The CAG repeat is extremely polymorphic and varies from 10 to 35 repeats on chromosomes of unaffected individuals and from more than 36 to 180 repeats on chromosomes of HD patients. Huntingtin Protein can cause loss of striatal neurons and can eventually lead to Huntington’s Disease. The goal of this is to study the effect of an amino acid substitution with different expansion repeats on the normal HTT protein by using different servers such as NCBI database, BLAST, SWISS MODEL, and JPRED. Expansion repeats of CAG are distinctive among people which is the reason I sought to perceive how much different expansion repeats of a substituted amino acid can be. My results suggest that expansion of Asparagine repeats may result in similar functions as the Normal HTT protein with a slight change in the secondary structure.
Huntingtin is a disease gene linked to Huntington’s disease, an inherited, autosomal dominant genetic disease that is progressive and lethal. The mutant gene can end up affecting movement by attacking nerve cells. It results from genetic mutations involving trinucleotide repeats of the huntingtin gene, which encodes the huntingtin protein. Everyone has a gene that codes for huntingtin protein, yet not everyone has the same number of glutamine repeats. The gene IT15 consists of a three letter codon sequence CAG (cytosine-adenine-guanine) which is repeated in the beginning of the sequence. A normal, functioning Huntingtin Protein consists of 10-35 glutamine expansion repeats. Whereas, a mutated Huntingtin Protein consists of 36 or more glutamine repeats. The exact function of huntingtin’s protein is unknown, however, expansion of the glutamine repeats causes increased polyglutamine (part of the protein consisting of several glutamine units in the sequence), PolyQ, in the huntingtin gene which plays a regulatory role in transcription and intracellular transport in which it dysregulates cell homeostasis and promote apoptosis (Schulte 2011). HEAT (Huntingtin, Elongated factor3, PR65/A regulatory subunit of PP2A, and Tor1) repeats are present throughout the protein. HEAT repeats are thought to mediate protein-protein interaction (Littleton 2011).
Since glutamine is a polar molecule, it causes links to form within and between proteins. HTT molecules “stick” to one another, forming strands that are held together by hydrogen bonds (Liou 2011). These hydrogen bonds between amino groups and carboxyl groups in neighboring regions of the protein chain end up resulting in misfolding patterns to occur. Most proteins comprise numerous alpha helices and beta sheets. It is known that any amino acid substitution will result in an adjustment in the protein structure but the function may or may not change. Similar structures are more likely to have similar functions.
In this research, I will test how an amino acid substitution in a certain protein can have an effect on the protein structure and function, if applicable. The amino acid being used will be Asparagine since it has a similar structure as glutamine. Asparagine is also a polar molecule; the side chains of Asparagine can form hydrogen bonds interaction between peptide bonds. Asparagine is required for the function and development of the brain. I will use the sequences of that gene in many tools to depict the gene structure and function by expansion of asparagine repeats. Since Asparagine is also polar, it should form hydrogen bonds similar to glutamine. For that reason, I would assume that number of beta sheets should be similar between the four structures that will be compared. I hypothesize that the function of the amino acid substituted protein would be the similar to the actual mutated huntingtin protein.
The National Center for Biotechnology Information develops and maintains molecular and biological databases. Through this I was able to obtain the FASTA protein sequence of the Huntingtin Protein in Homo Sapiens, discover the iC3dN protein structure and basic background information about the protein. My entire methods were dependent on that sequence. Using NCBI was certainly easy. Depending on what you’re looking for, you click which database you want to get results from. In my case, I clicked on Protein and typed “HTT homo sapiens”. There were 20 of 103 that were based on my results. Some sequences were full and some were partial. I picked the full amino acid sequence which was 3144 aa with Accession Number: NP_002102. On the top of the page, there is a FASTA button and by clicking that I got the FASTA sequence. On the bottom of the page with the Huntingtin Protein results, there is a short summary about the disease gene.
BLAST
BLAST (Basic Local Alignment Search Tool) searches for areas of similarity among sets of sequences (Anon n.d.). This tool is used to compare nucleotide or protein sequences to the sequences in a database, and analyze quality of matches. The tool can also be utilized to conclude evolutionary as well as functional associations among sequences (Altschul et al. 1990).
BLAST was performed on the genes to find the homologous proteins and determine their function and structure similarity. Using this was kind of complicated, I had to compare the mutated HTT protein sequence, the amino acid substituted sequence that consisted of 23 repeats, and the sequence that consisted of 56 repeats to the normal HTT protein. When I first opened the BLAST page, I chose Protein BLAST (proteinprotein). I put the FASTA sequence of the normal HTT protein under “Enter Query Sequence” and then chose “Align two or more sequences”. The first time I put the mutate HTT sequence under “Enter Subject Sequence” and ran BLAST. I ran BLAST 3 different times (3 different sequences, mutated HTT, and the 2 substituted sequences) to see the similarity.
SWISS-MODEL
SWISS-MODEL is a server for automatic comparative modeling of a three-dimensional protein structures. (Anon n.d.). It predicts a template and the 3D model of the protein along with a statistical graph of the model quality. All the described techniques in the methodology were applied on all the allocated genes to obtain their function and structure as well as understanding the principles of the tools and databases used for the analysis. Using this was not difficult, however the results given were not sufficient enough. The first thing that appears on the SWISS-MODEL page is a “Start Modelling” button so I clicked that. Then I pasted my target sequence which was the normal HTT protein sequence and then clicked Build Model. After that I had to wait a little in order for my model to be built and the templates to be tested. I repeated the same steps for the other sequences as well. By just looking at the model structure you would not think that there was any change which was why I also decided to use JPRED.
JPRED
JPRED is a protein secondary structure prediction server which provides predictions by the JNet algorithm. With this I was able to determine the number of alpha helices and beta sheets in each structure. The limit for JPRED to run is 800 residues. One problem I faced while using this server was that I had to cut down my sequences because they were too long for the program to run. I could have split my sequence into separate domains but since I was looking at the 1st domain, I just stuck to comparing those. The red tubes are alpha helices and the green arrows are beta strands.
I acquired the Huntingtin Protein sequence of homo sapiens form NCBI which consisted of 3144 aa and had 23 repeats of glutamine in the beginning of the sequence, which means that the protein is considered normal. Then that sequence was put in JPRED and SWISS-MODEL to determine the number of alpha helices and beta sheets in it as well as the protein structure. Then 33 glutamines were added on to make that protein mutated which then consisted of a total of 56 glutamine repeats. With that being said, now I have a mutated Huntingtin Protein. The same steps were repeated and the sequence was put in both JPRED and SWISS MODEL as well.
Now that I was able to compare the normal HTT protein the mutated one, I wanted to compare the amino acid substituted sequence to them as well. I started off by adding 23 Asparagine repeats to the normal HTT protein and repeated the steps. After getting my results for that sequence, I wanted to see how the expansion of Asparagine repeats can change therefore, I added on 56 total Asparagine repeats.
Results:
I first obtained the HTT protein sequence from NCBI. That sequence showed 23 glutamine repeats meaning it is a normal functioning protein. I based off all substitutions and repeats on this sequence. I put the normal sequence in JPRED and SWISS-MODEL to further analyze my results.
The functional and homology analysis of gene provide plenty of information about the gene, its purpose, its expression, its domains, and biological process of the gene. The structure was constructed through Swiss Model (Fig.1a) where the structure clearly shows that the protein contains maximum alpha-helix and only few beta-sheets (Fig 1b).
The structure in (Fig. 2a) had a small change compared to the normal HTT protein structure (Fig. 1a). Based on fig. 2b, there was a reduction in the number of alpha helices and beta sheets. However, the alpha helix strand was longer when more repeats were added. Just as the normal HTT protein, it had a similar QMEAN.
There were several changes in the protein structure of the normal HTT protein, the mutated protein, and the two sequences that were substituted with Asparagine. The normal HTT protein in JPRED appeared to have a total of 20 alpha helices and 7 beta sheets and the structure showed 3 domains. Whereas, the mutated HTT protein had a total of 17 alpha helices and 4 beta sheets. There was an obvious reduction in both numbers which explains why the domains were a little clearer in the mutated HTT protein.
Moving on to the substituted sequences, there was a total of 19 alpha helices and 6 beta sheets with 3 domains showing in the 23 expansion Asparagine repeats. Whereas, there were 16 alpha helices and 7 beta sheets with 4 domains showing when more expansion repeats of Asparagine were added. The trend I saw was that the more expansion repeats, the less alpha helices, beta sheets and domains that will appear. With that being said, the structures were different from one another, however, the mutated HTT protein was similar to the sequence with more expansion repeats of Asparagine.
The Qmeans is the qualitative model energy analysis that analyze to local geometry of structure on pair wise residual level. The Qmean scores that are -4.0 or below is an indication of models with low quality. The values for all 4 models show that the models are significant. The local quality estimates the similarity between models and native structure which means the local geometrical structure of local quality also gives good plot that confirms the accuracy of the prediction. (Fig 1,2,3,4 c,d,e)
JNetCONF is the confidence estimate for the prediction; high values mean high confidence. The normal HTT protein showed 2-4-7 continued for the glutamine repeats whereas the values for the expansion repeats of glutamine was 1 which is very low, therefore low confidence. Both Asparagine repeat substitution sequences had values of 7 for the JNetCONF which is very high.
BLAST showed a 99% similarity between the sequences compared to the normal HTT protein. Questions such as what happens if another amino acid that is not polar is substituted can be answered by obtaining similar tests as the above methods and looking at the same results.
Since the structures have significant changes, the functions may not be similar.
Conclusion
Even after several studies made on Huntingtin Protein, the actual function of the gene IT15 is unknown but the protein structure is known. Through this study I was able to determine the protein structure of the mutated protein with and without the substitution of the amino acid Asparagine. I was able to determine if there were any protein structural changes by looking at the number of alpha helices and beta strands. Expansion repeats of Asparagine are very similar to the mutated HTT protein therefore it might have a similar affect and also lead to Huntington’s Disease.