Secondary Structure Changes caused by ADAR Protein in the Development of ALS, Leukemia, and Schizophrenia

By Kavya Anjur

Metea Valley High School, IL

Abstract

This experiment compared specific protein sequences affected by the misediting  of  ADAR  which then  resulted in specific misalignments on the protein’s structure and folding. The information was analyzed for relationships between different mutations to help regulate genes and prevent diseases such as ALS, leukemia, and schizophrenia related to the misediting  of the ADAR protein. The sequences of the GluA2 Isoforms, PTPN6 protein, and 5-HT(2C) protein were obtained from the NCBI Database. These sequences were then altered to represent the region of the proteins that were unedited by ADAR. Next, these normal and unedited sequences were entered into RaptorX, where data about the structure of these proteins was obtained. There was not a significant difference in the secondary structures of the edited and unedited proteins. Alpha helices, beta sheets, and coiled regions were secondary structures of the proteins studied. There was a notable increase in the average alpha helix composition of most of the proteins. Alpha helices had the most considerable effect on the unedited protein structure and function; therefore, these helices have the most significant impact on the development of human diseases.

INTRODUCTION

Humans could not survive without proteins. Proteins are the basis of all living creatures, yet they can also cause a variety of diseases. When proteins malfunction, they throw off a lot of systems in the body, which in turn causes one problem after another. One protein in particular, ADAR (RNA-specific adenosine deaminase), has been related to ALS, leukemia, schizophrenia, epilepsy, cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders (Nishikura & Slotkin, 2013). There are potentially strong connections between RNA misediting by the ADAR protein in specific genes and human diseases, and differences in RNA editing are responsible for the development of these diseases. Scientists aspire to find ways to better regulate these genes; consequently, their research will aid in the prevention of these diseases.

To begin with, transcription and translation are important processes that precede RNA editing. RNA is different from DNA in that RNA has the nucleotide uracil instead of thymine. RNA is also made of ribose, not deoxyribose, and RNA is mostly single stranded, not double stranded. The function of RNA in a cell is to provide the means to make proteins. It does this through the processes of protein synthesis, which consists of transcription and translation. Transcription produces mRNA from DNA and takes place in the nucleus. The three types of RNA are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). An mRNA molecule, which conveys information from the nucleus to the cytoplasm, is synthesized from a gene in the DNA. RNA polymerase is a transcriber in the cell that binds at the promoter and then moves down the rest of the DNA strand. It pairs each nucleotide with its RNA complementary nucleotide. An mRNA strand is built when adenine binds with uracil and guanine binds with cytosine. The RNA polymerase moves down the DNA strand in steps that are three nucleotides long. Each sequence of three nucleotides on the mRNA, which are known as codons, corresponds to a particular amino acid. After transcription, the mRNA goes out of the nucleus and into the cytoplasm through pores in the membrane. RNA splicing is the process of the non-coding nucleotides (introns) being removed before translation.  This process prevents the creation of non- functional proteins. Translation is when ribosomes use the mRNA from transcription to direct the production of proteins. The tRNA contains anticodons, which are the complementary sets of three bases for the codons from the mRNA. The tRNA attaches to the mRNA and releases its amino acid, which is then linked to other amino acids by peptide bonds. These bonds form polypeptides, which then chain together and make protein molecules. Thus, a protein is created from this process of protein synthesis.

All proteins fold when created and have primary, secondary, tertiary and sometimes quaternary structures.  Secondary structures develop when polar atoms on amino acids form hydrogen bonds. These bonds will fold the chain into configurations called ? helix and ? pleated sheet. Secondary structures are usually judged for accuracy based on the percent of residues of H (? helix), E (? sheet), and C (coil) that match the predicted amount. This method is known as Q3, or the proportion of the amino acids that have accurate measures for C, E, and H. On average, the residues of protein structures are 30% in H, 20% in E, and 50% in C. The coils, or loops, connect ? helices and ? sheets. ? sheets are made up of ? strands that are connected through hydrogen bonds. These unidirectional strands usually amount to six for each ? sheet. The hydrophobicity of ? helices, or the measure of how much they repel water, depends upon the residues that make up their structures. Usually, hydrophobic residues are compatible with ? helices. This is because hydrophobic residues allow for the condensed packing of hydrophobic cores and enable one side of each helix to be hydrophobic; moreover, they also permit packing for remaining regions of the protein structure (Singh, 2005). The measures of C, E, and H are a way to show the quality and accuracy of predicted secondary structures of proteins. They also show some traits and characteristics of the protein from the hydrogen bonds and the residues.

Additionally, mutations can have very negative effects on protein structure. Amino acids, the building blocks of proteins, can be mutated in genetic diseases.  It is possible for amino acids to have a positive or a negative electrical charge. Some nonpolar and uncharged amino acids are asparagine, glutamine, and cysteine, while arginine, histidine, and lysine are examples of positively charged amino acids. Negatively charged amino acids include aspartic acid and glutamic acid. Aromatic amino acids are relatively nonpolar. Size, shape, and electrical charge are aspects of amino acids that influence their interactions within a protein. For example, a positively charged amino acid can react with water because of the hydrogen bonds that can be formed. Nonpolar amino acids, on the other hand, are hydrophobic as they do not interact with water (From Genes to Genetic Diseases: What Kinds of Mutations Matter?, 2014).

ADAR is a protein that aids in many human processes. The ADAR enzymes edit double stranded (ds) mRNA after its transcription by selectively converting adenosines (A) to inosines (I), which causes diversity in RNA. This A to I RNA editing is mostly directed towards repetitive RNA sequences located within 5’ and 3’ untranslated regions (UTRs). Thus misediting by ADAR leads to the formation of dysfunctional proteins. ADAR enzymes are mainly found in the nucleus of nervous system cells where they modify specific adenosines in pre-mRNAs of proteins involved in electrical and chemical neurotransmission.  The RNA editing sites on ADAR genes usually have a mutation or deletion which leads to a change in the codons coding for the protein.  ADARs also regulate gene expression by modifying double-stranded RNA (Savva et al., 2012).  Through this gene expression, the RNA editing by ADAR influences thousands of genes.   ADAR regulates this, in addition to transcript stability, through its interaction with other proteins associated with the processing of RNA (Cheung et al., 2013).

Furthermore, ADAR is a catalyst. In mammals, there are a total of three ADARs, although only two of these enzymes are catalytically active. In the central nervous system, the editing by ADAR is very important because it prevents the system from deteriorating. When binding with other proteins, ADAR plays a role in RNA interference. Complications arise from the RNA not being properly edited by ADAR. This leads to diseases such as ALS (Hogg et al., 2011). Between the two types of ADAR, ADAR1 is a necessity in the process of editing while ADAR2 is involved in gene expression (Cheung et al., 2013). A reduced amount of ADAR1 causes changes in the RNA editing of the AMPA receptors. The AMPA, a membrane receptor for glutamate, mediates the movement of nerve signals in the central nervous system (Kubota-Sakashita et al., 2014).

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In order for ADAR to edit properly, the pre-RNA must also have coordinated “editing and splicing” (Bratt & Öhman, 2003, [Online]). Sometimes problems occur in the RNA itself, thus making the job of ADAR even harder. Recent discoveries of ADAR and its role in RNA editing emphasize the importance of editing for normal body physiology and also suggest strong links to human genetic diseases.  This could lead to  new approaches for the treatment of RNA editing?related disorders possibly by  the development of RNA?directed gene therapy technologies (Maas, 2013, [Online]). It shows the strong relationship between ADAR and RNA.

The shape of ADAR, specifically related to editing, can be seen in Figure 1. In this picture, “adenosine deaminase includes a DNA binding domain,” which contributes to the overall complexity of the structure and function of ADAR in editing genetic material (Schwartz, Rould, Lowenhaupt, Herbert, & Rich, 1999, [Online]).

Furthermore, Amyotrophic Lateral Sclerosis (ALS) is a disease greatly impacted by the ADAR gene. Hideyama & Kwak (2011) conducted experiments to try to find the cause of sporadic ALS (SALS). Motor neurons have unedited GluA2 mRNA at the Q/R site. ALS greatly affects motor neurons. GluA2, which regulates the calcium permeability of the AMPA receptor after the genomic Q codon is replaced by the R codon in mRNA, is a subunit of the AMPA receptor, which is needed for fast synaptic transmission. This replacement is mediated by Adenosine Deaminase acting on ADAR2. ADAR2's activity is not enough to edit all GluA2 mRNA expressed in motor neurons of ALS patients. One experiment involved destroying the ADAR2 gene in a group of mice, which resulted in the mice exhibiting ALS symptoms, showing that motor neurons without ADAR2 cannot survive (Hideyama & Kwak, 2011). Another group of mice had the GluA2 allele replaced with GluR-BR allele, which coded for an edited GluA2.  These mice survived. The expression of unedited GluA2 causes death of motor neurons in SALS. It was hypothesized that ADAR2 activity plays a very important role in the development of SALS disease, which starts when motor neurons express unedited GluA2 (Hideyama & Kwak, 2011).

Additionally, it was discovered that oxidative stress prevents the release of neurotransmitters through pre-synaptic machinery (Pollari et al. 2014). This is also caused by the mitochondria not working properly and the increased concentration of calcium in muscle cells. When pre-synaptic transmitters do not work properly, it allows inflammatory agents to degrade the nerves. This, in addition to the death of motor neurons, increases the bodily damage that occurs from ALS. It is hypothesized that antioxidants combined with anti-inflammatory agents can act as medicine to counteract oxidative stress and to prevent pre-synaptic dysfunction and nerve degradation. Gender dependence of ALS and oxidative stress is such that men will be more affected than women (Pollari et al., 2014).

2   The shape of GluA2 can be seen in Figure 2. X-ray diffraction was used to show the crystal structure of this molecule (Armstrong & Gouaux, 2000, [Online]). The red, green, and blue areas of the graphic each represent a copy of GluA2.  The authors suggest that since zinc ion, glutamic acid, and water are the only non-polymeric parts of  this molecule, it may possibly have more than  one quaternary state (Armstrong & Gouaux, 2000, [Online]).

Moreover, in schizophrenia and mood disorders, it has been found that the 5-HT(2C) receptor protein does not work efficiently because of the lack of proper editing by ADAR. The RNA of this receptor is edited to create different forms of the receptor. After an experiment was conducted that compared 5-HT(2C) in people with and without schizophrenia, the conclusion was drawn that decreased RNA editing greatly influences the disease (Sodhi et al., 2001).

Also, the 5-HT(2C) receptor regulates the amount of dopamine released into the brain. Dopamine is a chemical that, when released into the brain, emulates feelings of happiness and joy.  These receptors are “localized in the dorsal striatum,” which is a part of the brain related to memory and the planning of movements (Alex et al., 2005, [Online]). Without these receptors working properly, not enough dopamine will be released into the brain, thus leading to mood disorders and schizophrenia in people.

Furthermore, 5-HT(2C) is a serotonin receptor protein. This protein, among other serotonin systems, is essential in managing behavior.  Tests were conducted to compare male mice that had this protein to other male mice in which this protein was removed. The 5-HT(2C) receptor has “been implicated in the pathophysiology and treatment of anxiety disorders, which are among the world’s most prevalent psychiatric conditions” (Heisler et al., 2007, [Online]).

Additionally, ADAR2 expression has been found to be diminished in people with mental disorders. This was observed in postmortem brains of patients (Kubota-Sakashita et al. 2014). After further experimentation was conducted with mice, it was discovered that the R/G site is the area of the AMPA receptor in which the ADAR2 editing of RNA was significantly reduced. This is where ADAR is involved in the functioning of mental diseases (Kubota-Sakashita, Iwamoto, Kato, & Bundo, 2014).

The three purple areas of Figure 3 show different forms of the 5-HT(2C) receptor which affect the binding of the receptor to G proteins. In turn, the change in binding causes dysfunctional proteins to be formed (Sodhi, 2013).

3     Another protein affected by ADAR is PTPN6, which plays a role in leukemia. This protein, also known as protein tyrosine phosphatase non-receptor type 6, is additionally involved in other cancers such as breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. PTPN6 has two promoter regions, both of which initiate transcription. One of these promoters is found in epithelial cells, which are found inside skin tissue, and the other promoter is found in hematopoietic cells, which aid in the formation of red blood cells. The PTPN6 gene codes for PTPN6 tyrosine phosphatase. Experimentation with mice showed that Shp-1 gene expression is affected when mutations occur in PTPN6, causing defects in hematopoiesis, or the creation of red blood cells (Beghini & Lazzaroni, 2012).  Shp-1 is one of the two N-terminal domains of PTPN6.  The mechanism of action of Shp-1 is to inhibit signaling through receptors for cell growth in red blood cells and immune cells. The regular function of PTPN6 is to prevent the formation of tumors; therefore, when PTPN6 is not expressed properly, it leads to many problems including overgrowth of myeloid and lymphoid cells (Beghini & Lazzaroni, 2012).

A study showed that there were large amounts of the ADAR protein in AML (acute myeloid leukemia) patients even though there were small amounts of ADARB1 and ADARB2 (Meduri & Huntly, 2014). This suggested epigenetic variation, which means that non genetic influences affected the ADAR gene expression, and it could be the reason for the development of the cancerous phenotype of leukemia (Meduri & Huntly, 2014).

PTPN6 has been associated with decreasing the amount of protein in the myeloid leukocyte signal transduction pathways (Beghini et al., 2000). Further research to look at PTPN6 gene expression in  CD34+/CD117+ blasts from acute myeloid leukemia patients showed that there were several places where A was edited to G in the RNA. For people with acute myeloid leukemia (AML) that went into remission, decreased amounts of CD117+ were shown in their bone marrow compared to the original amounts.  The conclusion was made that PTPN6 edited the mRNA after transcription, which in turn led to the development of leukemia (Beghini et al., 2000).

           4 Figure 4 displays the shape of the human protein tyrosine phosphatase Shp-1 (Beghini & Lazzaroni, 2012, [Online]). The crystal structure of this protein shows its folding in humans. The N-terminal, which is the blue area, contains the free amino group of the protein. The C-terminal, which is the red area, contains the carboxylic acid group of the protein. Interactions between these terminals and PTPN6 affect the editing of the mRNA (Beghini & Lazzaroni, 2012).

The normal functions of ADAR proteins are to catalyze the editing of adenosine to inosine after transcription within double stranded RNA. Because most cellular processes read inosine as guanine, this causes different kinds of dysfunctional proteins to be coded from the same gene, which eventually leads to health problems in humans (Barraud & Allain, 2012).

The purpose of this experiment was to compare the sequences of GluA2, PTPN6, and 5-HT(2C) proteins, before and after they are affected from misediting by the ADAR protein, to find the specific misalignments of the alpha helices, beta sheets, and coiled regions in the protein structure that cause human diseases. This information can be analyzed to find relationships between different mutations to help regulate genes and prevent diseases related to misediting by the ADAR protein such as ALS, leukemia, schizophrenia, epilepsy, cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders.  It will facilitate the development of a quantitative model of the potential connections between RNA editing by ADAR protein in specific genes of human diseases. The hypothesis for this experiment is that changes in the alpha helices will cause specific misalignments in the protein structure which will, in turn, develop human diseases. The misediting by ADAR results in the change of glutamine, a nonpolar and uncharged amino acid that is normally buried in the hydrophobic core of the alpha helix, to arginine, a polar amino acid that is positively charged. Arginine is not hydrophobic, as it can participate in hydrogen bonding, so it does not support the hydrophobicity of the alpha helix.  The AMPA receptor, which is the area that is affected from the misediting by ADAR in GluA2 and 5-HT(2C), needs to have the right structure so that it can properly regulate calcium transport in brain cells. If the amino acid sequence of its subunit is changed, the overall structure of the AMPA receptor and calcium regulation will change, causing protein malfunction which further leads to diseases (SALS and schizophrenia).  In the unedited region of PTPN6, 6 amino acid residues are changed in the tyrosine protein kinase Shp-1(Src homology region 2 domain containing phosphatase-1) leading to AML.

 

 

METHODS

 

We used the NCBI Database [http://www.ncbi.nlm.nih.gov/] to determine the protein sequence [Homo sapiens] of GluA2 Isoform 1, GluA2 Isoform 2, GluA2 Isoform 3, PTPN6 and 5-HT(2C). These sequences were entered into RaptorX [http://raptorx.uchicago.edu/] to predict the protein structure.  The specific amino acid in position 607 of the GluA2 Isoform 1 protein was changed from R to Q to represent the sequence of the protein when it has been unedited by ADAR. This will change the amino acid back to its original form, before it went through RNA editing.  This new sequence was entered into RaptorX to predict the structure of the unedited protein.  The structure of the unedited version of the protein was compared with the normal version of the protein to identify any changes in the folding and structure. This was accomplished through the predicted images of the protein and the H [alpha helix], E [beta sheet], and C [coiled region] percentages of the structure for each amino acid.  The amino acid in position 607 and 560 were changed from R to Q for the unedited GluA2 Isoform 2 and GluA2Isoform 3 sequences respectively and the procedure was repeated.  For the unedited protein sequence of PTPN6, G will become D in position 117, S will become N in position 138, C will become Y in position 160, E will become K in position 179, R will become Q in position 207, and G will become E in position 219.  The unedited sequences of 5-HT(2C)   were obtained by changing V to I in position 107, G to N in position 109, and V to I in position 111.  The data for all the proteins was analyzed using two-way factorial ANOVA analysis [vassarstats.net] to compare the mean alpha helix, beta sheet and coiled region percentages of the edited and unedited forms of the three GluA2 isoforms, PTPN6, and 5-HT(2C).  The independent variable of this experiment was the structure of the proteins that were unedited by ADAR. The dependent variable was the change in structure caused from the misediting by ADAR. This was measured by the change in percentage of H (alpha helix), E (beta sheet), and C (coiled region). The control of this experiment was the structure of the proteins when they were properly edited by ADAR. Things were kept constant in this project to avoid errors. The constants of this experiment include the structure prediction software (RaptorX), the resource for the protein sequences (NCBI Database), and the species of the proteins (Homo sapiens).  The Tukey one way ANOVA was used because Vassar stats can only compare up to four samples, and this experiment required the comparison of ten samples (proteins). Alpha helices were chosen to be compared because they comprised the largest portion of both edited and unedited structures of the proteins studied. The beta sheets and coiled regions were not compared because they made up a smaller fraction of the proteins studied and had less significance on the overall structures and functions of the proteins

 

RESULTS

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Figure 5: Comparison of folding patterns of GluA2 isoforms, PTPN6 and 5-HT(2C) edited and unedited proteins

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Figure 6: Comparison of folding patterns of GluA2 edited and unedited isoforms 1, 2 and 3

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Figure 7: Comparison of folding patterns of PTPN6 edited and unedited proteins

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Figure 8: Comparison of folding patterns of 5-HT(2C) edited and unedited proteins

This data explains how the protein structures were not significantly changed in the unedited form, as only a few amino acids were mutated for each protein sequence. Although the protein appearance may not have been dramatically changed, the evidence shows that even a slight difference in the structures of the proteins (resulting from the misediting by ADAR) is sufficient in causing human disease. This is represented in the experiment by the altered compositions of the diseased protein (unedited forms) and “normal” protein (edited forms).

The three components of the isoforms compared were alpha helices, beta sheets and coiled regions. All isoforms were composed of higher levels of alpha helices than beta sheets and coils. Additionally, all the isoforms had an increase of alpha helices, a decrease of beta sheets, and a decrease of coils in their unedited form, except for Isoform 2. This had a decrease of alpha helices, an increase of beta sheets, and an increase of coils. Curiously, the P value of isoform 2 when compared to that of Isoform 1 and 3, was 0.89 and 0.35, respectively, indicating an increase in alpha helices was common for all of the isoforms.

The average compositions of the unedited isoforms were very similar to those of the edited isoforms. In all the isoforms, only one amino acid was different in the unedited form, which accounts for the very slight differences in secondary structure. Glutamine is a nonpolar uncharged amino acid that is edited to arginine, which is a positively charged amino acid. In the unedited forms of these proteins, glutamine was not changed to arginine, which led the protein to malfunction and cause disease.

There was a decrease of alpha helices and beta sheets and an increase of coiled regions in the structure of unedited PTPN6.  In the unedited form of PTPN6, six amino acids were not properly edited by ADAR. The properties of these six amino acids, when compared to those in the edited form, all are the same except a negative amino acid found in the unedited form instead of nonpolar, as in the edited form.

In the unedited form of 5-HT(2C), the percentages of alpha helices and coiled regions increased while the percentage of beta sheets decreased. In the unedited form of 5-HT(2C), three amino acids were not properly edited. These amino acids all have the property of being nonpolar, both in the edited and unedited protein.

The overall trends of this experiment show that in the unedited form of the proteins there was an increase in alpha helices, a decrease in beta sheets, and an increase in coils. A few exceptions include the following: Isoform 2 and PTPN6 decreased in alpha helices, Isoform 2 increased in beta sheets, and Isoforms 1 and 3 decreased in coils. Alpha helices were the structure that changed by the greatest percentages from the edited to the unedited proteins.

Tables S1, S3, and S5 show the composition (percentages of alpha helices, beta sheets, and coiled regions) of the edited GluA2 isoforms 1, 2, and 3, respectively. The isoforms represent different forms of the same protein. The folding patterns of the three edited isoforms can be seen in Figures S1, S3, and S5. All isoforms had higher levels of alpha helices than beta sheets and coiled regions. Isoform 1 had the highest percentage of alpha helices and the least percentage of beta sheets. Isoform 3 was composed of the most coiled regions. A two-way factorial ANOVA for independent samples, indicated that there was a significant difference in the percentages of alpha helices, beta sheets, and coiled regions in the area edited by ADAR protein for the three isoforms (P < 0.0001, df=2). As expected, the analysis showed a value of 1 for P (df=2) for the three isoforms indicating that the folding patterns composed 100% of the proteins.  It is to be noted that these results refer to the specific regions of the GluA2 isoforms edited by the ADAR protein.

Tables S2, S4, and S6 represent the composition of the unedited GluA2 isoforms. These averages are very similar to those of the edited isoforms (Figure 6). There was not enough variance in the mutation of the unedited form of the protein to develop a radically different structure in the isoforms, which are evident in the protein structures shown in Figures 1-10. Figure 13 represents the slight differences in secondary structure caused by the change of one amino acid. Glutamine is a nonpolar uncharged amino acid that is edited to arginine, which is a positively charged amino acid. For edited isoforms 1 and 2, glutamine was changed to arginine in the 607th amino acid of the protein sequence (Figure S12). In the edited form of isoform 3, glutamine was changed to arginine in the 560th amino acid of the protein sequence (Figure S12). In the unedited forms of these proteins, glutamine was not changed to arginine, which led the protein to malfunction and cause disease.

The same components (alpha helices, beta sheets and coiled regions) of PTPN6 were analyzed using two-way factorial ANOVA for independent samples.  It was observed that there was a significant difference between the average percentage of alpha helices, beta sheets, and coiled areas between the edited and unedited forms of PTPN6 (P<0.0001, df=2).  Similarly, there was a significant difference in secondary structures between the edited and unedited forms of GluA2 isoforms 1, 2, and 3 (P<0.0001, df=2) and the secondary structures of edited and unedited forms of 5-HT(2C) (P<0.0001, df=2).

Tables S7 and S8 show the composition of the edited and unedited forms of PTPN6, respectively. As shown in Figure 7, there are slight differences between the edited and unedited forms of the protein. There was a decrease of alpha helices and beta sheets and an increase of coiled regions in the structure of unedited PTPN6. It can be seen in Figures S7 and S8 that there were more alpha helices and beta sheets in the protein structure when it was properly edited. In the edited form of PTPN6, amino acids in the region around the 141st amino acid of the protein sequence were changed. Glycine, a nonpolar amino acid, was edited to aspartic acid, which is negatively charged, in position 117 of the protein sequence. Serine is a nonpolar uncharged amino acid that was edited to another nonpolar uncharged amino acid, asparagine, in position 138 of the protein sequence. Cysteine, a nonpolar uncharged amino acid, was edited to tyrosine, a relatively nonpolar and aromatic amino acid, in position 160 of the protein sequence. In position 179, glutamic acid, which is negatively charged, was edited to lysine, which is positively charged. In position 207, arginine, a positively charged amino acid, was edited to glutamine, a nonpolar amino acid. Glycine, a nonpolar amino acid, was edited to glutamic acid, a negatively charged amino acid, in position 219 of the protein sequence (Figure S13). A total of six amino acids were changed in this protein from proper RNA editing. In the unedited form of PTPN6, these six amino acids were not edited by ADAR. As a result, these six amino acids stayed in their original form instead of being edited to their respective amino acids.

Tables S9 and S10 show the composition of the edited and unedited forms of 5-HT(2C).  The structures of the edited and unedited proteins, as shown in Figures S9 and S10, look very similar. Figure 8 represents the average composition of the edited and unedited forms of 5-HT(2C). In the unedited form of the protein, the percentages of alpha helices and coiled regions increased while the percentage of beta sheets decreased. In the edited form of 5-HT(2C), amino acids before the 112th position of the protein sequence were changed. In position 107 and 111, a nonpolar amino acid, valine, was edited to another nonpolar amino acid, isoleucine. Glycine, a nonpolar amino acid, was edited to asparagine, a nonpolar uncharged amino acid, in position 109 of the protein sequence (Figure S14). A total of three amino acids were changed in this protein by proper RNA editing. In the unedited form of 5-HT(2C), these three amino acids were not changed.

Using Tukey one-way ANOVA (Figure 11) to compare the mean alpha helix percentages of the edited and unedited forms of the three GluA2 isoforms, PTPN6, and 5-HT(2C), it was observed that P=4.1E-11 (df=9).  Mean alpha helix percentages of the edited GluA2 isoforms 1(P<0.0001, df=1), 2 (P<0.0001, df=1) and 3 (P<0.0001, df=1) significantly differed from those of the PTPN6 edited proteins.  There was also a significant difference between the mean alpha helix percentages of the unedited GluA2 isoforms 1(P<0.0001, df=1), 2 (P<0.0001, df=1)and 3 (P<0.0001, df=1) and those of the PTPN6 unedited proteins. Furthermore, there was an important difference between the mean alpha helix percentages of the edited GluA2 isoforms 1(P=0.001, df=1), 2 (P=0.002, df=1)and 3 (P=0.002, df=1) and those of the  5-HT(2C) edited proteins. Similarly, there was a significant difference between the mean alpha helix percentages of the unedited GluA2 isoforms 1(P=0.003, df=1), 2 (P=0.024, df=1) and 3 (P=0.003, df=1) and those of the 5-HT(2C) unedited proteins. A major difference was also noted between the mean alpha helix percentages of the edited PTPN6 protein and those of the edited 5-HT(2C) protein (P<0.0001, df=1) and unedited 5-HT(2C) protein (P<0.0001, df=1) and between the mean alpha helix percentages of the unedited PTPN6 protein and those of the edited  5-HT(2C) protein (P<0.0001, df=1) and unedited  5-HT(2C) protein (P<0.0001, df=1). These significant differences suggest that changes in secondary structure result from misediting by ADAR..

Tables S1, S3, and S5 represent secondary structure percentages of the edited GluA2 isoforms. When these data were compared, it became apparent that the differences of the secondary structure percentages were very low between the edited isoforms; they differed, on average, by three percent or less. When the data from Tables S2, S4, and S6 was compared, it became apparent that the unedited isoforms had an average difference of over ten percent. Table S17 represents the edited and unedited forms of all the proteins and shows how the misediting by ADAR caused slight, yet significant, changes within the secondary structures of the proteins. These relationships between protein composition and misediting by ADAR are compared in Figure S11.

In Figures S1-6, the edited and unedited isoforms appear very similar, but there are small differences caused by changes in secondary structure. Table S17 shows a comparison of the average percentages of each secondary structure in the composition of each protein. These data represent how the protein structure itself was not significantly changed, as only a few amino acids were mutated for each protein sequence, but the slight changes in secondary structure composition of the proteins was significant enough to cause diseases in humans.

 

 

CONCLUSION

 

In conclusion, the hypothesis for this project was supported.  There was a significant difference between the alpha helices in the secondary structure conformations of the proteins.  Although the difference in protein folding does not seem to be significant, there was a change in both the percent compositions and the secondary structures of the proteins in their unedited and edited forms. However, since the region chosen to be unedited was one of supreme importance to the respective proteins, it is possible that if more samples were to be studied, or more proteins, a significant difference might be found.  The hypothesis was that the alpha helices will cause the most detrimental changes to the proteins because glutamine is a nonpolar and uncharged amino acid and arginine is a polar amino acid with a positive charge. Since arginine is not hydrophobic, as it can participate in hydrogen bonding, it does not support the hydrophobicity of the alpha helix. The AMPA receptor, which is the area that is affected from the misediting by ADAR, needed to have the right structure so that it can properly regulate calcium (Ca2+) transport in brain cells. Changes in the amino acid sequence of the AMPA receptor affected its overall structure, though this may not be clearly shown in the appearance of the protein. This information was shown through the data, which exemplified the changes in the secondary structure of the protein. Changes in Ca2+ regulation causes proteins to malfunction, which leads to diseases such as sporadic ALS.  Likewise, the adenine residues are changed to guanine in the tyrosine protein kinase Shp-1(Src homology region 2 domain containing phosphatase-1), the unedited region of PTPN6, which leads to Acute Myeloid Leukemia.

All the unedited proteins, except for GluA2 Isoform 2 and PTPN6, had higher concentrations of alpha helices and lower concentrations of beta sheets. Because ADAR misediting did not change amino acids from hydrophobic to hydrophilic, alpha helices were favored in the unedited proteins. GluA2 and 5-HT(2C) had the same mechanism of action (AMPA receptor) and property (nonpolar) for all the unedited amino acids. Thus, they supported the hydrophobic alpha helices as being completely nonpolar prevented them from making hydrogen bonds. PTPN6 resulted in a negatively charged amino acid, thus making it hydrophilic and unsupportive of the alpha helix structure. GluA2 Isoform 2 had a P Value greater than 0.05, which shows that the data is not significant. Thus, the most common trend found in the unedited proteins is an increase in the amount of alpha helices.

Based on the evidence gathered from the data, it can be concluded that the three proteins have similar compositions of alpha helices, beta sheets, and coiled regions; however, all the proteins are composed mainly of alpha helices and coiled areas. The region studied in this experiment was the specific region (Q/R site of the AMPA receptor for GluA2, A/G site in Shp-1 gene for PTPN6, and R/G site of the AMPA receptor for 5-HT(2C)) unedited by the ADAR protein that resulted in diseases such as ALS, leukemia, and schizophrenia.

This project was done on the computer, so the errors were very limited. There were some opportunities for human error, though. This included typing in the sequences into RaptorX. This error was avoided by the careful checking and rechecking of the entries into Raptor X.  Raptor X was very slow in producing results due to a backlog of jobs, which might be a factor to consider in future studies. The computer simulation may not have predicted the true changes in protein folding with complete accuracy. This is why the Q3 system was used to test the computer predictions for accuracy through the predicted protein secondary structures. Additionally, only ten amino acids above and below the affected area were tested in this experiment. It is possible that if a wider range of amino acids are tested, then different data may be collected. Thus, more amino acids should be tested in the future.

It was only possible to misedit one sequence per protein in the interest of time for this project. Although this data showed that protein folding was not much affected from it, the misediting by ADAR led to changes in the secondary structure of the protein that were significant enough to cause disease. It would be very interesting to find out what happens if more sequences are unedited.  It would also be in the further interest of this project to compare the unedited sequences of the proteins studied to see if there are any similarities between them.  The figures of the folded proteins generated from Raptor X (Figures S1-10), show that their folding is only slightly affected by ADAR misediting. In order for there to be a significant difference in the folding of the protein, there seems to be a need to misedit more isoforms, more specific regions of the protein, more amino acids, and more proteins.

These results suggest that there was a significant change in secondary structure (alpha helices, beta sheets and coiled regions), of which the alpha helices seemed to play the most important role, judging from their abundance in the secondary structure of the proteins studied.  Since the function of the alpha helices is to stabilize the protein, it is important to mention that in the region studied of GluA2, the amino acid glutamine was changed to arginine. This could potentially explain why the protein becomes dysfunctional and causes disease.

It is well known that amino acids which favor the alpha helix include alanine, cysteine, glutamine and glutamic acid.  Amino acids such as branched amino acids (valine, isoleucine, phenylalanine), charged amino acids (arginine) and aromatic amino acids (tryptophan and tyrosine) interrupt alpha helical structure.  The reason for a charged amino acid disrupting the alpha helix is because the hydrophobicity of the core is changed, the hydrogen bonding within the same polypeptide is interrupted, and the overall function of the protein changes.  The lack of change of glutamine to arginine in the unedited form of the protein results in dysfunction of the protein and causes ALS.  The reason for the increased percentages of alpha helices could be because in an alpha helix, hydrogen bonds occur within the same polypeptide. Peptides can stretch in an alpha helix, but they are inflexible in a beta sheet as they have already been stretched. Hydrogen bonds occur between different polypeptides in beta sheets.  Since peptides are rigid in beta sheets, it is easier for the protein to fold into alpha helices instead.  This explains the higher percentages of alpha helices for all three GluA2 isoforms in this experiment.

It is interesting to note that all the unedited isoforms (which were present in disease conditions) of the protein, except for GluA2 isoform 2, had higher concentrations of alpha helices and lower concentrations of beta sheets. This indicated that the changes in amino acid sequences resulting from the misediting by ADAR changed the hydrophobicity and polarity of the protein. Due to the change from hydrophobic amino acids, which repel water, to hydrophilic amino acids, which attract water, the secondary structures of the proteins were altered in such a way that the alpha helices were no longer favored. Instead, more beta sheets were formed in the proteins.  It is also interesting to note that GluA2 isoform 2, when unedited, had a slightly lower concentration of alpha helices and a slightly higher concentration of beta sheets than the edited form.  This could mean that hydrogen bonding is favored in the beta sheet conformation over the alpha helix confirmation in the secondary structure of the unedited GluA2 isoform 2 due to the change in the amino acid caused from misediting. When the Q/R site of the AMPA receptor in GluA2 is not properly edited by the ADAR protein, it leads to sporadic ALS.  Further research will be necessary to determine why this does not seem to happen in the other two GluA2 isoforms.

ADAR enzymes have also been implicated in the development of cancers such as leukemia.  The A to I RNA editing catalyzed by ADAR changes the nucleotide sequence of specific RNAs, causing A to I mutations. PTPN6 was interesting in that, like GluA2 isoform 2, it had a lower concentration of alpha helices when unedited. PTPN6 was different from GluA2 isoform 2 in that it had a lower concentration of beta sheets and a higher concentration of coiled regions when unedited by ADAR. Because ADAR enzymes are so important to the function of normal proteins, their expression is very well controlled in cells.  When this control is breached, ADAR no longer has any restriction to change A to I, and this results in cancers. PTPN6 has been identified as a tumor suppressor gene because it modulates the growth and function of cells that produce blood cells. In patients with AML, PTPN6 has a longer length compared with the normal protein and the protein found in remission patients. It was found that the changing of A to I caused the retention of an intron (noncoding sequence) and led to the production of a non-functional PTPN6 protein. The results from this experiment show a change between the edited and unedited PTPN6 proteins, which was significant enough to cause leukemia. Due to the dysfunctional protein produced, the unedited form of PTPN6 is not able to regulate the growth and function of hematopoietic cells.

PTPN6 misediting by ADAR results in the changing of 6 residues in the tyrosine protein kinase Shp-1(Src homology region 2 domain containing phosphatase-1), leading to AML. Each of these unedited amino acids was not edited to the proper amino acid. Though these amino acids had different individual properties, some of the edited amino acids had the same individual properties. In PTPN6, misediting by ADAR resulted in two negatively charged, two nonpolar uncharged, one aromatic (nonpolar), and one positively charged amino acid. When compared to the amino acids of the edited form of PTPN6, the amino acids of the unedited form of PTPN6 included one more negatively charged amino acid (which takes the place of a nonpolar amino acid). This negatively charged amino acid affected the hydrophobicity of the alpha helices in the unedited structure. PTPN6, unlike GluA2 and 5-HT(2C), resulted in a negatively charged amino acid rather than a nonpolar amino acid. Since this is the only protein with this property, it could be a reason why it is the one of the two only proteins that had a decreased percentage of alpha helices when it was unedited.

The 5-HT(2C) receptor is necessary to transmit the 5HT (serotonin) signal in the central nervous system. Any misediting by ADAR in the structure of the 5-HT(2C) receptor would potentially cause psychiatric disorders that are dependent on the function of this neurotransmitter. Serotonin drugs are given to patients with psychiatric disorders including depression and schizophrenia. Thus, even the slight changes seen in this experiment between the edited and unedited forms of the 5-HT(2C) protein could have a great enough impact upon the neurons to cause schizophrenia and other psychiatric disorders.

It was interesting to note that both 5-HT(2C) and GluA2 are involved with G protein receptor regulation at the AMPA receptor while PTPN6 is involved in hematopoietic function.  The results of this experiment showed a significant difference between the edited and unedited forms of 5-HT(2C) and the other two proteins (PTPN6 as well as the GluA2 isoforms).  This was to be expected because the mechanism of action of PTPN6 was different from that of the other two proteins.  It was also interesting to note here that there was a significant difference between alpha helices of the GluA2 isoforms and 5-HT(2C) although they had the same mechanism of action.  Since there was a change of one amino acid in GluA2, seven in PTPN6, and three in 5-HT(2C), and the regions involved were the Q/R site of the AMPA receptor for GluA2, the R/G site of the AMPA receptor for 5-HT(2C), and the A/G site of the Shp-1 gene for PTPN6, it was possible that there was a correlation between the secondary structures of these proteins and the number of amino acids unedited by ADAR. The single amino acid change that occurred in GluA2 misediting might not have had as significant an effect on the secondary structure and formation of alpha helices as the change in three amino acids in 5-HT(2C). However, this single amino acid change was potent enough to cause ALS. The misediting of 5-HT(2C) changed residues 107, 109, and 111 from isoleucine, asparagine, and isoleucine to valine, glycine, and valine. This resulted in a large change in size of the amino acids (the former three are much bigger than the replacements). The misediting also changed the 5-HT(2C) from a very hydrophilic (asparagine) core to a very hydrophobic (valine) and neutral (glycine) core. This explained the significant difference between the structures of GluA2 and 5-HT(2C) and the correlation between the amount of misediting and the resultant mutations. Although ADAR misediting of both GluA2 and 5-HT(2C) caused changes in the AMPA receptor, the resultant diseases were different because of the differences in mechanisms of disease development.

Based on the evidence obtained from this experiment, it was observed that there was a significant difference between secondary structures of the edited and unedited GluA2 isoforms, PTPN6, and 5-HT(2C). Upon comparing the average alpha helix percentages of all the proteins, a significant difference was observed between those of the GluA2 isoforms and those of PTPN6 and 5-HT(2C). The significant difference between the GluA2 isoforms and the other two proteins suggested that the isoforms were not considerably different from each other. PTPN6 and GluA2 isoform 2 were unique in that, when unedited, they had a lower concentration of alpha helices. All the other proteins had higher concentrations of alpha helices after they were unedited.

There was no significant difference between the edited and unedited forms of each protein (or each of the isoforms).  One would have surmised that changing the sequence (usually done by ADAR upon misediting) even slightly would have changed the outcome (structure) of the finished protein. There was, however, a considerable difference between the mean alpha helix percentages of both the edited and the unedited GluA2 isoforms, PTPN6, and 5-HT(2C) proteins. The beta sheets were significantly increased in GluA2 isoform 2, and the coiled regions were significantly increased in PTPN6. This reinforces the conclusion that there is a considerable difference between the secondary structures, especially alpha helices, of GluA2, PTPN6, and 5-HT(2C).

This is a notable finding because now scientists have a direction for further research.  Comparing the unedited sequences could lead to the possibility of correlations between the diseases coded for by the unedited proteins (namely sporadic ALS (GluA2), leukemia (PTPN6), and schizophrenia (5-HT(2C)). This, in turn, could lead to the discovery of a common pathway of action for these diseases and other related diseases. This would enable the creation of a possible common cure for these diseases.  It would also help people learn more about why there seems to be a significant difference between proteins with the same mechanism of action (PTPN6 and GluA2 isoforms).  Further research can also show the reason for the natural variance between the GluA2 isoforms.  Understanding the mechanism of action of misediting by ADAR and similar proteins would also increase the current knowledge of human diseases and further advance the possibility of treatment and prevention.

 

 

BROADER IMPACT

 

The impact of this project is attributed to the conclusion that misediting by ADAR causes significant changes in the secondary folding of the alpha helices, which further leads to a variety of diseases including ALS, leukemia, schizophrenia, epilepsy, cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders (Nishikura & Slotkin, 2013). These findings will enable scientists to know the specific area targeted by these diseases, so they can develop a quantitative model and find common cures for all of them. This also allows for other possibilities of disease prevention such as monitoring the structures of secondary structures in proteins to detect any changes possibly caused by misediting. Many people can be helped by these findings because it affects the pharmaceutical industry.  Billions of people will be impacted from this wide range of diseases. Over 14.5 million people have been affected by cancer in some way today (Learn about Cancer, 2015). Additionally, 50 million people and 24 million people are affected by epilepsy and other dementias - including Alzheimer’s disease – respectively (Bruttomesso, Miglino, & Quaroni, 2007).  About fifty million people in America alone have been diagnosed with metabolic syndrome (What is Metabolic Syndrome?, n.d.). Also, approximately fifty million Americans also suffer from autoimmune diseases (Autoimmune Info, n.d.). These are just a few of the neurological disorders that affect people all over the world. The data analysis and conclusions from this experiment can easily be utilized. Furthermore, this could lead to the discovery of a common pathway of action for these diseases and a common cure. Ultimately, this project advances human understanding of diseases and increases the possibility of treatment and prevention so that billions of people around the world may have greater hope of surviving from the diseases that too often claim the lives of many.

 

ACKNOWLEDGEMENTS

There are many people for whom I owe my utmost gratitude and thanks for guiding me through the course of this experiment. I would like to thank my Mom for being an endless source of support and encouragement to me. I would also like to thank Mr. Finkle, my mentor throughout this project, for the time and effort that he put into helping me and Mrs. Naughton, my teacher, for looking over all my work for this experiment, cheering me on, and guiding me along the way. Lastly, I would like to thank IJAS for giving me the opportunity to participate in the science fair. Once again, I would like to thank all the people who helped me because I could not have done my experiment without them!

 

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APPENDIX 1

Appendix 1 contains the protein sequences of both the edited and unedited forms of the proteins. The red letters represent the specific bases that are changed when edited, and which are responsible for altered function.

 

glutamate receptor 2 isoform 1  [Homo sapiens]

EDITED

mqkimhisvl lspvlwglif gvssnsiqig glfprgadqe ysafrvgmvq fstsefrltp

hidnlevans favtnafcsq fsrgvyaifg fydkksvnti tsfcgtlhvs fitpsfptdg

thpfviqmrp dlkgallsli eyyqwdkfay lydsdrglst lqavldsaae kkwqvtainv

gninndkkde myrslfqdle lkkerrvild cerdkvndiv dqvitigkhv kgyhyiianl

gftdgdllki qfgganvsgf qivdyddslv skfierwstl eekeypgaht ttikytsalt

ydavqvmtea frnlrkqrie isrrgnagdc lanpavpwgq gveieralkq vqveglsgni

kfdqngkrin ytinimelkt ngprkigyws evdkmvvtlt elpsgndtsg lenktvvvtt

ilespyvmmk knhemlegne ryegycvdla aeiakhcgfk ykltivgdgk ygardadtki

wngmvgelvy gkadiaiapl titlvreevi dfskpfmslg isimikkpqk skpgvfsfld

playeiwmci vfayigvsvv lflvsrfspy ewhteefedg retqssestn efgifnslwf

slgafmrqgc disprslsgr ivggvwwfft liiissytan laafltverm vspiesaedl

skqteiaygt ldsgstkeff rrskiavfdk mwtymrsaep svfvrttaeg varvrkskgk

yayllestmn eyieqrkpcd tmkvggnlds kgygiatpkg sslrtpvnla vlklseqgvl

dklknkwwyd kgecgakdsg skektsalsl snvagvfyil vgglglamlv aliefcyksr

aeakrmkvak naqninpsss qnsqnfatyk egynvygies vki

 

UNEDITED

mqkimhisvl lspvlwglif gvssnsiqig glfprgadqe ysafrvgmvq fstsefrltp

hidnlevans favtnafcsq fsrgvyaifg fydkksvnti tsfcgtlhvs fitpsfptdg

thpfviqmrp dlkgallsli eyyqwdkfay lydsdrglst lqavldsaae kkwqvtainv

gninndkkde myrslfqdle lkkerrvild cerdkvndiv dqvitigkhv kgyhyiianl

gftdgdllki qfgganvsgf qivdyddslv skfierwstl eekeypgaht ttikytsalt

ydavqvmtea frnlrkqrie isrrgnagdc lanpavpwgq gveieralkq vqveglsgni

kfdqngkrin ytinimelkt ngprkigyws evdkmvvtlt elpsgndtsg lenktvvvtt

ilespyvmmk knhemlegne ryegycvdla aeiakhcgfk ykltivgdgk ygardadtki

wngmvgelvy gkadiaiapl titlvreevi dfskpfmslg isimikkpqk skpgvfsfld

playeiwmci vfayigvsvv lflvsrfspy ewhteefedg retqssestn efgifnslwf

slgafmqqgc disprslsgr ivggvwwfft liiissytan laafltverm vspiesaedl

skqteiaygt ldsgstkeff rrskiavfdk mwtymrsaep svfvrttaeg varvrkskgk

yayllestmn eyieqrkpcd tmkvggnlds kgygiatpkg sslrtpvnla vlklseqgvl

dklknkwwyd kgecgakdsg skektsalsl snvagvfyil vgglglamlv aliefcyksr

aeakrmkvak naqninpsss qnsqnfatyk egynvygies vki

 

 

glutamate receptor 2 isoform 2  [Homo sapiens]

EDITED

mqkimhisvl lspvlwglif gvssnsiqig glfprgadqe ysafrvgmvq fstsefrltp

hidnlevans favtnafcsq fsrgvyaifg fydkksvnti tsfcgtlhvs fitpsfptdg

thpfviqmrp dlkgallsli eyyqwdkfay lydsdrglst lqavldsaae kkwqvtainv

gninndkkde myrslfqdle lkkerrvild cerdkvndiv dqvitigkhv kgyhyiianl

gftdgdllki qfgganvsgf qivdyddslv skfierwstl eekeypgaht ttikytsalt

ydavqvmtea frnlrkqrie isrrgnagdc lanpavpwgq gveieralkq vqveglsgni

kfdqngkrin ytinimelkt ngprkigyws evdkmvvtlt elpsgndtsg lenktvvvtt

ilespyvmmk knhemlegne ryegycvdla aeiakhcgfk ykltivgdgk ygardadtki

wngmvgelvy gkadiaiapl titlvreevi dfskpfmslg isimikkpqk skpgvfsfld

playeiwmci vfayigvsvv lflvsrfspy ewhteefedg retqssestn efgifnslwf

slgafmrqgc disprslsgr ivggvwwfft liiissytan laafltverm vspiesaedl

skqteiaygt ldsgstkeff rrskiavfdk mwtymrsaep svfvrttaeg varvrkskgk

yayllestmn eyieqrkpcd tmkvggnlds kgygiatpkg sslrnavnla vlklneqgll

dklknkwwyd kgecgsgggd skektsalsl snvagvfyil vgglglamlv aliefcyksr

aeakrmkvak naqninpsss qnsqnfatyk egynvygies vki

 

UNEDITED

mqkimhisvl lspvlwglif gvssnsiqig glfprgadqe ysafrvgmvq fstsefrltp

hidnlevans favtnafcsq fsrgvyaifg fydkksvnti tsfcgtlhvs fitpsfptdg

thpfviqmrp dlkgallsli eyyqwdkfay lydsdrglst lqavldsaae kkwqvtainv

gninndkkde myrslfqdle lkkerrvild cerdkvndiv dqvitigkhv kgyhyiianl

gftdgdllki qfgganvsgf qivdyddslv skfierwstl eekeypgaht ttikytsalt

ydavqvmtea frnlrkqrie isrrgnagdc lanpavpwgq gveieralkq vqveglsgni

kfdqngkrin ytinimelkt ngprkigyws evdkmvvtlt elpsgndtsg lenktvvvtt

ilespyvmmk knhemlegne ryegycvdla aeiakhcgfk ykltivgdgk ygardadtki

wngmvgelvy gkadiaiapl titlvreevi dfskpfmslg isimikkpqk skpgvfsfld

playeiwmci vfayigvsvv lflvsrfspy ewhteefedg retqssestn efgifnslwf

slgafmqqgc disprslsgr ivggvwwfft liiissytan laafltverm vspiesaedl

skqteiaygt ldsgstkeff rrskiavfdk mwtymrsaep svfvrttaeg varvrkskgk

yayllestmn eyieqrkpcd tmkvggnlds kgygiatpkg sslrnavnla vlklneqgll

dklknkwwyd kgecgsgggd skektsalsl snvagvfyil vgglglamlv aliefcyksr

aeakrmkvak naqninpsss qnsqnfatyk egynvygies vki

 

 

glutamate receptor 2 isoform 3  [Homo sapiens]

EDITED

mvqfstsefr ltphidnlev ansfavtnaf csqfsrgvya ifgfydkksv ntitsfcgtl

hvsfitpsfp tdgthpfviq mrpdlkgall slieyyqwdk faylydsdrg lstlqavlds

aaekkwqvta invgninndk kdemyrslfq dlelkkerrv ildcerdkvn divdqvitig

khvkgyhyii anlgftdgdl lkiqfgganv sgfqivdydd slvskfierw stleekeypg

ahtttikyts altydavqvm teafrnlrkq rieisrrgna gdclanpavp wgqgveiera

lkqvqvegls gnikfdqngk rinytinime lktngprkig ywsevdkmvv tltelpsgnd

tsglenktvv vttilespyv mmkknhemle gneryegycv dlaaeiakhc gfkykltivg

dgkygardad tkiwngmvge lvygkadiai apltitlvre evidfskpfm slgisimikk

pqkskpgvfs fldplayeiw mcivfayigv svvlflvsrf spyewhteef edgretqsse

stnefgifns lwfslgafmr qgcdisprsl sgrivggvww fftliiissy tanlaafltv

ermvspiesa edlskqteia ygtldsgstk effrrskiav fdkmwtymrs aepsvfvrtt

aegvarvrks kgkyaylles tmneyieqrk pcdtmkvggn ldskgygiat pkgsslrtpv

nlavlklseq gvldklknkw wydkgecgak dsgskektsa lslsnvagvf yilvgglgla

mlvaliefcy ksraeakrmk vaknaqninp sssqnsqnfa tykegynvyg iesvki

 

UNEDITED

mvqfstsefr ltphidnlev ansfavtnaf csqfsrgvya ifgfydkksv ntitsfcgtl

hvsfitpsfp tdgthpfviq mrpdlkgall slieyyqwdk faylydsdrg lstlqavlds

aaekkwqvta invgninndk kdemyrslfq dlelkkerrv ildcerdkvn divdqvitig

khvkgyhyii anlgftdgdl lkiqfgganv sgfqivdydd slvskfierw stleekeypg

ahtttikyts altydavqvm teafrnlrkq rieisrrgna gdclanpavp wgqgveiera

lkqvqvegls gnikfdqngk rinytinime lktngprkig ywsevdkmvv tltelpsgnd

tsglenktvv vttilespyv mmkknhemle gneryegycv dlaaeiakhc gfkykltivg

dgkygardad tkiwngmvge lvygkadiai apltitlvre evidfskpfm slgisimikk

pqkskpgvfs fldplayeiw mcivfayigv svvlflvsrf spyewhteef edgretqsse

stnefgifns lwfslgafmq qgcdisprsl sgrivggvww fftliiissy tanlaafltv

ermvspiesa edlskqteia ygtldsgstk effrrskiav fdkmwtymrs aepsvfvrtt

aegvarvrks kgkyaylles tmneyieqrk pcdtmkvggn ldskgygiat pkgsslrtpv

nlavlklseq gvldklknkw wydkgecgak dsgskektsa lslsnvagvf yilvgglgla

mlvaliefcy ksraeakrmk vaknaqninp sssqnsqnfa tykegynvyg iesvki

 

 

protein tyrosine phosphatase PTPN6 [Homo sapiens]

EDITED

QDGEVVSPRPQWAGCRDPAQGPRCPR LPGSAQSQEPG LLALRQVGGPPATPGILATLLCHPGPEPLIPGSPWQCLPV

CSLAPNPHTPHPCLCPPMPMCAPTQDLSRSLPSCLYSCTG

WPHRLVPCRVGDQVTHIRIQSSGDFYDLYGGEKFATLTE

LVECYTQQQGVLQDRDGTIIHLEYPLNCSDPTSERWYHG

HMSGGQAETLLRAKGEPWTFLVRGSLSQPGDFVLSVLSD

QPKAGPGSPLRVTHIKVMCEGGRYTVGGWRPSTASRTW

WSISRRRGLRRPQAPLSTCGSRTMPRG MRLTLRTECWN TRSRSPRIQPKAGFWEEFESL

 

UNEDITED

QDGEVVSPRPQWAGCRDPAQGPRCPR LPGSAQSQEPG LLALRQVGGPPATPGILATLLCHPGPEPLIPGSPWQCLP

VCSLAPNPHTPHPCLCPPMPMCAPTQDLSRSLPSCLYSC

TDWPHRLVPCRVGDQVTHIRIQNSGDFYDLYGGEKFAT

LTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERW

YHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVL

SVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGWRPS

TASRTWWSISRRRGLRRPQAPLSTCGSRTMPRG MRLT

LRTECWN TRSRSPRIQPKAGFWEEFESL

 

 

serotonin receptor 5-HT(2C) [Homo sapiens]

EDITED

DGVQNWSLAQIVVMIIITIGLINGVIMAMSVLKKEHNA

TNYFSMLLAIAVLMDGLLLPMVALLSILYDYVWPLPR

YLCPSIWVLDVTSFLASICLHMAISLYRDVAVRGPVEH

SRFNSRTMIAKI

 

UNEDITED

DGVQNWSLAQIVVMIIITIGLINGVIMAMSVLKKEHNA

TNYFSMLLAIAVLMDGLLLPMVALLSILYDYVWPLPR

YLCPSIWVLDVTSFLASICLHMAISLYRDVAIRNPIEHS

RFNSRTMIAKI

10

 

11

12 13 14 15 16 17 18 19 20 21 22 23

 

 

 

 

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