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Creation of Novel alpha-Synuclein Mutation A53E Truncations 123 and 120 for Future Study in Budding Yeast
Alex Dunn & Logan Graham
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045
*This author wrote the paper as a part of BIOL221: Molecules, Genes, and Cells under the direction of Dr. DebBurman.
Parkinson’s disease (PD) is a neurological disorder resulting from the loss of dopaminergic neurons in the substantia nigra. ɑ-Synuclein, a PD-associated protein, clumps and forms aggregates, or Lewy bodies, in PD patients. Recently, a novel familial mutation of ɑ-synuclein, A53E, was discovered. Our study aimed to create two truncated versions of the familial mutant A53E by removing amino acids from the C-terminus of the protein in order to use to analyze in future studies compared to the more well studied wild-type and full-length versions of ɑ-synuclein. We hypothesized that the A53E mutation, as well as the C-terminus truncations, would cause a more aggressive pathology than wild-type or full-length ɑ-synuclein. As more amino acids were removed from the C-terminus of ɑ-synuclein, we predicted solubility to decrease and aggregation to increase. We successfully PCR amplified truncations A53E 123 and 120, displaying correct protein length, later subcloning and transforming the purified fragments into E. coli. Bacterial transformation produced positive colony growth, which was used for transformation into S. cerevisiae. The two truncations of ɑ-synuclein created, A53E 123 and 120, will be used in future studies to study the properties ɑ-synuclein displays in yeast, including patterns in localization and toxicity.
Parkinson’s disease (PD) is a progressive movement disorder characterized by neuronal loss and Lewy body formation (Spillantini et al., 1997; Polymeropoulos et al., 1997). Symptoms include tremor-at-rest, bradykinesia, and stiffness of limbs (Fahn, 2008). Parkinson’s disease is characterized by a loss of dopaminergic neurons in the substantia nigra (Polymeropoulos et al., 1997). In the diminished substantia nigra of PD patients, clumps of protein, called Lewy bodies, are present (Zabrocki et al., 2005). Lewy bodies are formed from the naturally occurring and abundant protein ɑ-synuclein. ɑ-Synuclein serves to release chemicals in the brain, has an unusually flexible shape, and is characterized by two main features: it likes to bind to membranes and form aggregates (Jin et al., 2011). In those with PD, too much ɑ-synuclein is being made or too little is being degraded, creating an imbalance. This imbalance is generated when ɑ-synuclein misfolds and accumulates to form Lewy bodies, leading to toxicity and neuron death (Petrucelli and Dickson, 2008; Jin et al., 2011).
There are two types of Parkinson’s disease, sporadic, accounting for 90 percent of individuals, and familial, accounting for 10 percent of individuals (Fahn, 2008). These two types are used to differentiate genetic from idiopathic forms of PD. There are eight known genes that, when mutant, can lead to genetic forms of PD. The first, SNCA, is the gene that encodes the protein ɑ-synuclein, the central problem in both genetic and sporadic PD. The other seven: Parkin, UCH-L1, PINK1, DJ-1, LRRK2, ATP13A2, and HTRA2, can also misfold ɑ-synuclein to form Lewy bodies (Ross, 2008; Polymeropoulos et al., 1997).
Past research has shown ɑ-synuclein can be mutated, modified by phosphates and nitrates, and shortened into smaller versions (Polymeropoulos et al., 1997; Fujiwara et al., 2002). Our research will explore mutated and smaller versions of ɑ-synuclein. We will focus on a recently discovered familial mutation of ɑ-synuclein, A53E, and how cutting this mutation into smaller versions will affect the membrane binding, aggregation, and solubility of ɑ-synuclein. Two truncated forms of ɑ-synuclein, 123 and 120, will be designed in this study. Truncation occurs by removing amino acids from the C-terminus of ɑ-synuclein, making the protein shorter. Truncations at the carboxyl and amino termini are naturally occurring in Lewy bodies, which are characteristic of PD. The C-terminus is crucial for maintaining the shape of ɑ-synuclein, which keeps the protein soluble (Hong et al., 2011). The C-terminus is rich in glutamic and aspartic residues similar to proteins with chaperone-like activity. It was found that C-terminal truncated ɑ-synuclein does not possess chaperone-like activity, showing the C-terminus plays a role in this activity and is important for maintaining ɑ-synuclein solubility as C-terminal truncated ɑ-synuclein aggregates faster than full- length ɑ-synuclein (Souza et al., 2000). Additionally, Kanda et al. (2000) found truncations at the 120 amino acid in the C-terminus are more susceptible to oxidative stress, which is hypothesized to influence neuronal loss in PD patients.
Several mutations in ɑ-synuclein have been found to cause genetic forms of PD, including A53T, E46K, A30P, H50Q, and G51D. Kanda et al. (2000) found the two well-studied familial mutations A53T and A30P are affected by oxidative stress, causing neuronal cell death associated with PD. A53T is known to cause ɑ-synuclein to bind to the membrane, while A30P localizes ɑ-synuclein in the cytoplasm (Sharma et al., 2006). A53T is more aggressive, suggesting that toxicity involves ɑ-synuclein binding to the membrane (Kanda et al., 2000; Sharma et al., 2006). Another familial mutant, E46K, also binds to the membrane, but shows no signs of toxicity (Volles and Lansbury, 2007). H50Q, a fourth mutant, does not bind tightly to the membrane, which causes it to aggregate faster. More work is being done to see if mutation G51D has the same effect as H50Q (Ghosh et al., 2013). A sixth mutation, A53E, has been discovered recently in one family. The patient experienced early onset pathology, age 36, which is typical of familial PD (Pasanen et al., 2014). An abundance of SNCA, the gene that encodes for ɑ-synuclein, was found in the patient’s brain and spinal cord. This suggests A53E makes ɑ-synuclein aggregate aggressively (Pasanen et al., 2014). Researchers suggest A53E is the cause of the patient’s PD, however more research is needed to investigate A53E’s role in PD. Designing this gene will help us gain a better understanding into the role A53E plays in PD, as well as the effects C-terminus truncations have on ɑ-synuclein.
It is known that the A53E mutation causes aggressive aggregation of the SNCA gene. Beyond this, the role of A53E in PD pathology is not yet understood. Also, the role of the C-terminus in ɑ-synuclein is still unclear. We hypothesize that the A53E mutation, along with the C-terminus truncation, will cause a more aggressive pathology than wild-type or full- length ɑ-synuclein. We hypothesize that solubility will decrease and aggregation will increase, as more amino acids are being removed from the C-terminus of ɑ-synuclein when the protein is truncated. Thus, the 120 amino acid truncation will show more aggregation than the 123 amino acid truncation, as more amino acids will be removed from the 120 version compared to the 123 version.
In this study, yeast will be used to explore the role ɑ-synuclein plays in PD. Yeast is the first eukaryote for which the entire genome was mapped. This allows for easy manipulation of genes; genes can be taken out and added to yeast at will. As PD is a protein misfolding and degradation problem and yeast make, fold, and degrade proteins similar to humans, yeast are an excellent model for which to study PD (Allendoerfer et al., 2008). Yeast are also inexpensive, therefore an appropriate model for undergraduate research. Yeast have provided significant insights into PD, showing toxicity patterns for ɑ-synuclein as well as localization patterns of the protein inside a cell (Sharma et al., 2006; Fiske et al., 2011). When expressed in yeast, ɑ-synuclein has been shown to associate with the plasma membrane in a highly selective manner (Outeiro and Lindquist, 2003).
In this study we aim to make two truncated versions of the familial mutant A53E of ɑ-synuclein (Figure 1C). We will make truncated versions 123 and 120 of familial mutant A53E, both of which will be tagged with green fluorescent protein (GFP) (Figure 1A). GFP will be used to show localization patterns inside the cell in future studies, in order to analyze the membrane binding and aggregation patterns of ɑ-synuclein compared to full-length and wild-type versions of the protein (Sharma et al., 2006).
Overall Project Design and Gene Fragment Amplification by PCR
Our aim was to create two truncated versions of ɑ-synuclein, syn-123-GFP and syn-120-GFP, of the familial mutant A53E (Figure 1C). This process required seven stages (Figure 1A), with the project outlined in this experiment beginning on the fourth stage. The last four stages, four to seven, were divided into six steps. The first three stages were previously completed by the DebBurman lab, who amplified and tagged the truncated versions of ɑ-synuclein with GFP.
In order to begin step one, stage five, primers were designed to amplify the gene region of interest from plasmid to be used in PCR. Both forward and reverse primers were designed to define the 5’ and 3’ boundaries of the syn-123-GFP and syn- 120-GFP fragments of ɑ-synuclein. The control plasmid, GFP- A30P 110, was previously designed for use in this project (Figure 1B). After designing the primers to be used in PCR amplification, ɑ-synuclein was amplified. This created many versions of GFP- tagged A53E 123 and 120, to then be purified.
Gene Fragment Purification by Gel Electrophoresis
A53E 123, A53E 120, and positive and negative controls were first predicted on a gel to compare with the actual gel results (Figure 2A). The product gel from PCR amplification with 10 KB ladder (Figure 2B) shows positive results for A53E 123, A53E 120, and the positive control. The positive control shows that PCR amplification worked, as there is a band present, demarking protein is present. The negative controls show no bands, which is to be expected. The negative controls are present to show that PCR amplification needs all its essential ingredients, without one the amplification step will not occur. Lastly, the PCR amplification gel shows bands for both A53E 123 and 120, which tells us that protein is present and we can move onto gel purification.
After amplifying the desired fragments of ɑ-synuclein, the PCR product was separated on an agarose gel by electrophoresis in order to remove the DNA fragment from the gel and clean it by removing agarose and other gel components.
This will ensure only the desired PCR product is present in the mixture. Again, the initial gel displays positive results for A53E 123 and 120 with 2 KB ladder (Figure 2B). After extracting the pure PCR product from the gel by manual excision (Figure 2C) and isolating the gene of interest through the GENECLEAN Turbo Kit, we then confirmed that the desired PCR product is present in the mixture. The next step was to subclone this into TOPO vector and transform into bacteria.
Subcloning into Plasmid Vector and Bacterial Transformation
The previously prepared and purified PCR product was then transformed into a plasmid vector for expression in budding yeast (S. cerevisiae), as shown in stage 5 (Figure 1A). After the TOPO cloning reaction, the vector was then transformed into bacteria and grown on plates containing ampicillin. Typically, bacteria die in the presence of ampicillin unless they have a plasmid vector that contains an ampicillin resistance gene. The only bacteria predicted to grow on these plates thus were the ones that were transformed with a closed circular PYES2 vector. Therefore, this method was used to ensure only bacteria that contain the vector with a properly subcloned PCR product, A53E 123 and 120, will multiply and survive.
Both positive and negative controls were plated, along with 30μL and 200 μL of A53E 123 and 120 (Figure 3). The positive control showed growth, as was expected, demonstrating that the TOPO vector was active, containing the ampicillin resistance gene allowing the bacteria to survive. The negative controls showed no growth, as per expected, showing that without vector, the cells will not grow. Both A53E 123 and 120 showed growth on both the 30μL and 200 μL plates. There was more growth on the 200μL plates for both truncations. No contamination was present, demonstrating successful transformation. The next step was to choose colonies to perform whole cell and plasmid-based PCR on, to check the orientation of the fragment.
Orientation Check I: Whole Cell PCR and Plasmid Prep and Plasmid Based PCR
Once the vector was transformed into bacteria, the orientation of the gene fragment needed to be confirmed. Genes can be inserted in the TOPO vector in one of two possible orientations, an incorrect or correct orientation. Therefore, in our transformation plates, we most likely had colonies of each possible orientation. In order to study the gene expression, it was important that our gene fragments be correctly aligned with the gene promoter on the 5’ end on the subcloning site of the vector. Thus, we had to identify which bacterial colonies had the correct orientation.
In order to test orientation, four whole cell PCR reactions and four plasmid-based PCR reactions were conducted by using cell colonies from the LB+amp plates. A forward primer that binds to the gene promoter and a reverse primer that binds to the 3’-end of the gene fragment was used. Positive control GFP-A30P 110 was also included to ensure the PCR reaction worked.
Orientation Check II: Bacterial and Plasmid PCR Gel Electrophoresis
Plasmid-based and whole cell PCR results were first predicted for A53E 123, A53E 120, and positive control GFP- A30P 110 on a gel to compare with the actual gel results (Figure 4A). The product from PCR amplification (Figure 4B) shows the positive control worked, demonstrating PCR amplification was successful as there is a band of protein present. 10 KB ladder was used for each gel. However, the other reactions for both A53E 123 and 120 were not successful. This is demonstrated by the absence of bands at the expected lengths. This signifies that whole cell and plasmid-based PCR were not successful for both the truncations. Therefore, before transforming into yeast, we had to choose more colonies and repeat whole cell and plasmid- based PCR until positive results were present. The DebBurman lab completed this process, providing us with correctly orientated A53E 123 and A53E 120 plasmids to proceed with onto yeast transformation. These plasmids were sent out for sequencing to the University of Chicago to ensure the fragments were in the correct sequence. The sequences came back with positive results for A53E 120 and negative results for A53E 123. Thus the DebBurman lab repeated the whole cell and plasmid-based PCR process to provide us with a correctly orientated A53E 123 plasmid to proceed with onto yeast transformation.
Plasmid Vector Transformation into Yeast
Plasmids were then transformed into yeast cells through LiAc transformation. A53E 123, A53E 120, positive, and negative controls were plated onto either SC-URA or YPD plates in 30μL or 200μL amounts. The positive control showed growth and the negative control showed no growth, as was expected. Both A53E 123 and 120 showed growth on both the 30μL and 200μL plates. There was more growth on the 200μL plates for both truncations. No contamination was present, demonstrating successful transformation. The photos of these yeast transformation plates were lost in the computer network, therefore there is no visual data to display these results. Refer to DebBurman Biology 221 papers for the plates for these data, as each truncation was done by two separate groups and two other groups have the photographs of these plates.
The goal of this experiment was to create tools to further study the role of ɑ-synuclein in Parkinson’s disease, to one day lead to treatment development. We hypothesized that the two C-terminus truncations 123 and 120, along with the A53E mutation would cause a more aggressive pathology, an increase in aggregation, and a decrease in solubility compared to the wild-type and full-length versions of ɑ-synuclein.
From this experiment, we were able to correctly isolate and purify our two desired gene fragments, A53E 123 and 120. This was confirmed by gel electrophoresis of the gene fragments (Figure 2B). The gel showed that correct primers were designed and were able to amplify the desired gene fragments. Protein bands at 120 and 123 base pairs confirmed that we were able to successfully truncate the C-terminus at the desired base pairs.
We were also able to correctly insert the gene fragments into vectors, which were transformed into E.coli cells. This was confirmed by successful cell growth on LB+amp plates. As the transformed vector included the Ura3 gene, which is resistant to ampicillin, the cells would be unable to survive without Ura3. Therefore, as growth was shown this meant the vector had been successfully inserted.
Inserting the gene fragments into the vector to be transformed into bacterial cells was not the most important aspect of this step. The crucial moment came when confirming the orientation of the gene fragments in the vectors. As demonstrated in Figure 4B, gel electrophoresis failed to produce any bands in the expected areas. Therefore we concluded that all eight of our tests, four whole-cell PCR and four plasmid-based PCR tests, had incorrect orientation of both gene fragments. The orientation in the vector is determined by chance, so while our results were unusual, there does not appear to be any significant error that caused this. However, as the orientations were incorrect, the plasmids could not be transformed into yeast cells because they would not survive. The DebBurman lab created new plasmids, one for A53E 123 and one for A53E 120, with correct orientations that we then used to transform into yeast.
Future experiments will be conducted to examine the properties familial mutant A53E displays in yeast. Specifically, exploring the properties of familial mutant A53E against the wild- type version and also the truncated forms, 123 and 120, against the full-length version will show how these modifications alter the ɑ-synuclein protein. Four assays will be used to examine how these modifications alter ɑ-synuclein in yeast.
First, localization will be studied using fluorescent microscopy (Sharma, 2006). As the versions of ɑ-synuclein created in this experiment were tagged with GFP, this assay will show where ɑ-synuclein is located in the cell and help to determine trends in localization. We predict that familial mutant A53E will be more toxic than the wild-type version of ɑ-synuclein, as one amino acid is being altered in the familial mutant compared to the wild-type. Thus, looking at fluorescent microscopy of A53E, we predict to see more accumulation, or aggregates, of ɑ-synuclein in the cell. When truncated versions 123 and 120 are taken into consideration, we predict there will be more accumulation of ɑ-synuclein as the amount of amino acids removed from the C-terminus increases. Thus, as we are removing amino acids from the terminus responsible for solubility, there will be more ɑ-synuclein accumulated throughout the cell than in full-length versions.
Second, toxicity will be studied using a spotting assay (Sharma, 2006). This assay explores toxicity patterns by examining cell survival. As previously mentioned, we predict there to be more toxicity for familial mutant A53E compared to wild-type versions of ɑ-synuclein and also more toxicity for truncated versions 123 and 120 compared to full-length ɑ-synuclein. This is due to the substitution of one amino acid in familial mutant A53E and by the removal of amino acids from the C-terminus in the truncated versions of ɑ-synuclein, leading to increased toxicity. Toxicity is detrimental to ɑ-synuclein, and leads to cell death as shown in PD patients.
Third, accumulation will be studied using a western blot (Sharma, 2006). This assay functions to separate proteins by size. We predict there to be more accumulation in both familial mutant A53E and also in truncated forms of ɑ-synuclein. Thus, we hypothesize to see bands of greater ocular density, showing more protein present, for both mutated and truncated versions of ɑ-synuclein compared to those of wild-type and full- length ɑ-synuclein.
Lastly, growth curve analysis will be used to measure growth of cells (Sharma, 2006). This assay will plot the increase in cell numbers compared to time of incubation, ultimately used to delineate stages of the cell growth cycle. It will be used to measure cell numbers and the rate of growth of yeast cells. This will be used to analyze cells with ɑ-synuclein compared to cells without ɑ-synuclein, to see how ɑ-synuclein will alter the cell growth rate. We predict for there to be less growth in the cells with ɑ-synuclein present, therefore in the two truncated forms of A53E, 123 and 120, there will be less growth compared to cells without these truncations present. This prediction is supported by our hypothesis that toxicity will increase for the truncated forms of familial mutant A53E. Toxicity alters the rate of cell growth, therefore if more toxicity is present, the less cells will grow.
Our aim was to create two truncated versions of the familial mutant A53E by removing amino acids from the C-terminus of the protein in order to use to analyze in future studies compared to the more well studied wild-type and full- length versions of ɑ-synuclein. We successfully PCR amplified both A53E 123 and A53E 120, subcloned, and then transformed the purified fragments into E. coli. We then transformed into S. cerevisiae from the positive colony growth from bacterial transformation. The two successfully created truncations of ɑ-synuclein, A53E 123 and 120, will be used in future studies to study the properties ɑ-synuclein displays in yeast, including patterns in localization and toxicity.
Materials and Methods
Primer Design and Synthesis
In order to create the specific truncations used in this lab, the primers were first designed according to the DebBurman (2014) manual. Forward and reverse primers were designed using the known 140 amino acid sequence for ɑ-synuclein, to truncate the C-terminus in the desired regions, 123 and 120. For the forward primer, 30 nucleotides were used on the coding strand going from 5’ to 3’. For the reverse primer, 30 nucleotides again were used on the template strand going from 5’ to 3’.
Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used
ɑ-Synuclein gene in a plasmid vector was used as the template DNA (DebBurman, 2014). PYES2.1 plasmid vectors were used when subcloning gene fragments. These plasmid vectors contained the gene Ura3, which is ampicillin resistant and makes uracil. This was included so the E. coli cells could survive on SC-URA (without uracil) plates, as E. coli would normally be killed by the ampicillin without this present. S. cerevisiae was the strain of yeast used in this study.
ɑ-Synuclein Gene Fragment Amplification by PCR
PCR amplifies the desired gene region from the plasmid, allowing the primers to bind more specifically to the gene fragment with every amplification. This process began by preparing one positive control, to ensure PCR works properly by using previously tested DNA template and primers, in this case GFP-A30P 110. A negative control was also prepared to demonstrate that the PCR reaction will not occur unless all the essential ingredients were included. The negative control removed the primer from the reaction. The materials used to prepare the plasmids to undergo PCR included master mix, sterile RNase-free water, forward primer, reverse primer, and purified plasmid. This process was done by repeating cycles of different temperatures 29 times for 30 seconds each. When the cycle reaches 95°C, the once double stranded DNA separates into two single strands. The temperature is then decreased to 55°C, when the primers bind to the DNA strands. The temperature is increased up to 72°C, where the polymerase binds to the primers and starts adding nucleotides. After all 29 cycles, the product is stored for 30 minutes in 72°C. At the end of the process, the products are stored in 4°C until gel electrophoresis is conducted.
Gene Fragment Purification by Gel Electrophoresis
After completing PCR, the products underwent gel electrophoresis in order to isolate the intended fragment from the rest of the plasmid. Once a gel was ran, the bands containing the desired gene fragments were cut out manually and prepared to be subcloned into the vector by purification according to the GENECLEAN Turbo Kit. Turbo salt solution, ethanol solution, and turbo elution solution were used to purify the gene fragments. The fragments were stored at -20°C once purified.
Subcloning into a TOPO Vector and Bacterial Transformation
The purified gene fragments were then subcloned into mammalian vectors tagged with GFP to be expressed in E.coli cells using the TOPO cloning reaction. After sub cloning, the vectors were transformed into bacteria cells and plated on LB+amp plates. The plates contained ampicillin, which would kill the bacteria cells if they were not given the Ura3 ampicillin- resistant gene. Negative and positive controls were also plated to ensure successful transformation. The growth of bacteria was monitored 24 hours later, incubating at 37°C.
Confirmation Gene Fragment Orientation in Vector in Bacteria
After monitoring the products of bacterial transformation, the plates displayed cell colony growth, but that did not mean the gene fragments were oriented correctly on the vector. The correct orientation would be the fragment aligning itself with the 5’ end of the subcloning region of the vector. To determine if the fragment was properly aligned, whole cell PCR was performed. This process will confirm orientation by creating PCR products if there was a correct orientation, and creating no PCR products if there was an incorrect orientation. The materials used to prepare the reaction included master mix, sterile RNase- free water, Gal forward primer, Syn reverse primer, and bacterial cells. A positive control was also prepared using the original plasmid instead of bacterial cells to ensure PCR worked. The products were stored below 4°C.
Plasmid Vector Purification
The plasmid containing the gene fragment was isolated from the bacteria cells by first vortexing and transferring to a microfuge tube. The cells were then pelleted by centrifuging the tube and pipetting off the supernatant. The cells were washed and buffers were added, vortexing between each step. The product was centrifuged again and transferred to another column, where it was repeatedly washed and buffers were added according to the QIAGEN QIAprep Miniprep Kit.
After purification of the plasmid, the products were run through gel electrophoresis. This was to confirm the orientation of the fragments in the vector. If there was correct orientation, the bands would appear in the correct location on the gel. If there was incorrect orientation, no bands will be produced. If bands are produced, they will be sent to the University of Chicago for sequencing.
Plasmid Vector Transformation into Yeast
The yeast initially did not contain the ampicillin- resistant gene Ura3, so the gene was included in the pYES2.1 vector. Transformation mix included PEG, LiAc, single-stranded carrier DNA, water, and plasmid DNA. The yeast (with the vectors) were then put onto plates. Three plates were used for fragment one: 200μL and 30μL on an SC-URA plate and 30μL on an YPD plate. The same process was used for fragment two. 200μL on SC-URA plates and 30μL on YPD plates were used for both positive and negative controls. The plates were stored in the refrigerator and monitored for the following week. The positive controls were expected to show growth, while the negative controls were expected to show no growth.
Thank you to Dr. DebBurman for teaching the Biology 221 course and instructing the laboratory section. Also thank you to the Biology 221 lab curriculum, Jyothis James for assisting with lab work and reports, the DebBurman lab for preparing plates and media, and Alex Roman for the truncations for which we began our research.
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