Difference Between Infectious Prion Proteins Biology Essay

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The objective of the research reported by Pan et. al. in the article "Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins" is to analyze whether the difference between infectious prion proteins (PrPSc in the case of scrapie) and the normal cellular prions (PrPC) is related to a difference in the conformation of these proteins: the ratio between the amount of α-helices and the amount of β-sheets. Previous research on prions showed that PrPSc is synthesized post-translationally from PrPC, but no process involving a chemical modification has been discovered, leading to the hypothesis of a conformational modification that would produce the PrPSc: the conversion of α-helices into β-sheets; this conformational difference hypothesis has been further supported by experimentally observed differences in properties between the two proteins: solubility in detergents, protease hydrolysis, accumulation, and others. In addition, research has shown the PrPSc has a protease-resistant core designated PrP 27-30 which has been discovered to have a predominantly LF (low frequency) β-sheet structure, characteristic of amyloids (prion protein aggregates associated with neurodegenerative disorders).

In order to test the hypothesis of the conversion of α-helices into β-sheets, the scientists devised an experimental procedure in which they isolated both PrPC and PrPSc (PrPSc was experimentally produced inside the tissue) from the brains of Syrian hamsters. The PrPC was purified through a procedure that avoided using low pH, urea or SDS/PAGE, all of which could denature the proteins. The purification procedure used for this experiment only employed zwittergent 3-12 (ZW), centrifugation, IMAC-CU2+, IMAC-Co2+ and wheat germ agglutinin (WGA) columns, and solutions containing buffer, sodium chloride, sodium phosphate and imidazole, used for washing the columns (all of the solutions were used in concentrations that would not interfere with the protein structure). PrPSc and PrP 27-30 were purified using procedures reported in other research articles, and were ultimately centrifuged and washed with PBSZ and PBSZ/H2O solutions.

The three proteins (PrPC, PrPSc, and PrP 27-30) were then subjected to various analytical techniques in order to compare them. The proteins were first denatured by boiling in buffer and their amino acid sequences were determined, for a prediction of the protein secondary structure: the analysis was first done by SDS/PAGE, followed by silver staining visualization, and were finally stained with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and with nitro blue tetrazolium (NBT), for amino acid analysis. Next, the FTIR (Fourier Transform Infrared) spectrum was recorded for all three proteins, followed by the CD (circular dichroism) of only PrPC. These spectra were necessary in order to get the exact secondary structure). For a visual analysis of the aggregation state (amorphous or amyloid aggregates) of the three proteins, electron micrographs were obtained by using gold particle conjugated goat antibody attached to the mouse IgG.

Before any of the previously listed analyses, the researchers first made sure that the purification method for PrPC was successful: both the lectin chromatography and the SDS/PAGE results showed that the second pool of fractions obtained from the purification procedures only contained the desired proteins, with a mass of 33-35 kDa. The amide I' band FTIR spectrum of PrPC showed a peak at 1653 cm-1 (characteristic of high α-helix content), while the spectra for PrPSc and PrP 27-30 were characteristic of a high β-sheet content. Upon deconvolution of the spectra, it was calculated thet PrPC had only 3% β-sheet content, while PrPSc and PrP 27-30 had a β-sheet content of 43% and 54%, respectively.

The data obtained from the FTIR spectrum of PrPC were confirmed by obtaining and analyzing the CD spectrum of this protein: the CD results were in close agreement with the FTIR ones. CD could not be performed for the other two proteins because they were not soluble. Class-dependent (α/ α, α/β, β/ β) and naïve secondary structure predictions were performed and the four α-helices showed both strong helix preference in the α/ α class and a strong β-sheet preference in the β/ β class: this supports the hypothesis of the conversion of α-helices into β-sheets. Finally, the electron micrographs obtained showed that PrPC and PrPSc appeared as amorphous, while PrP 27-30 polymers formed a rod-shaped amyloid.

Form the results reported in the paper, the researchers concluded that the formation and propagation of PrPSc from the normal protein PrPC mainly involves the conversion of α-helices into β-sheets, but no conclusions can be drawn as to whether this process is triggered by some kind of chemical modification in some of the PrPC molecules, or by some other type of event. All previous findings do not say anything about the cause for the conversion, butpoint toward the fact that prion diseases are caused only by the PrPC to PrPSc conversion, and toward the fact that this conversion is the main event that leads to prion diseases. In addition, past articles have proven that the conformational change can be triggered by the presence of a single PrPSc molecule. Previous findings have also shown that this conformational change is a very complex process which requires additional proteins (other than PrPC), which would catalyze the change, acting similarly to chaperones. Some researchers even suggested that of PrPSc formation might involve amyloid formation as its central component, but this idea is disproved in the present article by Pan et. al., which shows that the PrPSc is present as an amorphous aggregate and not as an amyloid. The findings from this article, along with all the previous findings mentioned here, raise a lot of questions, but at the same time open new doors toward a better understanding of prion diseases.

Critical Review:

In my opinion, the entire experiment was very well conducted and all the procedures (from purifying the three proteins to performing FTIR spectrometry, circular dichroism and electron microscopy on them) were very well chosen so that they would provide the information necessary to verify the hypothesis, and also, to prevent the denaturing of the proteins. The experiments followed a logical sequence, starting with the production of the proteins inside the mice brains, followed by the extraction and purification of the proteins, and ending with the analysis of the secondary structure of each of the three proteins and the interpretation of the experimental results.

Even though the experimental procedure was very well constructed and the research provided some very important answers regarding the process of infectious prion formation, there were a few minor shortcomings throughout the paper. First of all, the researchers discuss the FTIR of PrP 27-30 in the introduction, specifying the previous findings along with the fact that two thirds of the β-sheets were low-frequency (characteristic of amyloids), but don't do the LF analysis themelves. Since this type of analysis was specified in the introduction as previously being performed, it would have made a lot of sense to perform the LF analysis for all three proteins in order to find the LF β-sheet percentage (could have uncovered some new information) and at the same time, verify the accuracy of the experiment against the past experiment.

Secondly, this experiment seems to be composed of a very tedious purification procedure, followed by a relatively simple analysis of the three proteins which only returns the amounts of α-helices and β-sheets. A better way to approach prion diseases would be to elucidate the structures of the three proteins (using methods like X-ray crystallography or nuclear magnetic resonance). Then, by comparing these structures to one another, one can observe exactly where the conformational transitions occur and what amino acids are involved in these transitions, which might eventually lead to an understanding of prion diseases.

Thirdly, the researchers talk about not being able to obtain CD spectra for PrPSc and PrP 27-30 (because of insolubility of these proteins), but say that some CD spectra have been obtained (with the proteins deposited as thin films). However, the researchers do not talk about these results and do not compare them to the FTIR results that they obtained. This CD vs. FTIR coprison was done in the case of PrPC, but was omitted for the other two proteins, leading one to wonder why the researchers decided to omit these results.

Lastly, the discussion section of the paper did not interpret the experimental results in great detail, which is probably because the conclusion was very clear and did not need too much explaining: the hypothesis was proven correct with no other additional findings. However, the researchers chose to present a great amount of previous findings on the subject of prions and prion diseases, but they forgot to give the reason for presenting all of that information: there was barely any connection with their experiment and no reason was given as to why that information should be useful, especially in correlation with the experiment presented in the article.

In conclusion, this experiment is very well built, but it contains too little experimental data considering the magnitude of the biomolecular events that the researchers are trying to study. It is clear that the researchers only searched to test the short, pre-existing hypothesis of conformational transitions without any attempt to state an original hypothesis and try to discover something new. Performing more complex experiments to try to at least elucidate the structure of the proteins and analyze the amino acid interactions that lead to the formation of prions would provide the grounds for a much more in-depth conclusion, producing a high-quality research article which could open the path toward solving the problem of prion diseases.

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