The Mutant Pabp 2 Protein Need example essay topic

Protein Binding Studies for Expanded Poly-A Repeats and Mutant PABP 2 resulting from Oculopharyngeal Muscular Dystrophy INTRODUCTION: Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular genetic disorder. It has an autosomal dominant pattern of inheritance (Fried et al. 1975) in that the abnormal gene can be transmitted from only one parent. A child of an affected parent has a 50% chance of being affected.

The disorder is found to be more prevalent among French-Canadians and is characterized by its late onset (approximately 50). Affected persons experience dropping eyelids (o ptosis), difficulty with swallowing (dysphagia), and some develop shoulder, hip or leg weaknesses (MDA publications 1998). Genetically, its mutation is quite unique. OPMD is caused by the expansion of a GCG (which codes for the amino acid alanine) 6 repeat (Brais et al.

1998), whereas most triplet repeat disorders are expansions of CAG (glutamine) repeats. Rare polymorphisms would be to have 7 consecutive GCG's, but the disease is mostly characterized by the mutation of having 8-15 consecutive GCG's. Other findings have shown that even the expansion of a 6 GCG repeat to 7 can also lead to OPMD (LaFontaine 1996). The severity of the disease depends on the number of extra alanines.

Quite recently, scientists have found that the mutation occurs on chromosome 14 and is in the gene coding for a poly (A) -binding protein 2 gene (PABP 2) (Brais et al. 1998). PABP 2 was considered a good candidate for OPMD because it maps to the same location as the diseased gene, its mRNA is highly expressed in skeletal muscle, and the PAB 2 protein is exclusively localized in the nucleus, where it acts as a factor in mRNA polyadenylation. The site of the additional GCG expansions in the PABP 2 gene is at the poly alanine tract at the N terminus. From these findings, one may ask why this disease targets preferential l the skeletal muscle cells of the eyes and throat when the protein of the wild- type form of the mutant gene (PABP 2) is expressed in all cells. In order to answer this probing question, binding studies involving the abnormal poly A stretches and the mutant PABP 2 protein need to be performed.

This will determine what other proteins (if any) are involved in this particular type of muscular dystrophy. In this project, I hypothesize that there are proteins from affected tissues which bind to the expanded poly A stretches as well as the mutant PABP 2 protein. These proteins may also bind to them in varying amounts depending on the length of the expanded GCG repeats... To find out if any proteins bind with extended repeats of the corresponding mutant PABP 2 protein, affinity chromatography experiments can be done. This type of experiment will involve polystyrene beads, coated with synthetically made poly-A peptide representing the mutant protein domain, and are packed with various homogenates human muscle tissues. Human tissues will be used because the disease only seem to affect humans.

The synthetic peptides will be of varying repeats of alanine, thus testing for the different severities of the disease. Other molecular studies investigating OPMD examined up to 13 repeated Gcg. Therefore, for the polystyrene beads experiment, repeats of 6 to 14 will be used. A test with 14 repeats will determine whether there is still a continual increase in severity after 13 repeats or whether the effects will be abolished past the 13 repeat mark. An experiment to elucidate potential proteins that bind to the mutant PABP 2 protein involved in OPMD is the yeast-two-hybrid system.

The method uses the transcription of yeast reporter genes as a synthetic phenotype to detect protein-protein interactions. The approach takes advantage of the modular domain structure of eukaryotic transcription factors. Many transcription activators have at least two distinct functional domains, one that directs binding to specific DNA sequences and one that activates transcription. This modular structure is best illustrated by yeast experiments showing that the DNA-binding domains or activation domains can be exchanged from one transcription factor to the next and retain function. A crucial consequence of the modular nature of transcription activators is that the DNA-binding and activation domains do not need to be covalently attached to each other for activation to occur. Because of this, yeast transcription could be used to assay the interaction between two proteins if one of them is fused to a DNA- binding domain and the other is fused to an activation domain.

The system contains three components: Yeast vectors for expression of a known protein fused to a DNA-binding domain, yeast vectors that direct expression of cDNA-encoded proteins fused to a transcription activation domain, and yeast reporter genes that contain binding sites for the DNA-binding domain. For our purposes, we can use this system to screen a cDNA library for clones expressing proteins that interact with the different forms of PABP 2 (wt and varying mut forms). METHOD: Previous studies on other triplet repeat disorders, such as Huntington's disease and fragile-X syndrome, have used a procedure called Repeat Expansion Detection (RED) method to detect and isolate the repeats (Rifugo, G. 1997). Although these studies examined glutamine repeats, the RED method can also be used for alanine repeats. The resulting isolated alanine repeats can then be used for our binding experiments. The affinity chromatography experiments using polystyrene beads separate proteins based on affinity for a specific ligand, in our case, the alanine repeat peptides.

As illustrated in Figure 1, the polystyrene beads in the column are first coated with the alanine peptide of choice. Experiments will first be performed with synthetic 6-alanine peptides as controls because this is the number of alanine repeats that correspond to the poly-A tract at the N terminus of normal PABP 2 proteins. Then, various homogenized muscle tissues are packed into the column. Since OPMD is known to affect mostly skeletal muscles of the eye, throat, hip, leg and shoulder, binding tests involving the alanine repeat peptides will be done on such muscles. As well, tests using smooth muscles are used as controls because it is known that the disease does not affect this type of muscle. After the tissues pass through the column, only the proteins with high affinity for the alanine peptides will bind.

All the nonbinding proteins will flow through the column. The bound protein will be dislodged from the alanine peptide coated beads and eluted with a concentration solution containing the alanine peptide. The experiments will be repeated using varying number of repeated-alanine peptides, from 7 to 14. These tests will represent the mutated forms for the disease. The expected results are shown on Tables 1 & 2. To investigate if there are proteins that bind to the OPMD induced mutated forms of PABP 2 proteins, the yeast-two-hybrid system is used.

As shown in Figure 2, the bait' plasmid will contain a DNA sequence encoding the Gal 4 DNA-binding domain fused to the coding sequence for the type of PABP 2 protein being examined. Again, different experiments will be performed using genes encoding for PABP 2 proteins with 6 (control) to 14 alanine repeats. The Gal 4 binding domain is used because it is efficiently localized to the yeast nucleus where it binds with high affinity to the well-defined upstream activating sequence (UAS) which is placed upstream of the reporter gene (lacZ). The prey' plasmids include individual cDNAs from a library fused to the coding sequence for Gal 4 activation domain.

The fused genes in both the prey' and bait' plasmids can be achieved by recombinant DNA technology. Each type of plasmid also contains a wild-type selection gene (TRP 1 or LEU 2) so as to provide a selection for cells that have taken up both plasmids. Firstly, both bait' and prey' plasmids are transfected into yeast cells with mutations in genes required for tryptophan and leucine biosynthesis and then grown in the absence of tryptophan and leucine. The cells also contain the reporter gene construct. Only cells that contain the bait plasmid and at least one prey' plasmid survive under these selection conditions.

If there is protein binding, it will bring the Gal 4 activation domain within reach of the start of the lacZ reporter gene and will activate transcription. If there is no protein binding, no activation will occur. A screen for the activation of the lacZ reporters is performed by plating yeast on indicator plates that contain X-Gal. On this medium yeast in which the reporters are transcribed produce beta-galactosidase and turn blue.

DISCUSSION: The results for the two types of protein binding experiments can address the hypothesis of the existence of proteins binding to the extended poly-A repeats and to the mutant PABP 2 proteins. In terms of the affinity chromatography experiments, there may be two possible outcomes. One set of experiments could demonstrate that there is in fact proteins that bind to the expanded poly-A peptides to cause clinical manifestations of OPMD. These results will therefore confirm the hypothesis. The results of this potential outcome are illustrated in Table 1. Under the assumption of protein binding leading to OPMD, we can expect no protein binding to all forms of the alanine peptide when testing with smooth muscles.

This is because the disease is known to only affect skeletal muscles. For eye and throat skeletal muscles, we can expect no protein binding for normal poly-A repeats but find protein binding for repeats of 7-14. The amount of bound proteins also increase from 7 to 14 alanine repeats, thus accounting for the disease's increase in severity with additional repeats. Because patients with OPMD show more effects on their eyes and throat as compared to their legs, hip and shoulders, protein binding for the latter three muscle tissues will be less than that of the former two. We would still expect an increase in protein binding for leg, hip, and shoulder muscles with increasing alanine repeats.

However, this increase is slower than that of eye / throat muscles. Table 1 also shows that for tests with normal repeats (6), there would not be binding if indeed there is protein binding for expanded repeats, no matter which muscle type. If these results occur, there will be a need for further characterizations of the newly found mutant PABP 2-binding proteins. Table 1 shows a situation where a specific protein is binding to extended Ala repeats and more of it binds with increasing repeat size. There may however, be different proteins that bind to the different repeats. This possibility will nonetheless still affirm the hypothesis.

The protein (s) that bind to the extended poly A stretches will be involved in the progressive destruction of muscle tissue over time (a characteristic of muscular dys trophies). They will also lead to weakness and loss of muscular function. Because of this senescence phenotype, it can be predicted that the extended poly-A-binding protein (s) may affect (s) proper functioning of telomerase's. The protein may also alter the proper functioning of PABP 2 and initiate a cascade which results in degradation of muscle tissues. The other possible outcome would be that the decrease in protein binding leads to OPMD (Table 2).

The two controls (tests with smooth muscles and tests with 6 Ala repeats) will show significant protein binding. With the onset of the disease, under this scenario, there will be less binding of the particular protein. For experiments with eye or throat muscles, binding is decreased as the number of extended repeats increase. That same pertains to leg, hip and shoulder muscle tissues, but the decrease will be to a lesser extent.

If these results are to occur, identification of the binding protein is needed via sequencing methods (ie. Edman degradation). A prediction is that the protein binds to the poly-A stretches for normal functioning of PABP 2, perhaps assisting it to act as a factor in mRNA polyadenylation. In affected tissues, some of these binding proteins may not be able to recognize the extended repeats thus decreasing PABP 2's function.

Under these results, the hypothesis would be rejected. For the yeast-two-hybrid experiments, blue colonies will signify the binding of proteins to the different forms of PABP 2. If blue colonies appear for tests with the gene encoding for mutant PABP 2's, it will indicate an acceptance of the hypothesis. These positive clones will need to be analyzed further by restriction analysis, PCR, or by sequencing. These analytical methods will show if the same protein binds to all forms of the mutant or if different proteins bind to the varying mutant forms. The proteins that bind to PABP 2 may bind specifically to the poly-A stretches or to other sites of PABP 2.

This system, however, cannot distinguish these two possibilities. On the other hand, if no colonies appear blue for tests with mutant PABP 2's, there are no proteins that bind to them. This result will help reject the hypothesis. It may also show that the lack of binding to a particular protein specific for wild-type PABP 2 results in the disease. In this case, blue colonies will need to appear when the gene that is fused to the gene encoding for Gal 4 BD is the wild-type form of PABP 2. CONCLUSION: These binding experiments will greatly enhance the understanding of OPMD.

They will find potential proteins which act downstream of the mutated PABP 2, causing the continual disintegration of muscle tissue over time. Indeed, as mentioned before, sequencing, PCR, or restriction analysis of the elucidated mutant PABP 2-binding proteins will need to be performed in order to characterize them. Also, point mutation studies of the genes encoding these proteins can help find regions that are essential for its function. Experiments involving mRNA expression can be done to see if these proteins are expressed in various muscle cells of normal vs. diseased individuals. This can also be achieved by in situ hybridization using raised antibodies for the protein.

Of course, even more binding experiments can be performed on these extended poly-A and mutant PABP 2-binding proteins using methods already described. In addition, a future experiment can be to screen for compounds that can directly affect the gene defects by, for example, destabilizing the DNA structure that forms with the extra repeat. Somewhere down the road, for genetic disorders of muscle, lies the prospect of gene therapy... Today, there has not yet been a single published report of a patient who was helped by gene therapy. These different experiments may someday propel the prospect of gene therapy for not only OPMD but for other genetic disorders of muscles. However, it will be a difficult task due to the problem of intractability of muscle as a tissue (ie. difficult to reach each individual fibre or even enough of them to make a difference).

Main problems of all muscular dystrophy disorders about how to get the correct version of a gene, with all its controlling elements, into muscle tissue remain to be solved. TABLE 1: Expected results for the possible outcome of protein binding leading to OPMD using affinity chromatography experiments. The +'s represent protein binding activity and -'s represent lack of protein binding. The number of +'s illustrate the amount of bound proteins.

6 7 8 9 10 11 12 13 14 Smoot Eye - ++ + ++ Throat - ++ + ++ Leg - + ++ ++ ++ Hip - + ++ ++ ++ Shoulder - + ++ ++ ++ TABLE 2: Expected results for the possible outcome of the lack of protein binding leading to OPMD using affinity chromatography experiments. The +'s represent protein binding and the -'s represent lack of protein binding.

Bibliography

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