Conformational Changes Transthyretin Acquisition Of Familial Amyloid Polyneuropathy Biology Essay

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Familial amyloid polyneuropathy (FAP) is one of the main neurodegenerative disorder associated with transthyretin. The hallmark of the disease is the systemic deposition of mutated TTR variants as amyloidogenic fibrils affecting the peripheral nervous system (PNS). To date, there are over 100 mutated TTR variants discovered. The most common amyloidogenic TTR variant is Val30Met, while the Leu55Pro variant is the most aggressive TTR variant. However, not all of the mutated TTR variants are amyloidogenic, variant Thr119Met is known to be non-amyloidogenic and non-pathogenic. Instability of the TTR tetrameric structure is believed to be the root cause or problem for the formation of TTR amyloidosis. Amyloid formation is the result of a disruption of the quaternary structure of TTR and changes to the monomeric form. Conformational changes such as 'D" strand dissociation coupled with the β-sheet to α-sheet dubbed the amyloidogenic intermediate were observed in molecular dynamics experiments. The aim of the therapeutic strategies is to be able to stabilise the native tetrameric structure of the TTR so that it is able to prevent tetramer dissociation and hence prevent amyloidosis.

Introduction

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The contents below were quite jumbled together, so I have tried to make the introduction more logical. You need to make sure that you define abbreviations the first time you use them.

The human transthyretin (TTR) gene located on chromosome 18 at position 12 is responsible for the production of the human transthyretin protein. The transthyretin protein is a tetrameric protein with a molecular weight of 55 000 Dalton with 127 amino acid residue per subunit. It is produced in the liver, the retina and choroid plexus. (Foss et al 2005) The liver is responsible for the secretion of TTR into the blood while the choroid plexus secretes TTR into the Cerebrospinal Fluid (CSF). The TTR secreted by the two organs appear to be the source of amyloidgenic transthyretin in the periphery and brain (ref).

Role of Transthyretin (TTR)

The two functions that are commonly associated with TTR are as a transport protein for thyroxine (T4) and for retinol through the binding of retinol-binding protein (RBP) (ref). The name given to the protein transthyretin is derived from such dual physiological role as a transporter of thyroxine and retinol.

TTR transportation of T4

It is suggested that one of the possible function of TTR in the human body is to transport thyroxine (T4), a form of thyroid hormone. Binding of T4 to the TTR would allow the hormone to cross the blood-brain barrier (BBB) (ref). Being a thyroid hormone, thyroxine proves to be an essential hormone in the early brain development as deficiency of the hormone in such stage of development would result in a damage to the neuronal connectivity and mental retardation if the hormone is further deprived (Legrand et al 1984)

The role of transthyretin being a major or an essential transporter of thyroxine to the brain remains to be a subject of controversy as laboratory test conducted using TTR KO mice showed that the absence of TTR does not affect the levels of thyroid hormone function (ref) how was this shown? briefly However, further investigations resulting from human study does reveal a correlation between thyroxine and TTR in the CSF. This contradicts the previous results obtained with the TTR KO mice. Thus is was concluded that distribution of T4 from the CSF into the brain was indeed carrier mediated, TTR-dependent (Kaseem at al 2006)

TTR indirect transportation of Retinol

TTR is established as an indirect carrier of retinol in the body. TTR is simply a carrier of the retinol-binding protein complex (RBP). The choroid plexus produce and secretes TTR and RBP to the CSF. RBP interacts with TTR and retinol to form a trimolecular complex which is responsible for 90% of retinol in blood to be in this form (Zheng et al 2001) Retinol is transported across the blood-brain barrier thru the RBP and that TTR only facilitates in the transportation.

Wild type TTR and several TTR variants are responsible for the deposition of amyloid fibrils under physiological conditions causing diseases such as Familial amyloid polyneuropathy (FAP) (Saraiva et al 2001).

TTR Mutations

To date, there have been over 100 TTR mutations reported (Connors et al 2003). Majority of the mutations are inherited in an autosomal dominant manner and most of the mutations are deemed amyloidgenic as it is related to amyloid deposition affecting the peripheral nerve and the heart (ref). Only a small portion of the TTR mutation is considered as non-amyloidgenic and non-pathogenic.

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I think you need something on the protein structure here, with a diagram. You need to explain about the tetrameric structure and the strands in the monomers early on so that the information below makes more sense

Most of the disease associated with TTR mutations, be it single base point mutations or deletion, exhibit little or no changes in terms of structure from the wild type TTR. This is based on the X-ray crystal structure analysis describe this in more detail and reference it. Disease associated TTR protein functions normally in transporting and binding of thyroxine as well as retinol in the plasma and the Cerebrospinal Fluid how do you know? Evidence and refs. Therefore, it can be concluded that the fault lies in the mis-folding ability and mis-assembly of the mutated TTR rather than its inability to fold that cause it to gain its toxic functions causing disease (Hurshman et al 2008). This conclusion is not clear for the little you have said above - you need to explain it more fully / clearly

The most common mutation in the TTR protein is the substitution of methionine for valine at position 30, thus resulting in the mutated variant of Val30Met of the TTR protein. Variant Leu55Pro is a mutated TTR protein where leucine is replaced by proline at position 55. It is known as the most aggressive form of amyloidogenic TTR reported. Unlike variant Val30Met, X-ray crystal structure revealed that the structure of variant Leu55Pro had significant structural difference when compared to the wild type TTR or even mutated variant Val30Met. The difference in the structure revealed the absence of strand 'D'. refer back to figure 1 above or describe where it is. This caused disruption of the hydrogen bonds that link strand 'D' to strand 'A' thus altering the overall conformational structure of the strand and bringing about aggregation (Yang et al 2003) why / how does this cause aggregation? Explain in more detail

However, not all of the TTR mutation are amyloidogenic and pathogenic. The mutated TTR variant Thr119Met is one of the known variant that is non-amyloidogenic. X-ray analysis revealed that alterations were seen in the thyroxine binding site. This conformational structural change causes hydrogen bonding interactions within the monomers, dimers and tetramers thus promoting the tetrameric state and preventing and protecting the variant from amyloidosis. DNA analysis showed that this variant is a result of a second allele mutation of the TTR gene. Thr119Met is termed an interallelic trans-suppressor as it is able to bind to the T4 binding site of the amyloidgenic Val30Met variant and stabilise its structure and prevent dissociation of its tetramers. Do you mean that the 2 variant forms interact? Need to be clearer, is this repevant / important? What does it show?

Are these the only 3 mutations you have found? If there are 100, I would expect more information in this section, maybe a summary table. You are well under on your word count, so you could include more information here.

Familial Amyloid Polyneuropathy (FAP)

Familial amyloid polyneuropathy (FAP) is one of the main neurodegenerative disorder associated with transthyretin. FAP is an autosomal dominant neurodegenerative disorder that was first described by Andrade in 1952. It is also known as the Corino De Andrade disease named after the neurologist who discovered it. The disease was first discovered in the northern area of Portugal. Since then, other incidences have been identified worldwide more information? Where? Is there any pattern? Same muations in each location? A large foci of the patients were reported from Japan and Sweden. The common characteristic of FAP is the systemic deposition of amyloidogenic fibrils affecting the peripheral nervous system (PNS)

There are four classification or types of FAP and they are classified depending on the precursor protein that is mutated. Such proteins include Transthyretin, Apolipoprotein A1 and gelsolin (ref) what are these proteins and how are they related to TTR and / or its function? Or are they distinct?. The first two class of Familial amyloid polyneuropathy, termed FAP I and FAP II are associated with the transthyretin protein. This is the group focused in this article. FAP III is known as the 'lowa-type' which involves Apolipoprotein A1 and finally, FAP IV which is also known as 'Finnish-type' that involves gelsolin (ref).

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Due to the fact that the liver is the main source of TTR production, liver transplantation has been proposed as the only means of treating this once thought incurable disease. This hypothesis was supported by favourable results from clinical trials (Holmgren et al 1993) and substantial research findings that saw a reduction in the abnormal TTR serum levels (Holmgren et al 1991). What did these findings show? However, the success of liver transplantation has its own set of limitations. Such limitations included low survival rate and reoccurrence of FAP symptoms how / why?

This led researchers to develop alternative methods of treatment to cure the disease. One such strategy is to prevent the dissociation of the tetrameric TTR structure to its monomeric form by stabilising the tetramers with small binding molecules which strengthens the kinetic barriers preventing amyloidosis. (Tojo et al 2006) This will be further discussed in depth later on. You should be discussing this in detail - if you do it later, refer to that here

Symptoms of Familial Amyloid Polyneuropathy (FAP)

The common trait of FAP is the systemic deposition of amyloidogenic fibrils throughout the connective tissue excluding the brain refs?. Such amyloidogenic depositions affect the peripheral nervous system (PNS) (Andrade et al 1952) More recent refs? and causes organ dysfunction and death. Patients suffering from FAP experience progressive as well as severe lost of sensory and motor neuropathy. This is often followed by autonomic dysfunction that leads to death within the mean period of 10.8 years (Coutinho et al 1980).

It was observed that one of the first symptoms of FAP is the impairment of sensory organs. This would be credited to the deposition of mutated TTR deposits in the nervous system. Motor involvement is next to be affected as the disease progresses on. FAP patients were reported having degenerative lost of reflex with in the upper and lower limbs thus making the ability to walk an impossible task without any aids (Lambert et al 1969). These are very old references - are there no more up to date studies? It was also reported that FAP patients experienced nausea and frequent vomiting. This could be due to hypomobility which was resulted by the amyloidogenic depositions in the stomach and degeneration of the autonomic nerves (Ikeda et al 1982).

Some of the TTR mutations associated with FAP experience similar types of clinical symptoms, however others give rise to new phenotypes of symptoms such as cardiomyopathy and carpel tunnel syndrome. One such case was reported by the division of neurology from the University of Malaya.

The patient mentioned in the report had the mutated TTR variant Ala117Ser which was uncommon in the Chinese ethnic community. Alanine was substituted with Serine at position 117. One of the major difference in terms or clinical symptoms compared to the Val30Met variant was that the patient exhibit was a late onset of the first symptoms which was seen at the age of 49 years old coupled with Carpel Tunnel syndrome (Khean et al 2008)

Peripheral neuropathy is the main symptoms in Familial amyloid polyneuropathy while cardiomyopathy is the main symptom for familial amyloid cardiomyopathy (FAC). In a condition that affects the old people, non-mutated TTR is also found deposited in the heart. The condition is termed as senile systemic amyloidosis (SSA) (Saraiva et al 2001).

More on these conditions? Mutations associated with them?

Aggregation Pathway

Before scrutinising into the details of the conformational structure changes of the wild type TTR and the mutated TTR variant, it is essential to have a summary of how the TTR amyloid formation occurs or give rise to amyloidgenic fibrils. Amyloid fibrils are formed by the dissociation of the TTR tetramer into native monomers. This is termed as the rate limiting step in the formation of the amyloidgenic fibrils refs - how was this determined?. Upon dissociation to its monomeric species, it unfolds to form the aggregation intermediates which are then followed by the process of self-assembly of polymerisation. Further structural changes within the monomers are required before the fibrils can become amyloidgenic as formation of the fibrils alone is not sufficient to make it amyloidgenic (Hurshman et al 2008) This should be the main focus of your dissertation, you need to discuss these changes in a lot of detail

Conformational Changes in TTR structure

TTR is a tetrameric serum protein consisting of four identical subunits. Two beta sheets (β-sheets) of each monomer are composed of strand named from 'A' to 'H'. They are arranged into inner and outer sheets. The inner sheet comprise of 'DAGH' while 'CBEF' forms the outer sheet. Both sheets interact by hydrogen bonding between the two adjacent 'H' and 'F' strands to form a dimer. The tetramer is formed by hydrophobic interactions between two dimers via a connecting loop that connects strand 'A' to 'B' and strand 'G' to 'H' strand (Steward 2008). What about the Jeff Kelly lab in America? They have done a lot of work on TTR conformational changes associated with amyloid formation.

Amyloid formation is the result of a disruption of the quaternary structure of TTR and changes in the monomer. In order to observe the conformational changes that causes such amyloid formation, the mutated TTR variants (Val30Met, Leu55Pro), non-mutated variant (Thr119Met) were subjected to different pHs. It is noted that there is some difficulty in concluding the amyloidogenicity of a TTR variant using X-ray crystal structure analysis there will be as this just gives information on 1 static crystallised structure, it does not tell you about changes that happen in real time. What about NMR, CD or FTIR studies? alone because the structures changes are to minimal to be considered as a conformational difference. This lead some research groups to use Molecular Dynamics (MD) to help in identifying the amyloidogenic intermediates (Steward 2008). This is not the only way that the TTR has been analysed

An intermediate in the amyloid formation is termed as α-sheet, which is no different from β-sheet. However , α-sheet is fused with all the carbonyl groups on both sides of the strand face. These α-sheets are also observed under the Protein Data Bank (PDB) (Daggett 2006)

At a low pH, strand D was dissociated from the β-sheet to expose strand A which caused aggregation. The two amyloidogenic variants recorded a conformational change in their β-sheet to an α-sheet via peptide bond flips. The DAGH sheets in the amyloidogenic variants showed changes in the adjacent peptide planes. This was not observed in the wild type at a neutral ph but the same change was observed at a low ph. It can be concluded that the formation or change of the α-sheet intermediate is observed for amyloidosis formation.

In order for aggregation to happen, the 'C' and 'D' strands should dissociate to expose 'A' and 'B' strands for aggregation to take place. In the mutant variant, it was observed that Hydrophobic and electrostatic interactions prevented the dissociation of the 'D' strand even thought there was a lost in hydrogen bonds. Similar to the 'D' strand, strand 'C' was also did not dissociate and expose strand 'B' for aggregation. Instead, Strand 'C' anchored itself to the structural loops connecting strand 'C' to 'B'.

The TTR Thr119Met is a non-amyloidgenic variant. It is able to avoid amyloidosis or degradation of its tetramers into amyloidgenic fibrils by prevention of α sheets in the DAGH region by rotating its peptide plane. This is OK, but also need to include other studies as well.

Therapeutic strategies

As mentioned earlier, liver transplant was the recommended treatment for patients with FAP as it proved that liver transplant was able to decrease the circulating mutated TTR in serum. However, there was a high mortality rate in FAP patients that underwent liver transplant (Suhr et al 1995). One of the main causes of the high mortality rate is credited to the post-operative operation infection. This is coupled with complications of neurogenic bladder dysfunction.

Apart from the high mortality rate that was reported, there were also reports of no noted improvement in the motor functions or walking capability of patients that underwent the liver transplant operation. The finding from the results obtained contradicts earlier literature published by Parrilla in 1997. In the article, it states that improvement of the motor functions were observed within two years after the liver transplantation. There was also the issue of lack of liver donors who were willing to donate their liver. This lead to the downfall of liver transplantation as the primary means of treatment.

Due to such limitations, new therapeutic strategies were developed to tackle the conditions facing FAP patients. Such alternatives aim to be more efficient and definitely safer from the outcomes liver transplantation offers.

Instability of the TTR tetrameric structures is believed to be the root cause or problem for the formation of TTR amyloidosis. Therefore, the aim of the therapeutic strategies is to be able to stabilise the native tetrameric structure of the TTR so that it is able to prevent tetramer dissociation and hence prevent amyloidosis.

As mentioned earlier, the mutated TTR variant Thr119Met is a non-amyloidgenic variant. It is able to avoid amyloidosis or degradation of its tetramers into amyloidgenic fibrils by prevention of α sheets in the DAGH region by rotating its peptide plane (Robert et al 2008) as a result, X-ray analysis showed alterations were seen in the thyroxine binding site. This conformational structural change causes hydrogen bonding interactions with in the monomers, dimmers and tetramers. Thus effectively increases the non-amyloidgenic variant's stability. Thr119Met is termed as an interallelic trans-suppressor as it is able to bind to the T4 binding site of the amyloidgenic Val30Met variant and stabilise its structure and prevent dissociation of its tetramers. (Hurshman et al 2004) With this knowledge, Thr119Met is used to act as the trans-suppressor subunit as it is to bind to other amyloidgenic variants. Small molecule inhibitors - Kelly group

Stabilisation of the native TTR tetramer can be achieved by preventing dissociation into its monomers by small molecule bindings has been research and shown to raise the kinetic barrier of tetramer dissociation in vitro. Two non-steroidal anti-inflammatory drugs (NSAID), Diflunisal and flufenamic acid were able to achieve such aims by binding to the T4 binding sites of the TTR protein and bring about kinetic stabilisation of the tetramer thus preventing amyloidosis (Tojo et al 2006).

Among the two NSAID, Diflunisal showed the most promising result. TTR tetramers are known to dissociate faster at lower ph (<7) Diflunisal was able to stabilise the tetrameric structure of the TTR variant Val30Met in all pH ranges that it was subjected to. This shows increasing potential of Diflunisal as a TTR kinetic stabiliser as it was able to increase serum TTR stability of an FAP patient.

However, as promising as Diflunisal may be, extensive research is still needed to evaluate the effectiveness of this drug since results obtained are only limited to in vitro testing. One disadvantage that it has is the fact that diflunisal may prove to be ineffective against central nervous system amyloidosis because it is not able to cross the blood-brain barrier (BBB) efficiently. (Nuernberg et al 1991)

International trials on the pharmological effectiveness of the drug on TTR amyloidosis is still ongoing. This treatment part should be a bit briefer, it is OK to include, but is not the main focus of your dissertation

Conclusion

For the past few years, researchers have tried to treat the once thought incurable Familial Amyloid Polyneuropathy disease. With the help of modern technology, researchers are able to utilise new ways to discover the illusive amyloidgenic intermediates. It is now known that kinetic instability of the TTR tetrameric structures is believed to be the root cause for the formation of TTR amyloidosis. Therefore, the aim of the therapeutic strategies is to be able to stabilise the native tetrameric structure of the TTR so that it is able to prevent tetramer dissociation and hence prevent amyloidosis. Liver transplantation provided a temporary answer to treat FAP. However, its disadvantages out weights its advantages lead to the discovery of small molecule bindings that has been proved to be able to raise the stability of the TTR tetramer. However, extensive research is still needed to evaluate the effectiveness of this drug since results obtained are only limited to in vitro testing.