In today’s paper we discussed the connection between protein folding and function. For the alleles discussed describe how the sequence changes affects the protein folding, localization, and function.
The paper also descibes a number of techniques that were utilized to determine the protein structures. Give an example of one of these techniques and explain how the technique used added to our understanding of the enzymes described.
Finally, why is important that we know what alleles of the TMPT gene an individual harbors before therapy? What are the frequency of the A/B/C alleles?
Gillespie Lab 
According to the paper, scientists believed that protein misfolding and aggregation are caused by a mutated allele, TPMT*3A. To test the hypothesis, scientists used a technique known as size exclusion chromatography demonstrated some of the similarities and differences between the variant alleles (3A, 3B, 3C) and the wild type. The results are as follows, while WT has proper folding of proteins and active enzymatic activities, TMPT*3A has misfolding proteins that couldn’t perform any enzymatic activities such as metabolizing 6-mecaptopurine or azathioprine. The technique also indicated that TPMT*3B has some misfolding yet no enzymatic activity whereas TPMT*3C has some misfolding and some enzymatic activities. These are important information to know because any person with a combination of these allele expressions might suffer no enzymatic activities, where thiopurine will not be metabolized and excreted resulting in the death of the individual.
Again it is important to know what alleles of TMPT gene an individual harbors before therapy because individuals with variant alleles cannot metabolize standard dosage of thiopurine drugs. Without knowing, individuals will have an extremely hard time metabolizing these therapeutic compounds and suffer death as the worst case. I feel that because of TMPT’s clinical significance, it should fall under the list of required valid biomarker and not simply suggested. We are talking about the alleles of normal genes here. These are genetic traits that medicine cannot alter. More importantly TPMT*3A is most common in Caucasians about 5% frequency, whereas TPMT*3C has a 2% frequency in East Asians.
This paper reminds me of something I saw on Grey’s Anatomy last night and that is the body’s natural ability at metabolizing drugs at different rates. In the episode, a patient woke up in the middle of her gastrointestinal surgery. Of course, that freaked everyone out and ultimately led the patient to lose trust in the doctor who performed the surgery. However, the reason she woke up was because of her body’s natural ability to metabolize anesthesia faster than normal people. Now if the doctors knew about her condition, they would have collectively agreed to give the patient a higher dosage and kept her unconscious till after the surgery is complete. Yet, there was no way of knowing that she had such condition. In the end, there was no happy ending. The patient was left traumatized, while Doctor Bailey lost the trust of her very first patient.
Overall I think this paper is very effective in narrating the hypothesis, the course of action and the result of the experiments. I really enjoyed reading this paper because it is concise and straightforward. And more importantly it enhances my understanding of the importance of protein structures and how a misfold in an innocent little protein can cause so many problems. I feel that the paper has definitely connected what I learned in class to real life situations.
The focus of this paper was on the TMPT allele and the detrimental effects misfolding of proteins can have in different alleles of TMPT. In normal protein folding, the sequence is transcribed from DNA and then translated into the amino acid chain, which is then folded into the three dimensional shape of the protein. The shape of the protein is what gives the protein its function. When it is misfolded; it tends to not express the intended function. In the TMPT allele, it has been discovered that the mutated forms tend to be misfolded, causing a problem for those who express this allele in their genome. Misfolding often occurs in proteins when there is a mutation in the amino acid. This mutation often switches a nucleotide base which can change the amino acid in the sequence completely. This change will therefore change the primary structure of the protein, and will cause the protein to fold up differently than it’s supposed to in its normal form. This is seen in TMPT, along with what occurs with the folding of hemoglobin in sickle cell anemia patients. Both TMPT and hemoglobin mutations include a switching of a base which changes the amino acid. When a protein is folding up, it relies on the structures that come before it, so if the primary structure is incorrect, the rest of the folding will be also.
This paper focused on trying to identify how badly misfolded the TMPT alleles are in four different types; TMPT a, TMPT b, TMPT c and TMPT wild type. One technique used to determine how misfolded the proteins were that came from the TMPT allele was the use of circular dichroism spectroscopy (CD spectroscopy).In CD spectroscopy, the shape of what is being studied is observed when a rotational spin is applied. This technique adds to our understanding of enzyme activity because it easily allows us to tell how misfolded or well folded a protein in. If the protein is well folded, the rotational spin will be greater because it will be more compact, allowing it to spin faster. If the protein is misfolded, chances are it will be less compact and more linear. This would cause the protein’s rotational spin to be slower because the linear parts will slow it down when spinning. By using this technique, we can predict that the higher the rotational spin, the higher the enzymatic activity occurring since proteins functioning as enzymes can only occur when it is in its proper folded form.
By studying the affects the different alleles have on protein folding, it gives an answer as to why certain people can’t take therapeutic drugs containing thiopurine. It is important to know of these mutations and the effect they have on people because thiopurine drugs can have an adverse effect for these people if they take them at normal dosages or at any dose at all. Those people who possess the TMPT an allele can die if given a dose of thiopurine because it is the allele that has the most misfolding. It is important to know this so people possessing these mutations aren’t exposed to dosages or medication that can be toxic to them.
Overall I enjoyed reading this paper because it presented primary research. It was very interesting to see all the techniques used. I found the methods section hard to follow though because of the complex terms and techniques. After reviewing the figures in class, it did make it clearer as to what methods were used in this research.
CD spectroscopy was utilized to stud structural differences between the bacterially synthesized allozymes. It is basically a version of light spectroscopy with UV rats being absorbed at very small wavelengths. It is known to provide valuable information about the secondary structure of macromolecules, commonly proteins. The data from this was shown in figure 5 of the paper. The graphs detail the folding, unfolding and shifts in protein structure based on the enzymatic activity. The peaks of the enzymatic activity and MAU were markers for this.
The importance of knowing what alleles of the TMPT gene before therapy is that the A allele of this gene, which is relatively rare in human tissue, can cause “life-threatening thiopurine
toxicity in patients treated with standard doses of these drugs.” Thiopurine drugs are commonly utilized in the treatment of many autoimmune disorders like rheumatoid arthritis. Also, understanding the differences of allele structure of proteins may lead to further research and understanding of other pharmacological issues.
The frequency of the alleles depends on how quickly they are degraded in human tissue. Figure 5 b shows that C is more present than B or A, with A, being the smallest amount remaining after two hours of degradation.
This paper shows how “common polymorphisms affects TMPT protein function and as a result therapeutic response.” The paper proves that patient who are homozygous for TMPT 3A can incur life threatening toxicity when they are treated with standard doses of thiopurines. This paper says that in previous studies when a patient has TMPT 3A there is hardly any enzyme activity, and it results in rapid protein degradation. TMPT 3A is folded and so it aggregates and becomes degraded.
One of the techniques that were used was size-exclusion chromatography for the purified bacterial recombinant WT (wild type), 3A, 3B, and 3C. Figure 5A showed that aggresome formation was the highest for 3A. The formation was as follows 3A > 3B > 3C > WT. This gave us a better understanding of the enzyme described because it compares them to each other so we know which one can be hazardous. The graphs in figure 5C show the protein elution profile and the TMPT enzyme activity. It depicts that 3A definitely has strong enzyme problems as compared to the wild type as does 3B but not as great and 3C is better than the other two but is still not as good as the wild type.
It is extremely important to know what alleles of the TMPT gene an individual harbors before therapy. “TMPT is a cytotoxic drug-metabolizing enzyme that catalyzes the S-methylation of thiopurine drugs…” What the TMPT 3A gene does is not allow the metabolization of that drug therefore if a person goes on the drug with this gene their body can become intoxicated by the standard dose that would normally be given to them. This standard dose can be extremely toxic to the patients’ health.
I think that this was a great paper in the fact that it correlated with what we were learning in lecture and lab that week and it was great to get a look at an application that people are actually using in the lab. Also it was great to see how these proteins misfoldings and aggregations can affect people outside of the lab. I would also like to comment that I am really enjoying the way you showed us to read research papers. I took research for 4 years in high school and I wish I knew your method while I was reading papers for that class it makes them a lot easier to understand.
Some of the different alleles of the protein TMPT can dangerous effects on the body. In order to make proteins, RNA is first transcribed from DNA, and then Translated to form a polypeptide chain of amino acids. Sometimes misfoldings can occur; such is thought to happen to the allele TMPT. Misfoldings in proteins can change their shape and conformation, which can in turn affect its function. Mutations can also occur in the sequence of amino acids, that can cause deletions, substitutions, and frame shifts, which in turn can change the whole sequence and form a new or unusable protein. Misfolded proteins are not needed in the body and are removed by the cell by degradation. Since TMPT 3A, 3B, and 3C are misfolded and do not perform the right function, they form aggresomes to be removed.
On technique used to determine the proteins structures was size exclusion chromatography along with CD spectroscopy. In the paper they show the different rates of misfoldings between the alleles TMPT 3A, 3B, 3C, and WT. This helped us to compare their aggresome formation, degradation, and behavior. By comparing these alleles in Figure 5, we can see that the allele 3A, forms aggresomes the fastest and is the most hazardous, compared to the other 3. From this we can also observe that the WT also has protein misfoldings and forms aggregates but is the safest one.
It is important to know the allele’s people have for certain genes, because drugs have different effects on certain people. If a person is given a drug like thiopurine, depending on the allele they have for TMPT, dangerous effects can occur, such as death because of misfolded proteins. By studying the alleles and effects of the drugs on them, we can be able to give people the right drug and dosage that will be safe for their bodies. The most common of these alleles is TMPT 3A, which 3C is the least.
I enjoyed reading this paper because it was very reasonable and effective. The experiments carried out were also very clear. I also liked how this paper corresponded to the lab and lecture topic we had been learning about this week.
Variant alleles of thiopurine S-methyltransferase, or TPMT, include TPMT*3A, TPMT*3B, and TPMT*3C. TPMT*3A is the most common variant allele amongst Caucasians. It encodes for a protein that makes two changes in the amino acid sequence. Rapid degradation by ubiquitin proteasome occurs and results in no enzyme activity or protein. This suggests that this allele resulted in a misfolded protein. TPMT*3A was also found to form aggresomes in the presence of proteasome inhibitor MG132. This allele has two common polymorphisms. Its SNPs disrupt the enzyme structure which leads to its degradation. Alleles TPMT*3B and TPMT*3C have one of these polymorphisms. TPMT*3B is misfolded and its monomer has no activity. TPMT*3C also is misfolded but has comparable Km values as WT. One of the methods used to determine the protein structure was CD spectroscopy. The alleles were purified by size exclusion chromatography. Using this technique, we discovered that the alleles WT and 3C were similar in that they had a trough at 224 nm and therefore may have a beta-sheet structure. 3B had a peak at 220 nm and a lower signal, which is a different structure that may be the structural reason for the lack in enzyme activity. 3A aggregates had a peak at 231 nm. CD thermal unfolding revealed that WT and 3C were able to unfold but 3A was not able to because it did not show an unfolding transition. 3B showed an unfolding transition in the opposite direction with little signal change, which was determined to not be a false unfolding transition. Tm values were calculated using fraction folding curves ad EM was done to observe aggregate structure. The WT enzyme aggregate was smooth before and after filtration whereas the 3A one formed aggregates. It is important to know what allele of the TPMT gene a person has before therapy because it may result in adverse outcomes. Those homozygous for 3A can suffer from severe toxicity when consuming thiopurines. Standard doses are recommended for those suffering from this. 5% Caucasians have the 3A variant allele. 2% frequency of East Asians have the 3C allele and 3B is rare.
Behind every properly functioning protein lies a gene which codes for its function. If the slightest change occurs in that gene, the protein it codes for will ultimately be affected. The paper we discussed today describes how an allele that undergoes a sequence change affects the proper folding of the TMPT protein. Alleles make up our DNA, which is then transcribed into RNA which then utilizes several forms of RNA in order to construct a polypeptide chain on the ribosome. A correctly assembled polypeptide chain will then fold to form a three dimensional shape. The shape of this protein determines its function. If a protein is not folded in the correct way, it will not function properly. This is the case with the TMPT gene and its protein. The TMPT allele has three common variants, 3A, 3B, and 3C. These three variants in the TMPT allele cause the TMPT protein to misfold and therefore cannot execute its function. The TMPT protein is a drug-metabolizing enzyme that is vital for patients taking thiopurine drugs. If the protein is not functioning, the thiopurine drugs cannot be metabolized and they then become lethal to the patient. The buildup of these misfolded TMPT proteins causes the formation of aggresomes. The aggresomes are vesicles that contain the non-functioning TMPT proteins until they are able to degrade.
One of the methods the researchers used in this paper to determine protein structure is known as circular dichroism spectroscopy. CD Spectroscopy determines how a sample absorbs left or right handed circular polarized light (left helices or right helices). The analysis of the CD spectra produced can determine the secondary structure of macromolecules such as proteins. The data will reveal if a protein’s secondary structure is an alpha helix, beta sheet, or random coil. CD Spectroscopy helped the researchers determine which allele (TMPT 3A, 3B, or 3C) produces the most misfolded TMPT protein.
It is important to know what alleles of the TMPT gene an individual harbors before therapy because if they possess a TMPT gene that leads to the formation of misfolded TMPT proteins, the thiopurine drugs the individual needs won’t be metabolized and will become lethal. With this information, doctors will know not to prescribe a thiopurine drug. Knowing which allele of the TMPT gene a patient has will also let physicians decide what dosage to prescribe them.
Of the three allele variants, 3A is the most common and occurs in 5% of the population. That is followed by 3C which is seen in 2% of the population. The least common is the 3B variant.
I really enjoyed this paper because it explained the study clearly and thoroughly in a language that was easy to understand. The materials and methods section was most complicated since I am not familiar with all of the lab techniques mentioned. I thought this paper was an excellent example of how genetics play an important role in drug therapy.
In this article, it discusses how thiopurine S-methyltransferase (TPMT)can affect the protein folding within the allele of blood cells.TPMT is described as a drug-metabolizing enzyme that acts as a catalyst for drugs used for leukemia, autoimmune disease, and transplant recipients. These variant alleles are known as TPMT*3A, TPMT*3B, TPMT*3C. The most common variant allele is TPMT*3A and people who are homozygous for TPMT*3A can suffer from life-threatening toxicity with the intake of TPMT. This enzyme degrades protein folding by targeting the aggresomes and misfolding the molecule within the cell. As a result, the protein function is disrupted due to the rapid degradation by a ubiquitin (Ub)/proteasome-dependent process.
One of the techniques utilized to determine protein structures was Wild Type. Wild Type is a mechanism that refers to the phenotype of the typical form of a species as it occurs in nature.This form of determination assisted in understanding the enzymes being described because the wild type was the standard allele that contrasted with the nonstandard allele. Through this process, it was determined that the TPMT*3A has a stronger sensitivity to the enzyme when compared to the other alleles.
It is very important for physicians to be properly informed of what allele of TPMT gene an individual harbors because it could assist in saving a life. TPMT is a metabolizing drug that is utilized by patients that consist of children, as well as adults. Since this drug is commonly used for a good deal of diseases, then physicians should be very aware of the TPMT gene in a patient because without this knowledge then their life will be at stake. The frequency of the *3A allele is 5% in Caucasians, *3C is 2% in East Asia and *3B allele is considered to be very rare.
I believe this was a very good paper because it helps students become aware of the importance of pharmacogenetics in drug therapy. This importance is a concept that is often heard about within lecture. But it isn’t until we actually read about specific situations that this intangible importance becomes a more concrete value.
Thiopurine S-methyltransferase (TPMT) is one of the striking examples of the clinical relevance of genetic variation in drug metabolism. Thiopurine drugs are used to treat childhood leukemia, autoimmune diseases, and transplant recipients. However, these agents have a narrow therapeutic index with potentially life-threatening drug-induced toxicity. TPMT *3A allele is the most common variant allele in Caucasians (5% frequency) and TPMT is most common in East Asians (2%). Therefore, scientists hypothesized that TPMT 3A alleles might result in protein mis folding and aggregation. Since this allele seems to be most common among its counterparts TPMT *3B and TPMT *3C alleles. It was necessary to act promptly and observe the results which might correlate with the proposed hypothesis.
To better understand the protein structure of TPMT *3A allele, scientists used various experiments to prove their initial hypothesis. One of them includes chromatography which which explained aggresome formation and the respective enzymatic activity in different alleles: WT, *3A, *3B, and *3C. Since mis folded proteins often are targeted for degradation, but when the protein degradation capacity is exceeded, they also may accumulate aggresomes. Aggresome is the proteinaceous inclusion body that forms when cellular degradation machinery is impaired leading to an accumulation of protein for disposal. It forms in response to a cellular stress which generates a large amount of mis folded or partially denatured protein. The chromatography process compares WT allele with mutant alleles and the results reveal that WT exhibits various properties required for normal functioning as well as producing appropriate protein structure, whereas TPMT *3A allele shows no signs of enzymatic activity and is perhaps the most dangerous kind of protein structure (analogous to prion structures). On the other hand, *3B also shows no enzymatic activity but is still better than *3A allele. Lastly, *3C allele shows some enzymatic activity but it still cannot be considered a normal protein structure.
It seems vital to acknowledge the fact about alleles of the TPMT gene an individual harbors before therapy because a person might endure adverse health problems which might lead to various diseases and also death due to inability to metabolize thiopurine drugs.
Overall I enjoyed reading this scientific research because it helped me broaden my knowledge about the different kinds of protein mis folding and adverse effects of mutant (variant) alleles. I think this paper served as a supplemental to the protein lab we performed this week, while elaborating on effects of aggresomes and distorted protein structure.
The paper “Human Thiopurine S-methyltransferase Pharmacogenetics: Variant Allozyme Misfolding and Aggresome Formation” suggests that a disruption in the amino acid sequence and/or the alleles of a protein that causes a misfolding to occur, is more than likely the cause of it not working. Proteins internally know which way they are supposed to fold. Structure determines function. When there is a mutation in the gene for how the protein should fold, it makes the protein lose its funtionality. Misfolded proteins are first marked to be sent to the proteosome where it will be degraded but when this structure becomes overwhelmed, the misfolded proteins are then stored in vesicles until it can be degraded.
The protein structures were determined through fluorescent lighting. The stain sticks to anti-bodies on the TPMT. This technique added to the understanding of enzyme structur because the wild type of the protein shows fluorescen light a little faded where it is spread out. The mutant displayed globs of light indicating how the anti-bodies are clotting together.
It is important to know what alleles of the TPMT an individual possesses before therapy because the drugs the person take are toxic until it can be metabolized into something useful for the body. If the body can not metabolize it or excrete it then the toxins inside of the body will build up and death may occur. The allele *3A is the most dangerous as well as the most common allele. It shows the least amount of enzymatic activity compared to the wild type and the other alleles. The *3B allele begins as a big aggregate but if the temperature increased the proteins would unfold. The *3C are less folded as the temperature increases but still sticks together.
The article serves a beneficiary role in the science for medical and experimental reasons. However, I am partial to the article because I have a short attention span and it is difficult for me to focus. Still, if drug companies took into consideration the genetics behind pharmaceutical drugs, they could utilize their knowledge to their advantage to avoid consumers from suffering dire side effects and make more effective products.
I feel that this article is trying to inform us of the importance of knowing pharmacogenomics because not everyone’s genes are the same and that means that they do not all take in drugs the same way. This case is true in the case of an enzyme called Thiopurine S-methyltransferase, also known as TPMT. The thiopurine drugs, 6-mecaptopurine and azathiopurine, need TPMT to metabolize them. Not everyone can catalyze thiopurines. It depends on the person’s genes. There are many variations of the alleles that control the function of TPMT: TPMT 3A, TPMT 3B, TPMT 3C, and the wild type. The frequencies for TPMT 3A, TPMT 3C, TPMT 3B are, respectively, 5%, which are found in Caucasians, 2%, which are found in people from East Asia and, ≤1%, which indicates that it is rare. All except the wild type misfolds the protein and consequently will form aggresomes. This means that these people may not metabolize as much or not as fast as the wild type. People with TPMT 3A is especially bad because it leads to no enzymatic activity. If people with TPMT 3A were given the standard dosage of thiopurine drugs, then they will be experiencing severe toxicity that could lead to death. If we knew the type of gene that the person has, then we would be able to know how much of the thiopurine to give, so that the patient would not suffer from adverse effects.
This study also shows us how just a small change in the sequence in the DNA can disrupt the function of a protein and prevent the person from metabolizing thiopurone drugs. Through the process of transcription, where the mRNA’s are made based on the DNA, and translation, where tRNA’s bring the correct amino acids to the ribosome based on the codons on the mRNA’s, proteins are created. The instruction of how the protein will fold now lies on the polypeptides that derived from the two processes. If a sequence was changed on a particular allele, then the folding will, therefore, be changed as well. The conformation of the protein is vital. It enables only a certain ligand or substrate of that shape and size to fit in there, so that the enzyme can be activated and function properly. If the folding changes, and as a result, the conformation too, then the protein will not be able to perform its’ job.
The paper describes a technique called size-exclusion chromatography that can be used to show and identify the structural differences amongst all four types of alleles for TPMT. The way that size-exclusion chromatography works is that molecules in solution will be separated according to sizes. The molecules in the solution will be shot through a column that contains numerous holes of different sizes. Since it takes longer for bigger molecules can not go through all the small holes, they will either be deflected or will exit the column faster. Small ones will go through more holes and therefore gets stuck longer in the column. Ones that got through the holes will appear on the graph. In the case of proteins, those that folded correctly will be smaller and therefore there will be a peak someone on the end of the graph. Those that have mutations and are not folded properly, meaning that it is not folded in the most stable conformation where it is all balled up, will either not appear on the graph because it is too big, or appear in the beginning of the graph. In figure 5c, you can see that the enzymes were folded correctly for those with the wild type allele. It has the highest peak and it appears towards the end of the graph. This means that there are enzymatic activities. For the graphs for TPMT 3A and TPMT 3B, there is either little or no peak. This indicates that the structural of the protein is large and misfolded. TPMT 3C, on the other, shows a peak at the end as well, but it is not as high as the one shown in the graph for the wild type allele. This indicates that the proteins are misfolded, but enzymatic activities appear. People with TPMT 3C can probably catalyze thiopurine, but not as effective as those with the wild type.
Overall, I felt that this paper was much more interesting. It was easier for me to understand what was going on and how it can be applied to our lives. It was a real life example that I can relate to and understand. It took the information I learned in class and lab and put it in a real life situation. It made me realize the complexity how proteins are made and folded and how the field of pharmacogenomics is important.
Therapeutic drugs are used commonly worldwide such as anti depressants, analgesics and antihistamines and this fact relates greatly to this paper because although very helpful in most cases therapeutic drugs can also be very deadly. The reason for this is because there are a number of drugs that need to be metabolized by processes within the body before they take their helpful form but unfortunately some humans lack the ability to metabolize certain drugs and this can be fatal. Not being able to metabolize certain drugs is commonly due to a mutation in certain alleles and that is the general idea of this paper. The papers main focus was to identify and demonstrate how the mutated allele thiopurine S-methyltransferase (TPMT) effected the folding of proteins in the body.
They were able to prove that TPMT does in fact cause the misfolding of proteins through the use of various techniques. One technique in particular was CD Spectroscopy which was used to determine the shapes of the proteins thus helping the scientists determine the severity of the misfolding. CD Spectroscopy works by a rotational spin and changes in the shapes of the subject being tested effects the speed of that rotational spin, to be more specific, proteins which are very misfolded spin slower and proteins which are very folded allow for a faster spin. As reference was made to it, this is very much like a person spinning on ice, if one wished to spin faster they would bring their arms in close to them forming a tight compact form and if one wished to slow down they would unfold their arms and spread them out. Through this test the scientists were able to determine that the TPMTa did not change as much as the others, TPMTb aggregated but was not as bad as TPMTc in which the proteins misfolded early.
It is very important that we know what alleles of the TPMT gene an individual harbors before therapy because as noted before individuals with mutated alleles do not have the ability to metabolize the amount of a therapeutic drug that is usually given. This is crucial because if these individuals are given standard dosage the drug may become toxic to their body, become life threatening and likely cause death due to these drugs being non excretable. However if we are able to identify the allele’s, if it is the case that the individual can metabolize but just at a slower rate they can be given a smaller dose of the drug and if it is the case that they cannot metabolize at all perhaps an alternative can be given. The most frequent of these alleles is the TPMT*3A allele which has a 5% frequency in Caucasians and second most is the TPMTb which has a 2% frequency in East Asians.
I viewed this as a very informative paper although considerably hard to grasp at first once I understood the concepts and methods behind it; it was fairly easy to understand the purpose of it. I believe that the test for these type of alleles should not be on the recommended test list, it should be mandatory although this is not a very common issue it does exist and the effect it has on certain people is drastic. Overall this paper helped me to better grasp and understand a number of scientific concepts and I was glad to have read and understood it.
Protein folding is important for selecting functions of proteins as amino acid sequence is the crucial indicator in how proteins fold. Normally, amino acid sequence is transcribed by mRNA from DNA and translated to chains then which it folds to give the polypeptides its shape and function. However, the genetic polymorphism of TPMT allele causes misfolding and this creates such outcomes as TPMT*3A. TPMT*3A is a variant allele for low activity that seems to affect protein misfolding and aggregation. To test this hypothesis, scientists did several studies such as size exclusion chromatography which is a test to explain the differences between the variant alleles: TPMT*3A, TPMT*3B, TPMT*3C and WT (wild type). After the TPMT allozyme aggresome formation and degradation, which showed that the TPMT*3A had two SNPs suggested that it might result in TPMT misfolding and aggregation. So the size exclusion chromatography showed that *3B eluted 73% aggregation, *3C eluted 67% and WT was 37% aggregated which showed that these three alleles can possibly aggregate. *3A however eluted entirely as aggregated protein which suggests that much alterations altered the TPMT structure and caused misfolding and aggregation.
It is very important for scientists and the person who harbors the alleles to know what alleles of the TMPT gene they carry because some variant alleles are harmful because they can’t metabolize the toxicity of some thiopurine drugs even the standard dosage. If one was to take a standard dosage of a thiopurine drug carrying the TPMT*3A allele, he or she is very susceptible to toxicity and very likely even death. The TPMT*3A encodes the protein with two changes in amino acid sequence which causes misfolding and aggregation and because of this it makes the TMPT protein act in low activity and thus makes the TMPT protein which metabolizes the thiopurine drugs almost undetectable. This will cause the spread of toxicity of the thiopurine drugs within the body without a functional metabolizing agent.
By reading this paper, I definitely get a sense of how one’s genetics plays a part in effects of drugs. It seems it’s all hereditary because for example “TPMT*3A is the most common variant allele in
Caucasians” and this shows that obtaining this allele is not something one can avoid if they’re Caucasian and born with it or receive from some kind of transmission like a virus. If that’s the case personalized medicine seems to sound very favorable as each person’s genes vary to an astounding degree. I personally liked this article because it shows the importance of consumer products and the difference in dire effects of pharmaceutical drugs from one person to another. This article shows that genetics play an important role with drug interaction as there are many things in our body that are still to be studied and tested so that each individual person can have medicine that really work for them.
In pharmacogenomics, the study of gene variants on drug response, we have to be able to discern the possible feedback consequences of such drug effects in response to doses of thiopurine drugs. One variant allele for Thiopurine S-methyltransferase (TMPT), TPMT*3A, can change two amino acid sequences in protein and lead to extreme levels of toxicity with few doses of thiopurines. This raises doubt and inquires a question on this matter, whether or if the misfolding of the variant allele is the cause for the direct observation of these effects. There were three types of variant alleles studied: TPMT*3A, the most common variant allele in caucasians, TPMT*3B, a rare allele that has only the codon 154 SNP, and TPMT*3C, the most prevalent allele in East Asia which contains the codon 240 SNP. The TPMT*3A allele has two SNPs, both of which can influence the disruption of protein function by decreasing the level of enzyme protein and the presence of TPMT*3A results in rapid protein degradation. These misfolded proteins formed aggresomes in COS-1 cells as well as in a rabbit reticulocyte lysate (RRL) and in vitro aggregation in Escherichia Coli. When misfoled proteins accumulate, aggresomes appear, but they can sequester potentially cytotoxic aggregates which are believed to be transported to the microtubule network via dynein. But in the presence of the drug vinblastine, aggresome formation was disrupted, but it did not affect microaggregate aggregation in protein. Similarly, HDACs had multiple effects on TPMT*3A by linking misfolded proteins to the dynein-microtubule complex or inhibiting aggresome formation thus hypothesizing that HDAC might connect TPMT*3A to facilitate the transport of TPMT*3A to the aggresome. To determine the effects of sequence changes on protein folding, localization, and function, TPMT variants *3A, *3B, and *3C were compared in regard to aggresome formation, and it was determined that the proteins encoded by TPMT alleles with two SNPs, or either alone, are misfolded. These misfolded proteins are therefore targeted for degradation and they accumulate in the aggresome. The TPMT*3A SNPs disrupt the structure of the enzyme, resulting in misfolding, aggregation, and adverse effects on patients.
To test the probability that SNPs in genes can alter the rates of aggresome formation, immunofluorescence with COS-1 cells was conducted. COS-1 cells were transfected with TPMT*3A constructs and HA-tagged WT. Results showed that about 40% of transfected COS-1 cells with TPMT*3A after 20 hours of MG132, a proteasome inhibitor, indicated aggresome formation. Less than 1% of cells tranfected with WT showed aggresome formation. In immunofluorescence techniques, cells or proteins are transfected with foreign genes into a cell’s genome by artificial methods. This helps to count randomly selected regions for the expression of the quantity of aggresomes.
It is crucial to recognize which type of allele for TPMT one harbors before being introduced to thiopurine drugs because TPMT*3A genes can be dangerous in patients with standard doses of these drugs due to toxicity which is induced by TPMT*3A SNPs that defect the process of folding, and aggregating. Compared to TPMT*3A which has two polymorphisms, *3B and *3C alleles each have one SNP, where individual SNPs harbor a less effect on protein aggregation and aggresome formation.
From the results of the conducted studies we can conclude that the two common coding SNP’s in TPMT result in protein misfolding and by doing so TPMT is not able to metabolize the drug. This drugs that stay inside the patient’s body then cause damage to the organism with possible lethal outcomes. By experimenting one wild type alleles (WT) and three mutated alleles (*3A,*3B,*3C) we determined that in two cases (*3A, *3B) The TPMT enzyme was not able to process the drug and with protein aggregation and aggresome formation.
One of the techniques used in testing this peculiar case is TPMT CD Spectroscopy. In this experiment the graphs of the three TPMT enzymes and the WT have different peak locations. Graphs of *3A and *3B have different peak types from the WT which shows that the structure of the enzyme has been changed in this two and the proteins can’t do their function, because they are something else and not TPMT.
It is important for us to know about alleles of TPMT because people with this mutated allele are not going to be able to metabolize the drug and the drug might end up to be lethal for them. TPMT is a very good example of diversity among people and the effects of drugs on people of different geographical ancestry and how drugs use enzymes in organisms.
To be honest in beginning when I started reading this paper I had major difficulties trying to ‘’decrypt ‘’ this paper. For me at least it was hard to get all the names as MG132, Cos-1… After several copies of torn up papers in minor misunderstandings with me and the paper, I finally set down and started to separate all the names that I don’t understand and after some careful reading I started liking it. Now I am glad that I read the paper because I loved reading about the experiments conducted and the results that showed us the reason of toxicity in TPMT.
Fatima Taylor
02/05/10
TMPT and Protein misfolding
When reacted with different forms of the drug Thiopurine, individuals who possess the alleles TPMT in the forms 3A, 3B and 3C experience protein dysfunction due to polymorphisms. There is a rapid degradation of the Ub/proteasome process, which causes an imbalance in the proper folding and function of proteins. When administered this drug, the people who possess this allele, experience allozyme degradation and misfolding. By the drug reacting with TPMT, the interference with the protein sequence process can cause serious biological effects. The proper sequencing of amino acids is crucial to the functioning of the protein. The sequence determines a proteins folding, which is essential to proper protein localization and functioning. The sequencing is the information that directs the protein on the path that it takes.
In the cases of TPMT, when exposed to thiopurine, this balance of sequencing is thrown, causing a misfolding of the proteins, which is seriously hazardous to the individual.
One of the techniques used to determine and describe these TPMT alleles was fluorescence microscopy. It was performed with polyclonal anti-HA antibody to detect TPMT and aggresome components. The scientists stained TPMT3A to locate the aggresomes. What they found that was that with the involvement of dyenin, that aggresomes are microtubule dependent. Also, that Ub and molecular chaperones play a major role in the process of protein degradation and misfolding by reacting with the aggresomes. By observing this, they concluded that when the misfolding of proteins due to TPMT toxicity, is an intricate process and one that involves several cellular structures including centrioles and microtubules.
It is extremely important to be aware of what the TMPT alleles harbor before therapy because by not knowing, we can cause serious complications. When individuals are administered thiopurine, it must be known whether or not people possess these alleles because if remained unknown, these people can experience serious toxicity if a large amount of the drug us administered. Not being aware of the allele can cause protein degradation which can lead to a multiple number of toxic consequences, and even death. To now about these markers is very important because it can mean the difference between life or death in many instances. By knowing this information before drug therapy, it will be insured that the patient will be safe and that their life is not placed in jeopardy. TPMT 3A is the most common variant allele in Caucasians, and has a 5% frequency. TPMT 3C is the most common variant allele in East Asia and has a 2% frequency. TPMT 3B allele is rare and contains only one codon.
Thiopurine S-methyltransferase (TPMT) is a cytosolic drug-metabolizing enzyme that catalyses the S-methylation of thiopurine drugs, which are drugs used to treat childhood leukemia, autoimmune diseases, and transplant recipients. However, TPMT*3A, the most common variant allele for TPMT, results in the virtual lack of TPMT protein and enzyme activity. For this study, the scientists tested the hypothesis that TPMT*3A might result in protein misfolding and aggragration. Through such techniques as size-exclusion chromatography and CD spectroscopy the scientist found that TPMT*3A is misfolded, aggregates, and is a target of degradation. The misfolding results in the very low amount of TPMT in patients that have TPMT*3A, which prevents TPMT proteins from functioning properly.
One of the techniques used in this study was CD spectroscopy. Circular Dichroism (CD) is observed when optically active matter absorbs left and right hand circular polarized light slightly differently and CD spectrometer is used for CD spectroscopy. In this study, the CD spectroscopy was used to study the potential structural differences among the variant alleles which helped us better understand the enzymes and alleles being studied. It suggested that WT and TPMT*3C primarily adopt b-sheet structure, TPMT*3B has a significant change in secondary structure (which might explain the lack of enzymatic activities in TPMT*3b, and that TPMT*3A aggregates with a shift 231 nm.
It is important to know what alleles of TMPT gene an individual harbors before therapy because those who harbor the variant alleles of TPMT, such as TPMT*3A , cannot metabolozie hiopurine drugs since they will lack TPMT enzymes. Without the metabolizing, these individuals will not be able to treat their health problem. Among Caucasians, TPMT*MA is the most common variant allele in with a 5% frequency while TPMT*3C is the most common variant allele in East Asia with a frequency of 2%. TPMT*3C is the least common variant.
In this paper “human thiopurine S-methyltransferase pharmacogenetics” mainly focuses on the problem of protein misfoldings. Proteins are the most important molecules in our bodies, because they are involve in almost all the bodily functions. Proteins are usually get rid of by proteasome, a long chain of polypeptides will go in and only amino acids will come out. In this particular paper, it describes the problem that is cause when give certain doses of the drug TPMT will actually cause life-threatening toxicity if that individual has mutated alleles on 3A,3B,3C. This serious disorder is primarily cause by the misfoloding of the protein structure in the body. As the proteins misfold themselves, they are being store in the vesicles of the cells and come together to form very large aggresomes. One of the techniques that are used to determine the protein structures is the TPMT CD Spectroscopy. In figure 6, they show us three different graphs that compare and contrast the effect of the variables had on various wild type and mutated 3A, 3B, 3C alleles and from these comparisons we can roughly determine the particular protein structure for each alleles. Based on the graph that was provided, the wildtype and the 3C had very similar spectra, it is around 224nm so they might have a beta- sheet for the secondary structure. 3B and 3A alleles’ spectra are very shifted to 220nm and 231nm, this means that their secondary structure is misfolded. The second graph is a representation of the influence under temperature factor. The 3A aggregates allele does not change as the temperature increases, the 3B monomer starts with aggregates but it breaks apart as the temperature of the system increases. The third graph compares the wildtyped monomer with its similar spectra 3C monomer, the 3C monomer tends to have fewer folds at the lower temperature.
It is important that we know what alleles of the TPMT gene an individual harbors before therapy because when individuals have mutated alleles of the TPMT gene then their bodies are not able to metabolize the drug fully, sometimes the overuse of the drug can be toxic to the body and even can cause death. In order to avoid life-threatening toxicity, these patients must be treated with from 1/10th to 1/15th of the standard dose. Caucasians have five percent of frequency of having 3A alleles on the gene and East Asians have two percent of frequency of having 3B and 3C alleles on the gene. I find this paper very interesting to reading, because it guided me through the process of research and how pharmacogenomics is significantly important to individuals with various genetic disorders.
The paper’s main point is to prove that the mutant allele TPMT*3A causes misfolding in the protein it encodes, and that the misfolding is what causes the proteins to be aggregated by a cell. The paper does not only focus on the mutant allele but on the TPMT*3C, the Wild Type and the really rare TPMT*3B. While the Wild Type gene is able to encode proteins that fold properly and therefore have enzymatic activity, the three alleles encode proteins that misfold and thus are aggregated by the cell without doing their required work. The mutant allele TPMT*3A is the gene with the lowest enzymatic activity, because it causes the most misfolded proteins. The TPMT*3B has the same effects as TPMT*3A but it has a less percentage of cells that grow these aggregates. The TPMT*3C has some aggregate but unlike the other two alleles it did show some enzymatic activity. In order for me to understand these results I had to look up the word aggregate. What I understood from the meaning is that when the protein degradation process is overwhelmed the cell puts all the destined proteins in vesicle until they are degraded. This helped me understand more what the paper meant when they mentioned that TPMT*3A is highly ubiquitinated. Ubiquitin is used as a marker for the degradation of proteins, so for me this meant that the change the TPMT*3A had on the protein was that a part of misfolding them they were already destined to be destroyed. And the more proteins it produced the more stress it put on the cellular degradation machinery, thus giving me a reason to agree with the results of this paper.
In order to prove these results there were many techniques employed, but the one I was able to understand the most was size-exclusion chromatography. This technique compared in all four alleles the amount of cells that: first had an outstanding amount of aggresome formation, second how fast the proteins were being degraded and third if there was any kind of enzymatic activity. This technique was used to test the hypothesis if the two SNPs, found only in TPMT*3A, were the ones altering protein structure. In size-exclusion chromatography the TPMT*3A allele showed the greatest aggresome formation, the least amount of proteins left after a certain time, and no enzymatic activity. Although TPMT*3B was second in rank, meaning that it had some similar results to TPMT*3A, the fact that it has only one SNPs proved that both SNPs are needed in order for the fast aggresome formation and degradation to occur.
The TPMT*3A gives the individual the inability to catalyze certain Thiopurine drugs, and if these drugs are not catalyzed properly can lead to toxicity. The most common allele for Caucasians is TPMT*3A with a 5% frequency, and TPMT*3C with a 2% frequency in East Asia. This amount of frequency is enough to always be aware of these alleles to be present in patients. If a patient having this TPMT*3A is given these Thiopurine drugs it can lead to death. Since these drugs are used in patients with certain cancers, autoimmune diseases, and transplant recipients, which are beginning to be the most affecting issues around the world, there should always be exams testing for these alleles.
Genes in our body have specific proteins which can be studied to determine the gene’s functions. However, if there is a change in the protein sequence, changes in folding, localization, and function of the protein may occur. If any one of these properties is affected, so is the true purpose of the protein. The orientation of a protein helps identify its true function , but when it’s structure is disrupted, this usually leads to misfolding and aggregation We find this problem in the TPMT gene, which has three common variants known as 3A, 3B, and 3C. The TPMT 3A allele has two SNP’s and is highly ubiquitinated and targeted for degradation. This specific allele is not as good in “co-localization”, so it is the worst allele, but also the most common. It rapidly degrades and forms aggresomes, which is why the level of this allele is so low in human tissues. Both TPMT 3B and 3C have one SNP. Allele 3B has virtually no activity, but like 3A, it might affect the catalytic ability of the protein. This helps us to prove that the number of SNP directly affects the way enzymes may be destructed, altering the structure, and further leading to misfolding, aggregation, and toxicity.
Aggresomes are structures that aid in the removal of misfolded proteins with the help of other molecular chaperones. Proteins that don’t obtain the correct structure through folding are sent by our bodies to be degraded, because they serve no true function. Often times, a proteosome is the designated structure that carries misfolded proteins to eventually degrade them. However, if the number of proteins becomes overwhelming, they are sent to the vacuole for a period of storage. While these vesicles hold the proteins, they aid in some degradation due to their acidic environment. Proteins in the vesicle stick together to prevent from over flowing.
In order to determine protein structures, we can use a number of techniques. CD spectroscopy uses wavelength to measure proteins at a specific temperature of 4 degrees Celsius and records protein concentrations. Figure 5 shows us the results of these alleles. We saw that the WT and 3C monomer had a similar spectrum. 3B shows a change in activity, and a shift in its peak which tells us that its enzymatic activity is affected. While it is understood that WT and 3C have unfolded, 3A did not show any evidence of doing so, because there was no sign of enzymatic activity.
Before contemplating any therapy using drugs containing the TPMT gene, it is important to know which of the alleles an individual has. Due to its varying affects, each allele reacts to these thiopurine drugs differently. These drugs can be used to treat leukemia and autoimmune diseases. If an individual who does not carry the appropriate allele which can metabolize this drug, there are life-threatening risks involved. Amongst fast, moderate, and slow metabolizers, it is very dangerous for slow metabolizers to consume this drug because they can easily be intoxicated by a single dose of TPMT. For these individuals a normal treatment can go terribly wrong. TPMT 3A has a frequency of 5% amongst Caucasians, whereas TPMT 3C has a 2% frequency amongst East Asians.
I think it was especially important that this paper provided us with information on the effect of drugs and different individuals, because it shows a wider spectra of factors to look out for in the medical field. Drugs have side effects of their own, but our bodies and what’s inside them determine how these effects take place. The alleles that our bodies carry strongly determine if we can “handle” certain drugs or not. Therefore, this article is important to learn how the allele can be studied through different mechanisms so that the patient receiving treatment is best served. The more we learn about a drug, the better control we have over the treatment.
The general answer as to why the sequence changes affect the protein folding, localization, and function for the alleles in this enzyme is because there would be a variation in the sequence of the DNA which calls for the changes in the protein folding, localization, and function. When there are misfolded proteins present in a cell they should be degraded afterward by a proteosome but sometimes there can be too much accumulation of these useless proteins and build up in aggresomes. The folding is affected by the sequence change between all of these alleles because they all have a different amount of polymorphisms. The two SNPs in *3A cause the misfolding to occur and because of the exons that are present influence the catalytic activity as well. The sequence changes in the alleles affect localization by how the. The overall function of the alleles leads to protein aggregation and aggresome formation because of the two SNPs in TPMT that disrupt the structure of the enzyme. This is because the catalytic activity of these alleles (TPMT*3A or *3B whom both have misfolding from presence of either or both SNPs) was slowed down or stopped. “The decreased level of enzyme activity is from rapid degradation by a ubiquitin/proteasome-dependent process involving chaperone proteins” which leads to the misfolding of the TPMT*3A variant allele for the dynamic balance of proper folding, degredation, and aggregation. These two polymorphisms in the alleles of the TPMT gene causes its own degradation and ultimately its misfolding and aggregation. All of these units (folding, localization, and function) influence each other greatly from the change of the alleles’ sequence.
I believe that reading this paper was very beneficial to us in many ways and most importantly because of its purpose. In the field of Pharmacogenomics, it is important to know each individuals genetic makeup because some people have the presence of certain enzymes that may cause mutations and ultimately death from treatment of certain drugs. It is important to become aware of these happenings, to prevent them, and to discover a safer way to distribute or administer drugs to someone.
Thiopurine Drugs, according to the OMIM website that we were provided with, requires an extensive amount of metabolic activity for cytotoxic action to occur. These drugs are used on people who are diagnosed with leukemia (children), autoimmune diseases, and transplant recipients. Thiopurine S-methyltransferase (TPMT) functions to inactivate these drugs by S-methylation and people have variant levels of this enzymes because of genetic polymorphisms, which mainly involves the genotype of the individual. It is important to know what alleles of the TPMT gene a person harbors before therapy because certain variants alleles lack the TPMT protein which means the enzyme activity is lowered. Thus, a person with this specific variant allele for low activity called TPMT*3A can cause life threatening toxicity when treated with standard doses of thiopurine drugs. Unfortunately, TPMT*3A is most frequent in Caucasians with 5% frequency with two SNPs, TPMT*3C is the most common variant allele in East Asia with 2%frequency with 240 SNP, and the most rare variant allele, TPMT*3B with 154 SNP. Other statistics in OMIM say that in both African Americans and Caucasian Americans, the frequencies of the overall TPMT mutant allele were 4.6% and 3.7% respectively where TPMT*3C was mostly present in African Americans by 52.2%, 4.8% in Caucasians and TPMT*3A is found amongst the Caucasians by 85.7% and 17.4% in African Americans!
The different techniques used to determine the protein structures were COS-1 Cell Expression, IP and Immunoblot Analysis, Immunofluorescence Microsopy, In Vitro Translation and Degradation, Bacterial Recombinant TPMT and CD Spectroscopy. One of the techniques that helped me get a clearer understanding of the enzymes described was the CD Spectroscopy. The structural difference of allozymes showed that *3B and wild types usually adopt the Beta sheet conformational structure but the *3B had a lower signal and a shift showing a change in secondary structure. There was a thermal denaturing of the bacterial synthesized allozymes show that the structural change is due to the lack of enzymatic activity present in *3B while *3A shows a significant shift. When the CD thermal unfolding was done the wild type and *3C were able to unfold but the *3A didn’t. The *3B allele showed very little change.
One of the greatest difficulties I faced with this paper was the abundance of unknown or new vocabulary that was presented to me. There is nothing I don’t like about this article because they are just presenting a problem that has been unrecognized for who knows how long so it is good that they are introducing it to the world. Just entering the world of toxicology, I am just getting used to the use of biological terms which I have a weakness for. However, reading these articles has reminded me how much of a lazy person I am because I found that I had no patience to research a word I did not know and research the definition of that word as well. I would end up with over five tabs of words from definitions of definitions of definitions and so on from one word. No wonder it is so difficult and time consuming just to find a resolution for a problem in this field. I will most definitely expect one entity to branch off to a myriad of other entities that get affected. Nomenclature is obviously very important in this field because of the complexity of the human body and what makes this matter more difficult is that every body is unique.
I am glad that we got the chance to read a research paper at this level because we have to realize the importance of everyone’s unique genetic sequences and also familiarize ourselves with the language used in the field to improve the faults in it. Enhancement in the field will occur by “avoiding adverse drug reactions, maximizing drug efficacy, and by selecting patients that respond to certain therapeutic classes of agents” just as this and the pervious article about personalized medicines have mentioned.
This paper demonstrates how therapeutic responses can cause polymorphisms within our body currently affect the TMPT protein function. A patient with this polymorphism is in danger of high toxicity levels but can be treated with mild standard doses of thiopurines. The importance of knowing which alleles of the TMPT gene are affected is because this gene is relatively rare in human tissue. The frequency of the alleles depends on how rapidly they can be degraded into the tissue. C is the most present, A being the smallest. If it is possible to understand the similarities and differences among different allele structure of proteins, it may also lead to the discovery and comprehension of many other diseases and pharmacological issues.
The shape of a protein is was designates the protein function. When it is misfolded, there can be an issue in expressing specific alleles resulting in a mutation. This mutation will cause a change in the primary genome sequence, altering the protein form and function all together. Enzymatic activity and markers can be used to monitor these changes.
This paper is interesting to me because I have found it intriguing that one slight change in one sequence can alter a protein and its function immediately. Mutations occurs spontaneously, and anything can cause this cause and effect chain reaction to start off.