As implied in its name, Fatal Familial Insomnia (FFI) is an inherited autosomal dominant condition that shows incomplete penetrance. (Benedini et. al. 2008) There have been 28 FFI family pedigrees documented worldwide and the one shown in Figure 1 is the first from the mainland of China. This pedigree nicely illustrates the non-Mendelian inheritance pattern of FFI. (Yu et. al. 2006)
FFI has been linked to a single residue mutation of the prion protein gene (PRNP) at codon 178, whereby an aspartic acid is replaced with an asparagine (D178N). In addition to the D178N mutation, a 129M residue gives the FFI phenotype, whereas the 129V residue promotes Creutzfeldt-Jakob Disease (CJD178). The different residues will affect the size of fragments of the cellular prion protein (PrPC) generated by proteinase K digestion. (Peterson et. al. 1996) PrPC is a soluble, monomeric protein attached to neuronal membranes, with specificity for binding copper and zinc cations. Specifically, PrPC is an extracellular glycosylphosphatidylinositol (GPI)-anchored glycoprotein that consists of a helical C-terminus domain and a partially structured N-terminus domain. (McDonald, 2014)
Wholly, PrPC may trap pre-amyloid-beta oligomers, associated with Alzheimer’s Disease (AD). It also stimulates NMDA receptors and promotes neuron growth and differentiation. In this sense, PrPC acts as a neurodegenerative defense, as well as a neurological development agent. (McDonald et. al. 2014) On the other hand, an infectious scrapie prion protein (PrPSc) is an insoluble monomer, prone to aggregation and proteinase K resistance. The conversion from PrPC to infectious PrPSc does not require covalent modification. (Requena et. al. 2004)
The prion protein is about 210 amino acids long and consists of five different domains. The domains are labeled Strand (S)1, Helix (H)1, S2, H2 and H3, as seen in Figure 3 below.
The residues closest to the N-terminus of PrPC are called the octapeptide repeat region. This area is involved in the binding of cations like zinc and copper. Human PrPC (HuPrP) typically has five octapeptide repeats, but scrapie mutants can contain anywhere from one to nine additional repeats (6-14 total). (Yin et. al. 2006)
Alpha- and beta-cleavage are shown below to separate the N-terminus from the C-terminus, and to cleave the octarepeat domain, respectively. (McDonald et. al. 2014)
Alpha-cleavage is performed by a disintegrin and metalloproteinase (ADAM) family of enzymes. For example, ADAM10 functions to remove PrPC from its GPI membrane attachment. (McDonald et. al. 2014) The fragments produced from alpha-cleavage have different cellular functions. Following alpha-cleavage, the N-terminus fragment is anti-apoptotic through caspase-3 inhibition, while the C-terminus fragment promotes apoptosis via p53-dependent caspase activity. In this proposed scenario, the effect of the N-terminus fragment outweighs those of the C-terminus fragment and overall functions to inhibit apoptosis. Cleavage of PrPC by enzymes in the ADAM family can be seen below in Figure 6. (McDonald et. al. 2014)
The ‘protein-only’ hypothesis proposes that the key event in the occurrence of FFI is the conversion from PrPC to the pathogenic PrPSc. Scrapie prion proteins can go on to form oligomers. In a study by Chakroun & others (2010), the H2H3 domains were able to form discrete oligomers themselves. This is to say that the rest of the protein, S1, H1 and S2, do not assist in the formation of oligomers. Specifically, the residues responsible for oligomerization are proposed to be 167-222. Mutating residue 222 to a stop codon allows for an enhanced rate of polymerization, which is thought to be due to destabilization of the C-terminus. This unfolding event is deemed a beta-sheet ‘seed’ that allows the rest of the protein to convert into a double-beta-hairpin connected by a loopy region. These oligomers have neurotoxic activity.
More so, two different oligomers were formed, denoted O1 and O3 in Figure 7, below. These different oligomers were induced by point mutations. An I208M mutation in the H3 domain will exclusively form the O3 oligomer. If the I208M mutation in H3 is accompanied by an H190A mutation in the H2 domain, the O1 oligomer will be formed. In the native structure, the S1, H1, and S2 functions to sterically hinder polymerization, but as a scrapie prion, the conserved S1, H1, and S2 domains act in a ‘dock and lock’ model whereby they assist in monomer connections within the overall oligomer.
The native structure of H2H3 is the result of stabilization by the rest of the protein and the disulfide bond found within these domains. This is to say that H2H3 do not preferentially fold into helices. The reliance on the remaining part of the protein to stabilize the helices of H2H3 was demonstrated by showing that the absence of S1, H1 and S2 favors polymerization over that of the full protein sequence. Furthermore, the cellular environment must energetically and thermodynamically favor a conversion from the native form. To this extent, the H2H3 domains have been dubbed the ‘prion domains’ and their change in conformation is the prerequisite to infectious prion formation. Lastly, these findings have implications in the McDonald (2014) paper because ADAMs may function to release the H2H3 domains from the stabilizing portion of the protein and initiate the conversion from native conformation to infectious oligomers. (Xu et. al. 2011)
Oxidative stress is the disequilibrium between creation and elimination of reactive oxygen and nitrogen species by antioxidant defense systems. Methionine residues are the most readily oxidized residues of proteins. (Requena et. al. 2004) PrP contains nine methionine residues at positions 109, 112, 129, 134, 154, 166, 205, 206 and 213. (Colombo et. al. 2009) Prion proteins can undergo direct oxidation through metals, named metal-catalyzed oxidation (MCO). (Pamplona et. al. 2008) Requena & friends (2004) have proposed that the high number of oxidizable methionine residues in PrPC give it antioxidant properties by allowing substantial oxidation to take place without significant structural implications. This would allow PrPC to comfortably reduce the amount of reactive oxygen species (ROS) available to the body and decrease oxidative stress.
Oxidation of the conserved methionines in H3 destabilizes the native fold and favors the shift toward a more flexible state. The oxidation and conversion are similar to ligand-based change or allosteric regulation that effects the conformation of the protein without a major transformation. (Colombo et. al. 2009)
In a study by Pamplona & associates (2008) scrapie infected hamsters showed higher levels of oxidation, lipoxidation and glycoxidation products in their brains. The authors conclude this study by saying that kinase activation and cytotoxic effects are the result of increased p38 phosphorylation and activation of extracellular signal-regulated kinases (ERKs). (Pamplona et. al. 2008)
Another study looked into a recombinant PrP with three extra octapeptide repeats. They found that it left the N-terminus more exposed, which allowed it to bind more effectively to glycosaminoglycan. The mutant was also more susceptible to oxidative attack. Furthermore, oxidative stress has been found to cross-link amino acids that results in the aggregation of proteins. Since the number of octapeptide repeat regions is proportional to the amount of oxidation occurring, the authors were able to conclude that increased octapeptide repeat regions and increased oxidative stress leads to earlier disease onset and shorter disease duration. (Yin et. al. 2006)
Oxidative stress insults may increase as a function of increased protein methionine oxidation. This further supports that an accumulation of mutant prion proteins must occur before the disease phenotype is observable. Furthermore, a decrease in efficiency of methionine sulfoxide reductase as a function of age allows for an increased accumulation of oxidized proteins. Since PrPSc and oxidized proteins inhibit proteasome function, the initial accumulation of these species allows for a chain reaction of amplification of infectious PrPSc. (Colombo et. al. 2009) Figure 8 below shows that coherent motions of the proteins are affected by methionine oxidation. The oxidation sites can be seen as spheres within the helices and the red lines show connections between atoms of various residues that affect its energy landscape, or its ability to deviate from the static equilibrium state in which proteins are typically viewed.
Another dynamic figure illustrating the difference between native and M213 oxidized prion proteins can be seen below. The image on the left shows the typical ‘breathing motion,’ or low frequency fluctuations in the protein, while the image on the right shows the initial elongation of the loop moving toward beta-sheet formation. (Colombo et. al. 2009)
The regions shown within Figure 10 are those undergoing major structural change. After conducting a simulation at 380K and oxidation of methionine residue 213, the final structure and its formation of the beta-sheet can be found in Figure 11. (Colombo et. al. 2009)
In order to observe the extent of oxidation by methionine residues and their proneness to aggregate once oxidized, HuPrP was treated with periodate (Figure 12) and analyzed using electrospray ionization-mass spectrometry.
To attribute the conformational change strictly to methionine residues, the authors did the same experiment but replaced methionines with norleucine (Nle) and methoxinine (Mox) residues. Seen below, Nle is hydrophobic, while Mox is hydrophilic; both of these residues are nonoxidizable. Results indicated that the Nle and Mox did not have a propensity to form aggregates and, thus, oxidation of methionine residues seems to play a direct role in the conversion from alpha-helices to beta rich conformations. (Wolschner et. al. 2009)
The manifestation of the disease state is a function of age even though infectious scrapie protein is produced throughout a person’s life. The thought behind this phenomenon is that there is a required concentration of scrapie protein to reach neurotoxicity and until that level is reached, there are no noticeable effects. (Peterson et. al. 1996) To reiterate, proponents of FFI propose that misfolding events are induced solely by the endogenous protein and are independent of external agents. (Jackson et. al. 2013)
FFI is unusual compared to other prion diseases in that the two most common markers, spongiform degeneration and proteinase K resistance, are not observed. (Jackson et. al. 2013) Interestingly, homozygosity at M129 is thought to accelerate the progression of the disease state, but not effect its time of onset. (Yu et. al. 2006)
Some of the most commonly affected pathways by FFI are Parkinson’s disease, AD, oxidative phosphorylation, lysosome coordination, viral myocarditis and protein export in the cortex and cerebellum regions. The interactions between infectious PrPSc and these pathways is indirect, as PrPSc is found in the cortex and cerebellum regions at lower than control concentrations. Confirming the results of Jackson & others (2013) and breaking the traditional paradigm, the FFI cases observed in this study had remarkably lower proteinase K resistance than did other TSEs. (Shi et. al. 2015)
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