Explaining and examining polyglutamine repeat diseases

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A significant group of genetic disorders are trinucleotide repeat disorders (or repeat expansion disorders) which as their name suggests, is caused by a trinucletide repeat expansion, a mutation where a certain trinucleotide repeats itself more frequently than the standard, steady state, threshold, characteristic for each gene. Numerous categories of this group of genetic disorders exist. Category I will be the focus of this mini-review. It includes nine diseases (at least) which are caused by the expanded CAG repeat located in exons of specific genes. Glutamine is coded for by the CAG codon: CAG repeats translate into a succession of glutamine residues which materialize into what is called a polyglutamine tract or polyQ tract. Proteins affected by Category I diseases are expressed throughout the body. However, these disorders mainly affect the brain: each one has characteristic symptoms and affect a specific area of the brain. These diseases seem to express gain-of-function mutations: mutations which result in a different and anomalous function at the level of an affected protein. However, they could also simply be caused by the fact that usual protein activity is reduced due to the polyglutamine tract expansion. As previously stated, affected proteins are expressed throughout the body. They are in fact unalike in their structure (secondary and tertiary), their size as well as their function to name but a few. The only common feature which they all share is the presence of the polyQ tract expansion. This expansion could be the cause of the anomalies in the affected proteins found in polyglutamine diseased patients. A number of experiments have been and are currently being conducted in order to identify close links between polyQ expansion and misfolding and aggregation of the diseased proteins. This mini-review will focus on explaining and enlightening the intimate relation between the polyglutamine expansion and the aggregation in addition to the misfolding.

Monomer comprising misfolded PolyQ sections

What could be responsible for the Category I diseases are monomers which contain misfolded polyglutamine sections. Chen and his team treated polyglutamine proteins with a novel substance which had the effect of mimicking the aggregation affinities of similar length polyglutamine peptides that have been biologically altered. This resulted in proteins which displayed a random coil conformation (notwithstanding the repeat length) with regard to the circular dichroism (a secondary structure will convey a specific circular dichroism repectively to its molecules).(9,21). Chen et al omitted the suggestion that polyglutamine peptides folded into an energetically favourable β-sheet conformation.(18,19). Further research using circular dichroism and nuclear magnetic resonance studies also demonstrated that synthetic proteins containing polyglutamine sequences are monomers exhibiting a random coil conformation (notwithstanding the repeat length).(22-24). However, in solutions with a lower temperature, α-helices can be observed (25).

The length of the polyglutamine repeat is not directly linked to the conformation of the polyQ segments. The possibility of there being proteins which have more or less affinity for polyglutamine consistent with the length of the polyglutamine repeat is to be questioned. In fact, Trottier et al(27) suggested that a certain antibody had affinity with a conformational epitope present within a populated polyglutamine region but their thesis was refuted when later studies demonstrated that it is more probable that the increased affinity is as a result of a linear lattice attaching to polyQ segments.

Regardless of the above. Aggregates of polyglutamine containing monomers fold only into a conformation abundant in beta sheets upon incubation.(Wetzel) However a recent study contrasts this. Polyglutamine folds into an α-helical structure when a synthesised thioredoxin binds to it. Then, when incubated, polyQ adopts a conformation in which the polyglutamine folds largely into a β-sheet structure. When passed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), these proteins with their respective conformations were perfectly normal, suggesting that they were rather stable. The results obtained in this study submit the hypothesis that specific proteins comprise folded domains which have the capacity to recruit and stabilize a polyglutamine segment. These results are based on the binding of thioredoxin and polyglutamine. However, the resulting protein is not a disease protein. Studies are on-going in order to identify domains capable of recruiting and stabilizing polyQ within the nine disease proteins.

Another hypothesis supported by growing evidence is that the stability of covalently binded (aggregated) folded domains is intimately related to the polyglutamine sections. In fact, these sections are believed to modify the conformation of neighbouring domains. This results in a monomer which folds differently or not at all. Numerous studies indicate that what is observed is an increase in the aggregation and decrease in the amount of folded, stable protein which accomplishes its role. However, numerous studies suggest that a polyglutamine expansion is associated with a reduction in protein activity. In addition, it has been revealed that a few polyglutamine expansion disorders exhibit indications which can be considered as deficit of normal function.(31-33)

Aggregation of simple polyQ sequences CHANGE NAME

As stated previously, polyglutamine disease proteins differ in size, structure and function. Their only common feature is the presence of the polyglutamine tract expansion. These diseases could be the result of a shared aggregation affinity of all polyglutamine segments that have been expanded. In vitro studies with artificially induced polyglutamine proteins suggest a strong inclination for unprompted aggregation: as the size of the expansion increases, the rate of aggregation also increases.(9). This aggregation process and more particularly the initial phase can be illustrated and further explained using a thermodynamic nucleation-dependant polymerization model of aggregation.FIG1


The polyglutamine aggregation process results in amyloidlike aggregates (fibrils)(9). These fibrils are less likely to undergo additional nucleation phenomena meaning they expose increased stability. Another system with different processes could have had the effect of falsifying and complicating the modelling of this particular initial reaction because of these so called additional nucleation phenomena which confuse results but the stability of the amyloid fibrils permits the use of this thermodynamic model which clearly illustrates the aggregation kinetics of polyglutamine.(38-39).

This thermodynamic model fits the aggregation process of polyglutamine proteins. It is based on the fact that when a nucleus develops, it is too unstable and therefore spontaneously decays into an elementary monomer or fuses with a polyglutamine chain which in turns elongates.(37).

This polyglutamine aggregation process appears not to enable other aggregated structures (which do not fit the simple thermodynamic model illustrated in fig. 1) to in a certain way, evolve. (34). Studies have demonstrated that it is possible to mimic recombinant polyglutamine proteins. This process consists of a thorough and accurate disaggregation procedure which develops stable aggregating and evenly distributed peptides although there exists a treatment with a specific non aqueous fluid which has revealed a fibril-free polyglutamine aggregation.(42). The model for nucleation presented may be a tool in order to assist the research for polyglutamine disease processes and even other maybe more general processes of aggregation systems.

The thermodynamic model for nucleation can be used to formulate the idea that upon addition of small segments of polyglutamine, the aggregation rate of nuclei is increased at the location of expansion of the polyglutamine protein. In fact, these small segments would accomplish this with the aid of the nuclei which would need to disintegrate at a higher frequency. This is a factor which may explain the phenomenon of aggregation within a cell. Studies have shown that the aggregation rate of a polyglutamine expansion (on the huntingtin protein of a drosophila) increased upon fusion with a 20 amino acid long polyglutamine segment. Another point can be made concerning inhibition: molecules which can prevent elongation could have an effect on slowing down nucleation.(46).

CHANGE NAME role of celullular environment

The Polyglutamine aggregation process is influenced by a whole range of environmental factors within the cell. Nollen et al conducted an experiment on specific transgenic Caenorhabditis elegans strains more particularly factors which influenced polyglutamine aggregation. In fact, they used genome-wide RNA interference to detect genes, that when inhibited, caused early development of protein aggregates.

To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the early appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.