and shows trembling and paralysis. Still with its function unknown, the normal form of alpha-synuclein looks like a stretched out piece of string.
In some families, a genetic defect leads to mutant forms of alpha-synuclein being made, which starts to build up in the inside of the nerve cells (neurons) of a region of the brain responsible for controlling movements and kills them. Another surprise: all the fibrils formed by alpha-synuclein broke down into their basic units, which are toxic for neurons. But the mutant forms would break down two or three times more quickly than the fibrils formed by the wild protein. Knowing the difference in behavior between the fibrils is important for outlining the most appropriate strategies for dealing with each one of the diseases.Republish