Prion and amyloid formation have significant outcomes in the cells which increased the significance of studying them. Prions are unusual proteins that have the capacity to change shape and to selectively template that change in shape to other proteins of the same type while Amyloids are misfolded proteins that usually have deleterious outcomes such as the inactivity of a crucial protein in a cell. These proteins are characterised by large contents of beta sheets in their structure. They are extremely stable and insoluble which entails loss of function. Prion formation is thought to be primed by a peptide in the protein rich in a particular amino acid residues (Asparagine and Glutamine). According to this hypothesis, the formation of prions and amyloids depends on the amino acid content of the sequence rather than the sequence itself.
A scientific literature published recently (September, 2016) challenges the idea mentioned above. The authors identified a sequence that is responsible for the formation of amyloids. This study uses four yeast proteins that contain the prion forming sequences: Sup35, Swi1, Mot3 and Ure2. In normal conditions, these proteins are soluble and are used in the cell to perform various activities. Sup35 is a translation termination protein that binds the stop codon. Swi1 is a chromatin remodeling protein and is responsible for the expression of 6% of the yeast genome. Mot3 is a transcription factor that regulates genes expressed during low oxygen periods. The last protein the authors looked at, Ure2, represses alternative nitrogen sources when nitrogen is in abundance in the cell which saves the energy used to harness the nitrogen from the alternative sources.
The first thing the authors did was to look at the structures of the above proteins in their amyloid states. They used biochemical techniques such as circular dichroism and Infrared spectroscopy (FTIR) to characterise the amyloids. A general shift in circular dichroism reading from 200 nm to 216 nm was observed as the proteins transformed from their functional and soluble form to an insoluble amyloid form. This indicated that there was a shift to the predominance of beta-sheets as the amyloids were formed. These shifts were confirmed as the infrared spectroscopy analysis of the proteins were performed. A broad peak at 1625 cm-1 was observed depicting the dominance of beta sheets in the structure of the amyloids.
From the above data, the authors have got that the proteins formed beta sheet dominated structures. To confirm that these proteins were indeed amyloids, staining was done with the dye Thioflavin T (or Th-T in short). An increased intensity in the detection of the stain meant the presence of amyloid proteins. The staining of the observed beta sheet protein with Th-T resulted in a significant increase in intensity when compared to that of a control protein. These amyloids were, then, characterised as fibrils with diameters of 5-10 nm and lengths of 2-10 micrometers after an inspection under an electron microscope.
Figure: Fibrillar structure of the amyloids as seen by electron microscopy. Upper panels are to a 1 micrometer scale while lower panels are to a scale of 0.5 micrometers.
Once the amyloid proteins were understood, the next step was to see if contact with the predicted prion forming sequence resulted in the conversion of functional proteins to insoluble amyloids. To test this the researchers measured the rate of amyloid formation of the NM domain of Sup35 protein. They saw that this transformation took a very longer time to take place spontaneously. This process was made quicker when the protein was put in contact with a preformed Sup35 NM amyloid. But the fastest transformation was observed when the prion forming sequence of Sup35 was added to the domain. It was concluded that the prion forming sequence has an effect in the formation of amyloid from a functional protein.
The next thing the authors looked at was the conversion of endogenous Sup35 protein in yeasts is facilitated by the prion forming sequences. To test this, the researchers transformed some yeast cells with a preformed Sup35 NM domain amyloid and Sup35 prion forming sequences. Red cells indicated functional Sup35 while white cell formation was associated with the transformation to the amyloid state. Upon transformation, an increase in the amount of white cells (12% increase for Sup35 prion forming sequences and 4% increase for the Sup35 NM domain) was observed which indicates the addition of the prion forming unit resulted in more prion formation. Then finally, to conclude that the action of these prion forming sequences is dependent on the whole sequence and not the mere content of amino acids, a collaborating laboratory introduced mutations to the sequences. These mutations affected the rate of prion formation: some increased the rate while other mutations decreased it.
All in all, by conducting these experiments the authors were able to conclude that the prion forming sequence plays a crucial role in prion formation. They also showed that the propagation of misfolding is also influenced by these sequences as the addition of a prion forming sequence increased the rate of amyloid formation.
SOURCES
Primary Article
Sant’Anna, Ricardo, Maria Rosario Fernández, Cristina Batlle, Susanna Navarro, Natalia S. De Groot, Louise Serpell, and Salvador Ventura. "Characterization of Amyloid Cores in Prion Domains." Scientific Reports. Nature Publishing Group, 2016. Web. 10 Dec. 2016.
Other Sources
Sabate, Raimon, Frederic Rousseau, Joost Schymkowitz, Cristina Batlle, and Salvador Ventura. "Amyloids or Prions? That Is the Question." Prion. Taylor & Francis, May-June 2015. Web. 10 Dec. 2016.
Verma, Ashok. "Prions, Prion-like Prionoids, and Neurodegenerative Disorders." Annals of Indian Academy of Neurology. Medknow Publications & Media Pvt Ltd, 2016. Web. 10 Dec. 2016.
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