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Acetylation Sites Lysine 6 and Lysine 10 are Potential Targets to Influence Alpha- Synuclein Toxicity in Six Mutants Known to Cause Familial Parkinson’s Disease: A30P, A53T, E46K, G51D, H50Q, and A53E

Alex Biel and Nicole Hedger
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045


Parkinson’s disease is a hypokinetic movement disorder marked by the loss of dopaminergic neurons in the midbrain. Its main pathological marker is the accumulation of Lewy bodies, which are made up of misfolded alpha-synuclein. In addition to the six known familial mutations, posttranslational mutations like glycation and acetylation have also been shown to influence alpha-synucle­in’s toxicity. To try and investigate the effects these mutations and modifications have on alpha-synuclein, we attempted to make a K6Q mutation on A30P, A53T, and a K6Q/K10Q mutation on G51D. These mutants were created by a step- by- step mutagenesis reaction. We hypothesized that all mutations would be created, successfully ac­cepted into E. coli for growth, and would be transformed into yeast to be prepared for further assays. The overall predictions for the success of these mutations were: 1) the K6Q mutation on A30P and A53T would result in less aggregation of familial mutant alpha-synu­clein, which leads to its toxicity and results in genetic Parkinson’s Disease, and 2) the K6Q/K10Q mutation on G51D would further exacerbate these effects. All mutations were successfully created, taken in by E. coli to grow more copies of the mutated plasmid, and transformed into yeast. Important knowledge has been gained from this experiment, but we are still far from finished. Only further assays will prove whether progress was truly made in the vastly uncertain world of Parkinson’s Disease.


Parkinson’s Disease (PD) is characterized by the loss of dopaminergic neurons in the midbrain. It is the second most common neurodegenerative disease after Alzheimer’s (Ross, Braithwaite, Farrer, 2008). There are currently more than four million cases of PD worldwide; the diagnosis is one that a general physician can make (Outeiro, 2003). Parkinson’s Disease is a hypokinetic movement disorder: “hypo” meaning less, “kinetic” meaning movement. The requirements for a diagnosis of Parkinson’s Disease is any combination of the following movement abnormalities: tremor-at-rest, bradykinesia, poor posture, rigidity, shuffling gait, poor balance, and the “freezing phenomenon” (Fahn, 2008). The di­agnosis, however, does require an individual to present with bradykinesia and tremor-at-rest as defining symptoms (Fahn, 2008). These symptoms do not necessarily lead to death in patients, but a secondary cause stem­ming from these symptoms may lead to mortality (Fahn, 2008).

Parkinson’s Disease and several other neurodegenerative diseases all fall under a broader category of diseases known as synucle­inopathies, in which the protein alpha-synuclein forms clumps (aggre­gates) and kills the cells (Dawson & Dawson, 2003). The key pathological marker that defines PD is the presence of Lewy bodies on the inside of neuronal cells. Lewy bodies are mainly composed of misfolded α-sy­nuclein protein (Petrucelli & Dickson, 2008). There are two main types of Parkinson’s Disease: sporadic and familial. 90% of the cases are sporadic, but it has been difficult to identify the causes of development for the sporadic forms. Parkinson’s Disease may be described as a neurode­generative disease with many possible etiologies. Possible etiologies of sporadic-type PD that have been suggested are altered metal homeosta­sis, environmental toxins, and mitochondrial dysfunction (Bossy-Wetzel et al., 2004). Certain gene mutations in α-synuclein have been discovered to play a role in the development of genetic, or familial, Parkinson’s Disease (Ross et al., 2008). When dopaminergic neurons are functioning properly, they give the substantia nigra in the midbrain bands of a characteristic dark pigment. The aggregation of α-synuclein protein in the dopaminergic regions of the substantia nigra results in a noticeable depigmentation in these regions. About 90% of these dopaminergic neurons die before the first symptoms are even present (Petrucelli and Dickson, 2008). There are many genes that can lead to the onset of Parkinson’s disease other than alpha-synuclein. Some disease-causing genes include Parkin, Pink1, DJ-1, ATP13, LRRK2, and VPS35. Some examples of risk factor genes include SAC1, VPS13, and SWA2. (Brás, Guerreiro, and Hardy, 2015). In addition, there are six familial mutations on alpha synuclein that are directly linked to genetic Parkinson’s Disease that will be described later in this article.

The function of α-synuclein remains mostly unclear. It is one of the most abundant proteins in the brain, and it works at the synapse level. Due to its abundance in presynaptic terminals and synaptic vesicles, there is evidence that α-synuclein plays a key role in vesicle transport and neurotransmission (Abeliovich et al, 2000). In mice, it has been shown that knocking out α-synuclein leads to a decrease of synaptic vesicles, synaptic responses upon stimulation, and a poorer recovery of synaptic strength from use-dependent depression (Chandra et al., 2004). One may argue that this is evidence suggesting that α-synuclein could be involved in brain plasticity, a phenomenon that invertebrates do not experience (Szego et al., 2012). Alpha synuclein is 140 amino acids long and has three domains, each with corresponding functions: membrane binding in the N domain, aggregation in the M domain, and solubility in the C domain (Bartels et. al, 2010). There are six familial mutations - implicated in PD that are concentrated in the N-domain, and posttranslational modifi­cation sites for glycation and acetylation have been found there. There is a common hypothesis that Parkinson’s Disease is a result of α-synuclein coming out of solution, misfolding, and aggregating, leading to its cellular toxicity. The influence of posttranslational modifications and the presence of a familial mutation may affect the localization, accumulation, or solubili­ty of the protein.

As mentioned previously, there are six mutations known to cause familial Parkinson’s disease, and much more is known about these 10% of PD cases. The six known familial gene mutations found in the N-terminus of α-synuclein are: A30P, E46K, H50Q, G51D, A53T, and A53E. A53T, E46K, H50Q and the wild-type α-synuclein mutations have an increased affinity for lipid membrane binding (Outeiro, 2003). This results in toxic levels of aggregation and what we know as Lewy Bodies. In addition, in the A30P and A53T mutants, the catabolic pathway by which wild-type α-synuclein is normally degraded is affected, which leads to further accumulation and toxicity (Ghosh et al., 2014). In the A30P and G51D mutants, protein aggregates are mainly cytoplasmic as they exhibit impaired membrane association. This is most likely due to a defect in endocytosis. In fact, these two mutations behave very similarly in yeast (Fares et al., 2014). One defining characteristic of G51D is that it enhanc­es mitochondrial fragmentation in primary neurons. It is believed that this is where its toxicity lies, given that it does not bind to membranes. H50Q produces toxicity by enhancing aggregation of α-synuclein in neurons (Fares et al., 2014). The A53E mutation has a mechanism that is yet to be delineated; however, it does show reduced aggregation in cells, indicating that, instead of generating large amounts of aggregation, the mutation has a different mechanism of toxicity (Rutherford and Giasson, 2015). Individuals who have any of the six known mutations will develop early-onset Parkinson’s Disease.

Glycation and acetylation are two post-translational modifica­tions that can occur in proteins. Glycation isan age-related post-transla­tional modification that has beenshown to enhance α-synuclein toxicity in Drosophila and mice (Miranda et al., 2017). Glycation influences the N-terminus of α-synuclein and has been shown to reduce membrane binding and impair its clearance (Miranda et al., 2017). In other words, glycation within the protein α-synuclein produces negative effects on the cells. Research has shown that α-synuclein can be acetylated and glycat­ed on lysines 6 and 10, and that these sites can be deacetylated by the protein sirtuin 2 (De Oliveira et al., 2017). A mutation- blocking acetylation in the substantia nigra in rats has been shown to decrease α-synuclein toxicity. This goes to show that acetylation may be a regulatory mecha­nism when it comes to the aggregation and toxicity of α-synuclein (De Oliveira et al., 2017). Acetylation has been shown to have a positive influ­ence on cells while glycation has a negative impact. While some research has been done on both post-translational modifications, the mechanisms of their interaction with the six known familial mutations on α-synuclein are unknown.

The most common and appropriate organism in which to test these mutations and others is budding yeast, or Saccharomyces cerevisi­ae. Budding yeast is a single-celled eukaryote, so it contains membrane-bound organelles likehuman cells (Duina et al., 2014). The genome of yeast has been completely sequenced and described because of its small size, not to mention that the genes and proteins in yeast are very similar in function to those in humans. Saccharomyces cerevisiae reproduces quite readily and very quickly by “budding”, which allows scientists to conduct research (Allendoerfer, 2008). The key feature that diversifies budding yeast as a model organism is the simplicity of adding or remov­ing genes from the cell, whether it be by means of a plasmid that has been replicated in bacteria beforehand, or by inserting new genes directly into the chromosomes (Duina et al., 2014). Since the important pathology of Parkinson’s Disease involves the misfolding of a protein, budding yeast is an excellent model organism to study it because they make, fold, and degrade proteins just like humans do (Sharma et. al, 2006).

To establish the interaction of glycation and acetylation and the six known familial mutations, we first plan to create a K6Q mutation in which lysine is being converted to a glutamine on the 6th amino acid. We will then combine this mutation with the six known familial mutations. We will also create a K6Q/K10Q mutation in which lysine is being converted to a glutamate on the 6th and 10th amino acids and combine this muta­tion with the G51D familial mutation. These mutations will block glycation and mimic acetylation on these particular sites. In doing this, we will be ultimately creating seven mutagenized products. To gain success, we have four aims: 1) create the mutants by PCR mutagenesis, 2) transform them into bacteria,3) purify the plasmid from the bacteria and sequence it, and 4) transform the mutants into S. cerevisiae for future assays. For the purposes of the K6Q mutation, we hypothesize that the mutation will be successfully created, accepted by both E. coli and yeast, and will be phe­notypically different from wild-type α-synuclein. We hypothesize that com­bining the K6Q or the K6Q/K10Q mutation with the six familial mutants will exacerbate the phenotype elicited by the K6Q or K6Q/K610 mutation alone because the familial mutations already have a negative impact on cells. Our hypothesis regarding the mutation is that the acetylation mutation on α-synuclein will be different in the way that it folds, which will cause it to be less “sticky than wild-type and familial mutant α-synuclein. By acetylating amino acid 6 and/or 10, this will result in steric hindrance due to the large acetyl group and will therefore prevent misfolding of the protein (Iyer et al., 2016). The normal “misfolding” will not be able to take place due to the conformational difficulty, preventing unfavorable folding and protein aggregation in the nerve cells of the brain.


Primer Design and K6Q Mutagenesis of A30P, A53T, and E46K Alpha-Sy­nuclein and K6Q/K10Q Mutagenesis of G51D Alpha-Synuclein

An alpha-synuclein cartoon in the Wild Type (WT) version was compared to our mutant plasmids in Figure 1A. The goal of the project was to create a K6Q mutation with the familial mutations A30P, E46K, and A53T, and a K6Q/K10Q mutation with the familial mutation G51D. The location of the amino acid mutation is indicated in Figure 1A. The primers for the mutations and WT sequences are depicted in Figure 1B. The over­all schematic of our experimental design is shown in Figure 1C. The goal was to create our mutants in Saccharomyces cerevisiae in a PYES2 DNA vector. To do this, we performed a PCR reaction using full length alpha-synuclein in addition to the crafted primers. When proven successful, we transformed the mutagenized alpha synuclein into the bacteria, E. coli. This destroyed the unwanted template DNA. After the mutant plasmid was isolated, we purified it from the E. coli and confirmed the results using gel electrophoresis. When the gel electrophoresis indicated that the plasmid was present, we sent it to the University of Chicago for sequenc­ing. When the sequence indicated that we had successfully mutagenized our desired mutants, we transformed them into yeast to perform future assays. Before beginning the project, our peer leader Chisomo Mwale ran a primer check using wild type alpha synuclein on the forward and reverse primers for the K6Q mutation and the K6Q/K10Q mutation using WT alpha-synuclein by running a gel electrophoresis. The results are shown in Figure 1D. Primers were designed based on the guidance of DebBurman, 19-21.
















Figure 1. Creating the Mutations and the Overall Schematic

(A) Alpha-synuclein cartoon (WT) vs. Mutant plasmids K6Q A30P, K6Q A53T, and K6Q E46K, including the GFP gene. We predict that mimicking acetylation by means of the K6Q mutation, the proteins containing the familial mutations will aggregate and “stick” less. Top right: A30P-familial mutation: Amino acid 30 is changed from alanine to proline. K6Q mutation is done on alpha-synuclein with the A30P mutation. K6Q: Lysine to glutamine. Bottom left: E46K-familial mutation: Amino acid 46 is changed from glutamic acid to lysine. K6Q mutation is done on alpha-synuclein with the E46K mutation. K6Q: Lysine to glutamine. Bottom right: A53T-familial mutation: Amino acid 53 is changed from alanine to threonine. K6Q mutation is done on alpha-synuclein with the A53T mutation. K6Q: Lysine to glutamine.

(B) PCR primer pairs for each mutation and for control reactions.

(C) Overall schematic of experimental design. Step 1: create primers and synthesize by PCR mutagenesis. Step 2: Transform into bacteria. Step 3: Amplify mutant DNA in bacteria. Step 4: Isolate mutant plasmid by plasmid purification. Step 5: Send to university of Chicago for sequencing. Step 6: Transform into yeast.

(D) This gel shows the results for the primer checks. All success is displayed by the presence of dark bands in lanes 2-7. In lane 2, The K6Q forward primer check was successful. In lane 3, the K6Q reverse primer check was successful. In lane 4, the K10Q forward primer check was successful. In lane 5, the K10Q reverse primer check was a success. Lane 6, which contained the K6Q/K10Q forward primer check and lane 7, which contained the K6Q/K10Q reverse primer check were also com­pleted successfully, identifiable by the dark bands between .8 and 1.5 kb.


K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D Gel Electropho­resis

Using the forward and reverse primers for K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D, mutagenesis reactions were done following the procedure in DebBurman, 28-34. Control reactions for the primers (positive and negative) were also created. This procedure included running a Polymerase Chain Reaction (PCR). A DNA Agarose gel was run to separate the PCR products. The ideal versions of the gels are shown in Figures 2A-2C and they indicate where a dark black band is expected to be. Results of the actual DNA gel are shown in Figures 2D-F. Successful mutations can be determined by comparing the ideal gel and the actual gel; if the bands are in the same location, they can be deemed successful.

K6Q A30P, K6Q A53T and K6Q/K10Q G51D Bacterial Transformations

The next step was to transform the PCR products for K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D into E. coli follow­ing the procedure in DebBurman 28-34. This was done to get rid of the original template vector and keep only the mutagenized product as well as replicate them. Lysogeny Broth (E. coli) + Ampicillin (LB+ Amp) Agar plates were used to allow only cells that contained a vector with Ampicillin resistance to grow. Both 20μl and 80μl of cells were plated onto the LB+ Amp plates. Chisomo Mwale created positive and negative controls for the class, shown in Figure 3A. These plates show a lot of growth for the positive control and no growth on the negative controls. Figure 3B shows the transformation of K6Q A30P. On both the 20μl and 80μl E. coli + Amp plates the white dots indicate the presence of bacterial colonies. Figure 3C shows the transformation of K6Q A53T. Few colonies are present on both the 20μl and 80μl plates. Finally, Figure 3D shows transformation of K6Q/K10Q G51D into E. coli.






Figures A and D Legend:

Lane 10: DNA Ladder (10Kb)

Lane 9: Mutagenesis Positive Control.

Lane 8: Familial Mutant A30P Negative Control.

Lane 7: Familial mutant A30P K6Q mutagenesis.

Lane 6: Familial Mutant E46K Negative Control.

Lane 5: Familial Mutant E46K K6Q mutagenesis.

Lane 4: Familial Mutant A53T Negative Control.

Lane 3: Familial Mutant A53T K6Q mutagenesis.

Lane 2: PCR Forward Primer Check Negative Control.

Lane 1: PCR Forward Primer Check.





Figures B and E Legend:

Lane 1: nothing.

Lane 2: K6Q –A30P.

Lane 3: K6Q +A30P.

Lane 4: K6Q/K10Q -A30P.

Lane 5: K6Q/K10Q +A30P.

Lane 6: K6Q/K10Q -A53T.

Lane 7: K6Q/K10Q +A53T.

Lane 8: K10R –A53T.

Lane 9: K10R +A53T.

Lane 10: MW Ladder.





Figures C and F Legend:

Lane 1: nothing.

Lane 2: High Mass Ladder.

Lane 3: A30P Background.

Lane 4: K6Q/K10Q/A30P.

Lane 5: E46K Background.

Lane 6: K6Q/K10Q /E46K.

Lane 7: A53T Background.

Lane 8: K6Q/K10Q /A53T.

Lane 9: H50Q Background.

Lane 10: K6Q/K10Q /H50Q.

Lane 11: G51D Background.

Lane 12: K6Q/K10Q/G51D.

Lane 13: A53E Background.

Lane 14: K6Q/K10Q /A53E

Figure 2. PCR Mutagenesis Gels

(A) This gel shows the ideal gel for the K6Q mutagenesis of A30P, E46K, A53T, and PCR FP check. Gel was run backwards. See figure legend.

(B) This gel shows the Ideal gel for the mutagenesis of K6Q A30P, K6Q/K10Q A30P, K6Q/K10Q A53t, and K10R A53T. This gel was run backwards. See figure legend.

(C) This gel shows the ideal gel for the K6Q/K10Q mutagenesis of A30P, E46K, A53T, H50Q, and G51D. See figure legend.

(D) This gel shows the results for mutagenesis of A30P, E46K, A53T, and PCR FP check. See figure legend. The presence of a band at approximately 6kb is indicative of a successful PCR product.

(E) This gel shows the mutagenesis of K6Q A30P, K6Q/K10Q A30P, K6Q/K10Q A53T, and K10R A53T. This gel was run backwards. See figure legend. A relatively dark band is present at about 6kb which matches our K6Q A30P mutation on the ideal gel.

(F) This gel shows the K6Q/K10Q mutagenesis of A30P, E46K, A53T, H50Q, and G51D. There is a relatively faint band in lane 12, but it is dark enough to transform into bacteria.










Figure 3. Bacterial Controls and Transformations

(A) This figure contains the bacterial transformation control plates. Top: Negative control (transformed with water) contains no growth. Bottom left: Mutagenesis neg­ative control. No plasmid, no growth. Bottom right: Positive control, which contains the positive PCR mutagenesis control product, so there is growth.

(B) Bacterial (E. Coli) transformation plates LB+Amp media. Our K6Q mutated A30P mutagenized vectors were successfully transformed into bacteria. Left: 80μL Right: 20μL

(C) Bacterial (E. Coli) transformation plates LB+Amp media. Our K6Q mutated A53T mutagenized vector was successfully transformed into bacteria. Left: 20μL Right: 80μL

(D) Bacterial (E. Coli) transformation plates LB+Amp media. Our K6Q/K10Q mutat­ed G51D mutagenized vector was successfully transformed into bacteria. Left: 80μL Right 20μL










Figure 4. Plasmid Purification Gels

(A) This is an ideal gel for the plasmid being purified: K6Q A30P. See figure legend.

(B) This is an ideal gel for the plasmid being purified: K6Q/K10Q G51D. See figure legend.

(C) This is an actual gel for the plasmid being purified: K6Q A30P. In lanes 2-5, there were very dark bands present at about 4kb. All the plasmids were effectively purified. See figure legend.

(D) This is an actual gel for the plasmid being purified: K6Q/K10Q G51D. Lanes 1, 3, and 4 contained dark bands at about 4kb, and lane 2 contained a relatively faint band. All appeared to be successfully purified. See figure legend.


K6Q A30P and K6Q/K10Q G51D Plasmid Purification

At this point in the experiment, we gave our K6Q A53T mutation to group 4. To send for sequencing, the plasmids for K6Q A30P and K6Q/ K10Q G51D needed to be purified. Plasmid purification protocol was followed according to DebBurman 35-39. Toconfirm that the plasmid was purified, a DNA gel was run. Figure 4A shows the ideal gel for the plasmid purification of K6Q A30P. The results of the gel are shown in figure 4B. Four plasmids were purified and appeared on the gel in lanes 2-5. Figure 4C is the ideal gel for the plasmid purification of K6Q/K10Q G51D. The ideal gel shows that dark black bands are found at around 5kb base pairs. The results of the real gel are shown in Figure 4D. Four plasmids were purified, and the gel shows bands in lanes 2-5.

K6Q A30P and K6Q/K10Q G51D Sequence Results

Two of the four plasmids for K6Q A30P (plasmid 1-1 and 1-2) and K6Q/K10Q G51D (plasmid 2-1 and 2-3) respectively were sent for sequencing to the University of Chicago. Plasmid samples were pre­pared according to DebBurman, 35-39. The samples were sent with the appropriate forward primers (GAL Forward), which allowed us to see if the mutagenesis was successful. Figures 5A and 5B show the sequenc­ing results of K6Q A30P. The sequence results of K6Q/K10Q G51D are shown in Figures 5C and 5D.









Figure 5. Sequenced Plasmids

(A) Figure A is the sequence we received back from the University of Chicago for plasmid 1-1 (K6Q A30P) shown in Figure 4A as a purified plasmid. Highlighted in red is the incorrect mutation.

(B) This is the second sequence we received back from the University of Chicago. The plasmid sequence shown here is for plasmid 1-2 (K6Q A30P) shown in Figure 4A as a purified plasmid. Highlighted in green is the correct mutation, and highlight­ed in blue is another mutation which should not have been there.

(C) This is the plasmid DNA sequence for plasmid 2-1 (K6Q/K10Q G51D) shown in figure 4D as a purified plasmid.

(D) This is the plasmid DNA sequence for plasmid 2-3 (K6Q/K10Q G51D) shown in figure 4D as a purified plasmid.


K6Q A30P and K6Q/K10Q G51D Yeast Transformation

The plasmids that were deemed successful for K6Q A30P and K6Q/K10Q G51D based on sequence results needed to be transformed into S. cerevisiae. SC-Uracil plates were used (one with 30μl of cells and one with 200μl of cells), as well as a YPD plate with 30μl of cells. Figure 6A shows SC-URA and YPD positive controls and a SC-URA negative control. Figure 6B show the yeast transformation for the plasmid at 4 days growth for K6Q A30P. Figure 6C shows the yeast transformation for the plasmid at 2 days growth for K6Q/K10Q G51D. Sufficient growth was present on all three plates. Transforming the mutants into yeast allows for future assays to be conducted to test their effects on alpha-synuclein. YFG stands for your favorite gene. In this case, K6Q A30P in trials 1-3.














Figure 6. Yeast Transformation Plates

(A) Yeast transformation control plates. Upper left: SC-URA positive control, which contains

growth but not as much as the YPD positive control has. Upper right: SC-URA nega­tive control, which contains no growth. Bottom: YPD positive control, which contains a lot of growth.

(B) Yeast transformation plates day 4. 3 plates on the left- Top left: SC-URA plate with 200

μL K6Q A30P. Top right: SC-URA plate with 30 μL K6Q A30P. Bottom: YPD plate with 30 μL K6Q A30P. 3 plates on the right- Top left: SC-URA plate 2 with 30 μLK6Q A30P. Top right: SC-URA plate 2 with 200 μL K6Q A30P. Bottom: YPD plate with 200 μL K6Q A30P.

(C) Yeast transformation plates day 2. Upper 2: YPD plates with K6Q A30P muta­tion. Bottom 4: SC-URA plates with K6Q A30P mutation.

(D) Yeast transformation control plates round 2. Upper left: Positive SC-URA control. Upper right: SC-URA negative control. Bottom left: YPD positive control. Bottom right: YPD negative control.

(E) Yeast transformation plates round 2 day 2. Top: 30μL K6Q/K10Q G51D on YPD media. Bottom left: 30μL K6Q A30P on YPD media. Bottom right: 30μL K6Q A30P on SC-URA media.

(F) Yeast transformation plates round 2 day 2. Upper left: 30μL K6Q/K10Q G51D on SC-URA

media. Upper right: 200μL K6Q/K10Q G51D on SC-URA media. Bottom: 200μL K6Q A30P on SC-URA media.



Acetylation sites on lysine 6 and lysine 10 are potential targets to influence alpha-synuclein toxicity, so it is important to gain a greater un­derstanding of this post-translational modification. Not only is it important to understand this modification in WT alpha-synuclein, but it is also crucial to understand it in the six mutants known to cause familial Parkinson’s disease: A30P, A53T, E46K, G51D, H50Q, and A53E. For the purposes of our project, we wanted to establish the interaction of glycation and acetylation and the six known familial mutations. We planned to create a K6Q mutation in which lysine would be converted to a glutamine on the 6th amino acid and combined with the six known familial mutations. We also planned to create a K6Q/K10Q mutation in which lysine would be converted to a glutamate on the 6th and 10th amino acid and combined with the G51D familial mutation. These mutations would block glycation and mimic acetylation on these particular sites. A series of steps were followed to create these mutations. The aims of the project were to 1) cre­ate the mutants by PCR mutagenesis, 2) transform them into bacteria, 3) purify the plasmid from the bacteria and sequence it, and 4) transform the mutants into S. cerevisiae for future assays and a potential new direction in Parkinson’s research.

K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D PCR Mutagene­sis and Gel Electrophoresis

The first round of PCR mutagenesis did not yield the results we had hoped for. The fragment in Figure 2D at approximately 6kb in lane 3 was a positive mutagenesis product for K6Q A53T. As a group, that is the only success we received in this round. Other members of the lab re­ceived similar results. Some members received positive results for A53T as well, and others received positive results for A30P. Based on that suc­cess, it was decided that those two mutations were the most likely to yield the best results, so we re-did the PCR mutagenesis for those mutations, as seen in Figure 2E. Since our group had already obtained success with K6Q A53T, we only performed mutagenesis on A30P (Lanes 2 and 3 in Figure 2E). Results indicate a band in lane 3. This means that the mutag­enized product for A30P was amplified compared to the negative control (lane 4) and in the right location compared to the DNA ladder in lane 10. This time, we received success on that mutation. As for the mutations on E46K, none of them worked. It is likely that the problem did not lie in human error, and there was nothing wrong with anyone’s primers, given that primer check results were all positive (Figure 1D). Not only that, but our primers worked on other mutations, so it was apparent that they were not the issue. We further performed a PCR check, to ensure that the machine was not the problem. The machine was functioning properly. Figure 2D shows a DNA ladder and a dark black band in lane 9 and a weak band in lane 3. This gel shows that the mutagenesis positive control and the K6Q A53T mutation have bands in the correct location. The mutagenesis product for A53T was amplified compared to the negative control. When comparing lane 3 and 4 respectively, it is evident thateven though the band is weak in lane 3, (A53T) it is still stronger than lane 4 (A53T negative control). The results indicate that there is a dark band in lane 12 at about 6kb.This proves that the K6Q/K10Q G51D mutation was successful.

K6Q A30P, K6Q A53T and K6Q/K10Q G51D Bacterial Transformations

We had great success in our bacterial transformations for K6Q A30P, K6Q A53T and K6Q/K10Q G51D. In Figures 3B-3D, we had growth of individual colonies of bacteria. Since there was Ampicillin in the media, nothing would have grown if mutagenesis had not worked because the cells would not have had the plasmid containing the Ampicillin resistance gene. On the negative control plates in Figure 3A, there was no growth because the plasmid DNA was not present. The positive control was successful because the E. coli on that plate was mutated with the positive PCR control plasmid, so it contained the gene for Ampicillin resistance. There were no contamination issues in our transformations. These results do not imply that alpha-synuclein is ready to be expressed into a protein in E. coli.

K6Q A30P and K6Q/K10Q G51D Plasmid Purification

The plasmid purification gels (Figures 4C-4D) looked nearly identical to the ideal gels (Figures 4A-4B). The purifications of the K6Q A30P and K6Q/K10Q G51D plasmids were successful. The dark bands at about 5kb, with respect to the MW ladder, were exactly where we expect­ed them to be, and they were very distinct for all the plasmids except for the K6Q/K10Q G51D plasmid 1-2 in Figure 4D, which was relatively faint. The reason that some of the dark bands were followed by lighter bands higher up on the gel is due to supercoiling in the plasmid DNA. The plas­mids were purified and sent for sequencing at the University of Chicago.

K6Q A30P and K6Q/K10Q G51D Sequence Results and Yeast Transfor­mation

The first sequence (plasmid 1-1) we received back (Figure 5A) had the incorrect mutation. The second sequence (plasmid 1-2) we received back (Figure 5B) contained the correct mutation, so we were able to transform into yeast. Figure 5C displays the first K6Q/K10Q G51D sequence we received back from the University of Chicago, (plasmid 2-1) which was incorrect. Figure 5D shows the successful sequencing of the K6Q/K10Q G51D mutation (plasmid 2-3).

All the yeast transformations were successful. For any growth to be present on the SC-URA plates, the transformation had to be suc­cessful so that the cells could have the gene for uracil. Without uracil, the yeast could not survive on these plates. For the YPD plates, growth would occur regardless of whether the transformation was successful because this is the preferred media for yeast growth. Given this information, it is apparent that the reason there is more growth on the YPD plates is because even yeast that is not transformed may grow in YPD media. The transformation into yeast is complete, which concludes our experiment. Pictures would have been taken on the6thday as well, but there was a power outage in the building for multiple days resulting in room tempera­ture exposure and overgrowth.

Future Experiments

Creating mutations is only the first step in studying the mutants’ effects on Parkinson’s Disease. K6Q A30P and K6Q/K10Q G51D have been transformed into yeast for them to be studied using various assays. In Parkinson’s disease, alpha-synuclein presents a certain level of protein expression, localizes in cells in specific ways, and expresses toxicity, which ultimately leads to cell death (Allendoerfer, Su, and Lindquist, 2008). Knowing this allows researchers to perform assays that test for these specific characteristics. One assay that tests for the toxicity of al­pha- synuclein is known as serial dilution spotting. Serial dilution spotting involves plating on media that induces the expression of α-synuclein and comparing it to a media that represses its expression. We can assess the toxicity of cells by comparing their growth rates (Sharma et. aI 2006). If K6Q A30P and K6Q/K10Q G51D were spotted compared to the wild type, we would expect slightly more growth than WT cells because mimicking acetylation is considered a “good” mutation; however, the addition of the familial mutations will still make it toxic. An assay that can be used to test the localization of alpha-synuclein with these mutations is florescence microscopy. Alpha-synuclein can be tagged at the C or N terminal with a green fluorescent protein. When a blue light is shined on these cells, the localization of alpha synuclein can be seen (Duina, Miller, and Keeney, 2014). If K6Q A30P and K6Q/K10Q G51D were tested using this assay we would predict the cells to localize in a diffuse manner instead of ag­gregating because of the positive effects acetylation has. A western blot can be used to determine alpha-synuclein protein level expression in the yeast cells. To perform a western blot, protein is extracted from yeast cells and loaded onto a gel. The gel is probed with antibodies that specifically reveal alpha-synuclein levels (Outeiro and Lindquist, 2003). For the K6Q A30P and K6Q/K10Q G51D mutations we can predict that protein levels would be less than WT because it is less toxic.

The K6Q A30P and K6Q/K10Q G51D mutations were success­fully made and transformed into yeast to be studied using future assays. Because these mutations would block glycation and mimic acetylation at particular sites (lysine 6 and lysine 10), we can look at their effects on the toxicity of alpha-synuclein. These mutations in particular are beneficial to learning more about anyone with the familial mutation A30P and G51D and their interactions with post-translational modifications like glycation or acetylation. Understanding theseinteractions could be beneficial in the long term when it comes to finding a potential cure or drug that could work by influencing alpha-synuclein toxicity.

Materials and Methods

Primer Design and Synthesis

We created primers that contained the desired mutation K6Q/ K10Q and K6Q, which was 100% complementary; the primer sequence can be seen in Figure 1. The original amino acid code that we manipulat­ed was AAA (lysine) to CAA (glutamine). The creation of a point mutation within the circular DNA was done using PCR. PCR mutagenesis was run, which involved combining the plasmid sample with a forward and reverse primer with a mutagenesis master mix. This is done to amplify copies of segmented fragments of DNA, therefore making many copies of a partic­ular sequence. An additional PCR reaction was run to see if our forward and reverse primers were able to bind to the template plasmid that was used. For the full procedure see citation below.

DebBurman, S. (2017). Design and Create your Mutation by PCR. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 20-27) Lake Forest, Il: Lake Forest College.

Plasmid-based PCR Mutagenesis and Gel Electrophoresis

To determine the success of the PCR mutagenesis, gel electro­phoresis was run. Gel electrophoresis was done to separate and purify all the fragments. It is first done by making an agarose gel, which is poured into a gel tray. After it is solidified, DNA is inserted into the wells of the gel and a charge is then run through the gel, which ultimately separates the DNA fragments by down the gel. For our specific gel we used 0.3 g of Agar powder and 40 mL of 1X TAE buffer solution. After we ran our gel, we imaged it to find out if we succeeded with our PCR product.

DebBurman, S. (2017). Gel Electrophoresis and Bacterial Transformation. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 28-34) Lake Forest, Il: Lake Forest College.

Bacterial Transformation

The mutagenized plasmid was transformed into bacteria to be grown on plates containing LB+Amp. Bacterial transformation is when DNA is cut from its origin source and then put into a plasmid by ligation That plasmid is then put into bacteria. Bacteria usually dies when it is in ampicillin (Amp) unless they have a plasmid that has an Amp resistance gene. The E. coli that was used during the experiment kills off the tem­plate plasmid that is methylated, which allows only the mutated plasmid to grow and divide in the E. coli plates. The peer teacher supplied us with the positive and negative controls of the E. coli cells. After we picked our four main colonies that grew the most, we stored the plates at 4 degrees Celsius.

DebBurman, S. (2017). Gel Electrophoresis and Bacterial Transformation. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 28-34) Lake Forest, Il: Lake Forest College.

Plasmid purification

Plasmids are separate from chromosomal DNA and they are abundant in bacteria, which is why it’s an easy way for researchers to replicate and insert genes into organisms to study. After the bacterial transformation, we had to purify the plasmid, which would demonstrate a successful purification by the gel electrophoresis. This step is done as a safety check to make sure all the methylated template DNA is destroyed during the first step of mutations and amplification. To maximize our chances, we purified plasmid from four E. coli colonies that grew the most on the plates.

DebBurman, S. (2017). Plasmid Purification and Sequence Confirmation. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 35-39) Lake Forest, Il: Lake Forest College.

DNA sequencing

Purified plasmid is sent to the University of Chicago for DNA sequencing where they use two Applied Biosystems 3730XL 96-capil­lary and one 3130 16-capillary automated DNA sequencers. The DNA sequence is sent back to us, and we compare it with the correct original alpha-synuclein DNA sequence to see if mutations are correctly made.

DebBurman, S. (2017). Plasmid Purification and Sequence Confirmation. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 35-39) Lake Forest, Il: Lake Forest College.

Yeast transformation

Yeast transformations involve transforming the mutated alpha-synuclein in the plasmid vector into budding yeast, S. cerevisiae. The yeast was incubated at 30 degrees Celsius for 30 minutes and then heat shocked at 42 degrees Celsius for 25 minutes. After we centrifuged and removed


the transformation mix, we plated the yeast. We transformed the yeast on YPD and SC-URA plates. YPD medium is used for normal cell growth and contains yeast, peptone, and glucose. SC-URA is a synthetic complete mixture without amino acids. Yeast grows more slowly, so we monitored it over a period of a week.

DebBurman, S. (2017). Plasmid Vector Transformation into Yeast. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 35-39) Lake Forest, Il: Lake Forest College.


Thank you Dr. Alexander Wilcox and Dr. Shubhik for your guidance and support throughout the project. Thank you to Chisomo Mwale, Rosemary Thomas and our classmates for your help with data generation, analysis, and lab report writing.


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