ANALYSIS OF THE EFFECTS OF PROTEIN FOLDING ON ALZHEIMER’S AND POTENTIAL APPLICATIONS OF PROSAAS
By John Paul Cross
Cell Biology
Professor Kierra Wilkins
Old Dominion University
Norfolk, Virginia
12/12/21
Analysis Of The Effects Of Protein Folding On Alzheimer’s And Potential Applications Of Prosaas
Protein folding is when ionic and hydrogen bonds present in amino acid chains interact to form structures such as alpha helices and beta-pleated sheets that in turn form what we know as proteins. Protein folding is one of the most important processes performed by cells during respiration, and when performed incorrectly causes a myriad of issues for the cell as well as the organism the cell resides within. These range anywhere from improper devotion of resources towards deleterious processes or outright shutdown of vital organelles such as mitochondria not being capable of completing cellular respiration and producing ATP. One of the most prevalent examples of protein misfolding is the conversion of Alpha helices into Beta-Pleated sheets, where the typically cylindrical helices are flattened into sheets allowing for more amyloid formation, and aggregation (Ashraf et. al, 2014). One process that exacerbates the issue of misfolded proteins is Protein Aggregation, where disorderly proteins spontaneously group up and become incapable of carrying out biological processes (Wang and Roberts, 2018). This is the driving force behind Alzheimer’s in which beta-pleated sheets clump together with beta-amyloid proteins such as Amyloid-Beta (Fluidic Analytics, n.d.) to form plaque buildups that causes extremely deleterious memory loss, and in many cases prevents new memories from being formed (Libretexts, 2021).
Background Information
One method that prevents the congregation of amyloid proteins is that of Chaperone proteins, where groups of highly specialized proteins form small chambers around protein substrates to properly fold a protein complex without amyloids interfering by providing a neutral zone free of the effects of PH and hydrogen bonding allowing for proper protein formation. (Bessinger and Buchner, 1998). One study performed by a group of scientists published in the Journal of Neurochemistry highlights how one anti-aggregant chaperones referred to as proSAAS could be utilized in order to prevent amyloid aggregation during protein formation and aggregation (Hoshino et al., 2013). [While it is normally produced in the body for the purposes of inhibiting Prohormone Convertase, the researchers noted that the protein was present in cells not related to suppressing Prohormone Convertase and hypothesized that proSAAS could be utilized in other fields] (Hoshino et al., 2013). [The study details how by utilizing ProSAAS in mice and human brains afflicted with Alzheimer’s that amyloid aggregation and fibrillation was widely prevented depending on the size of the dose in question] (Hoshino et al., 2013). This breakthrough highlights how with more time and funding that future research into the function of proteins could unlock knowledge on how to prevent and possibly reverse the deleterious effects of protein based neurodegenerative diseases such as Alzheimer’s.
Experiment
Procedure
The researchers used a combination of human samples (one from a willing donor afflicted with AD,) Bovine Serum Albumin, Rabbit anti-proSAAS, and monoclonal mouse antibodies, incubated with “goat anti-rabbit” and “Cy2-conjugated donkey anti-mouse” within blocking solutions (Hoshino et al., 2013). ProSAAS antiserum was incubated within rabbits and slides were doused in Fluoromount G. 12 month male mice were used as models for the purposes of studying amyloid plaque. Mouse brain sections were then treated with many solutions such as Aqua DePar, incubated with blocking kits and rabbit proSAAS, and then stained with “…pan-b-amyloid (Ab) monoclonal mouse antibody,” (Hoshino et al., 2013). before being viewed under microscope. Mice proSAAS plasmids were prepared via generation by pQE30 templates, and were subsequently processed and expressed via E. coli samples. Cells were lysed, put under centrifuge and examined, before proSAAS was concentrated with lyophilization and placed in 5 mM acetic acid (Hoshino et al., 2013).
Data Analysis
The researchers found that proSAAS co-localized within mouse models within the Hippocampus as well as the cortex. Co-localization of proSAAS was discovered after finding co-localization of 7B2 within mouse models, and the researchers wagered that due to its overabundance compared to 7B2 that proSAAS would also co-localize. Then the researchers went on to test if proSAAS could prevent the formation of misfolded Beta-sheet fibrils from forming. Further testing revealed that 21 kDamproSAAS demonstrated effective prevention of Beta-sheet fibril formation depending upon the given dose size, as well as that proSAAS drastically reducing the length of fibrils formed, in some cases up to a 75% reduction in length (Hoshino et al., 2013). Following this development, the researchers attempted to understand what areas of the proSAAS molecule were responsible for the prevention of misfolded protein amylation. By inhibiting protein expression in certain Alpha-helices present in the proSAAS compound, the scientists were able to isolate certain Alpha-helices for the purposes of testing their amylation prevention capabilities. Finding that it is likely that Alpha-helix groups 2 and 3 are both required in order to prevent fibrillation, they also attempted to see what prevented Alpha-Beta cytotoxicity by using AB oligomers to induce amylation within Neuro2a cells, and observed two sets, one with lactalbumin and carbonic anhydrase, the other with proSAAs. The cells with proSAAS were found to successfully prevent cytotoxicity while those with only lactalbumin and carbonic anhydrase were incapable of regulating cytotoxicity. The researchers also experimented on the Neuro2a cells by injecting them with lentiviruses saturated with proSAAS with the hopes of overexpressing the protein and found that by endogenously exposing cells to the virus the levels of proSAAS were almost doubled, and that viable cell counts were dramatically increased following these injections. They concluded that in Neuro2a cells, exogenous and endogenous exposure to proSAAS lead to a drastic decrease in cytotoxicity when compared to cells not exposed (Hoshino et al., 2013).
Results
Following these findings, the researchers then proposed several explanations and further hypotheses based off of the data collected. Some of the more interesting developments in the data suggest that proSAAS prevented amylation and plaque formation depending on the stage of said plaque formation, and that overexpression of APP in mice brains triggered co-immunoprecipitation of both proSAAS and Abeta, suggesting that there is a physical association between amyloidogenic peptides and proSAAS while in vivo. Another study cited in the paper found that the direct binding of chaperone proteins to oligomers aids in preventing toxicity within cells, and the research team suggest that a similar event may have occurred with the introduction of proSAAS in the respective cells used in the experiment.
Reflection
These findings suggest that through the insertion of proSAAS within human nervous cells via virus-template carriers or other delivery methods that misfolded protein amylation could be prevented, leading to a reduction in the formation of plaque within the brain, and possibly preventing Alzheimer’s from occurring altogether. The research is still extremely experimental and repeated trials must be conducted in order to determine if these methods are truly safe for human testing or use. If these tests are found to be successful, the potential medical and pharmaceutical applications for this technology are immense. With further knowledge on the human brain and the processes involved in its degeneration coupled with more testing, scientists could potentially find ways to altogether remove the risk of humans ever developing certain misfolded protein-related neurological diseases and pave the way for a bright future for neuroscience.
References
Ashraf, G., Greig, N., Khan, T., Hassan, I., Tabrez, S., Shakil, S., Sheikh, I., Zaidi, S., Akram, M., Jabir, N., Firoz, C., Naeem, A., Alhazza, I., Damanhouri, G. and Kamal, M. (2014). Protein Misfolding and Aggregation in Alzheimer’s Disease and Type 2 Diabetes Mellitus. CNS & Neurological Disorders – Drug Targets, 13(7), pp.1280–1293. https://doi.org/10.2174/1871527313666140917095514
Beissinger, M., & Buchner, J. (1998). How chaperones fold proteins. Biological chemistry, 379(3), 245–259.
Hoshino, A., Helwig, M., Rezaei, S., Berridge, C., Eriksen, J., & Lindberg, I. (2013). A novel function for proSAAS as an amyloid anti-aggregant in Alzheimer’s disease. Journal Of Neurochemistry, 128(3), 419-430. https://doi.org/10.1111/jnc.12454
Wang, W. and Roberts, C.J. (2018). Protein aggregation – Mechanisms, detection, and control. International Journal of Pharmaceutics, 550(1-2), pp.251-268. https://doi.org/10.1016/j.ijpharm.2018.08.043
Fluidic Analytics. (n.d.). Protein aggregation—why it matters, and how to study it. [online] Available at: https://www.fluidic.com/resources/protein-aggregation-and-why-it-matters/ [Accessed 29 Nov. 2021].
Chemistry LibreTexts. (2013). Protein Folding. [online] Available at: https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Protein_Structure/Protein_Folding [Accessed 28 Nov. 2021].
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