This webpage was produced as an assignment for Genetics 677, an undergraduate course at the University of Wisconsin - Madison.
Conclusions and Future Directions
The purpose of my research was to use bioinformatics programs to analyze the CFTR gene and its protein to understand the molecular underpinnings of the genetic disease Cystic Fibrosis.
Nearly all homology and phylogeny analyses for both the mRNA and amino acid sequence of CFTR gave the same results. Pan troglodytes has a nearly identical sequence and is evolutionarily the closest to Homo sapiens, thereforethese results were expected.
One finding that was somewhat unexpected was that the homologs of both Saccharomyces cerevisiae and C. elegans had more than 50 extra amino acids at the N-termini and shortened C-termini. No other homologs displayed such great modifications. Although, the fact that these homologs even existed in such distant relatives of Homo sapiens shows a great deal of evolutionary conservation of the CFTR gene.
I also used gene ontology (GO) to gain a better understanding of purpose and localization of the CFTR protein in well-differentiated cells. The GO results were expected, given its classification as an ABC transporter. Most of the GO terms confirmed CFTR's involvement in transmembrane ion transport, lung development, and ATP binding; and CFTR’s localization to the apical plasma membrane and endosome.
The research into protein domains supported CFTR’s GO terms. CFTR has an ATP-binding cassette and a six-helix transmembrane domain and when correctly folded, is transported to the plasma membrane as a chloride channel. Many mutations within the CFTR gene can lead to a misfolded protein. Thousands have been documented, but the most common mutation (fΔ508) and the majority of others are found in the most highly conserved domain, the ATP-binding cassette. A defect here leads to the most severe phenotypes of CF, even something as small as a single amino acid deletion (such as fΔ508).
Because misfolded CFTR proteins do not allow for the passage of chloride, I wanted to further explore the process for correct folding of CFTR. I found that the CFTR protein has a regulatory domain situated between the two ABC transporter domains. The regulatory domain must be phosphorylated by PKA or PKC (which are stimulated by cAMP) in order to promote correct folding of the entire protein. After researching the regulatory domain of CFTR specifically, I found that it has over 20 serine residues which are phosphorylated primarily by PKA and enhanced by PKC. For my hypothesis to resolve CF symptoms, I postulated that the upregulation of PKA and PKC in CFTR -/- organisms would increase the functionality of mutant CFTR proteins, thereby reducing the severity of the phenotype.
The main conclusions that can be drawn from this project are that CFTR's role in CF is through loss of its function as a chloride ion transporter, caused by the misfolding of the protein. While researching, I noticed this became the principal focus due to its role as the primary cause of the CF phenotype and its predominance in the literature. CFTR, when mutated, leads to a well-defined and well-known set of symptoms unique from any other disease. CFTR is a highly researched protein and therefore has become well-understood. Although there has yet to be a cure discovered for this debilitating and often fatal genetic disease.
I believe that even though CFTR is well-studied, that several areas of research should be focused on to further examine the role of CFTR and several promising drug targets. The exact mechanisms of CFTR folding, transport, and destruction are unknown. Through knowledge of these detailed mechanisms, I believe several drug targets could expose themselves, leading to a substantial increase in the standard of living for severely affected CF patients.
First, it is not well-understood how the folding of CFTR occurs or exactly how phosphorylation affects CFTR. The correct folding of CFTR is necessary for the protein to function as a transporter. Misfolded CFTR does not allow chloride to pass through, leading to an imbalance of water in mucus. It is known that the regulatory domain of CFTR is necessary for correct folding, via phosphorylation of its serine residues by PKA and PKC. But it has not been found how exactly PKA and PKC work together (or separately?) to phosphorylate the correct amount and exact serine residues. I propose that further studies examine the roles of PKA and PKC in CFTR phosphorylation to determine if 1) phosphorylation by PKA and PKC reaches mutant CFTR protein, 2) PKA and PKC could be supplemented to increase phosphorylation of CFTR, and 3) if this increase in PKA and PKC leads to increased phosphorylation of mutant, not just wild type CFTR. If this is possible, it would result in increased functionality of mutant CFTR proteins in CF patients, leading to a decrease in the thickness and stickiness of their mucus.
Secondly, the transport of CFTR beginning from nascent polypeptide and ending in apical membrane localization is not well known. The transport of CFTR is critical if CFTR is to be of use. A perfect CFTR protein is unable to perform its job of ion transport across membranes if it never becomes localized to the membrane. I suggest that more research be done to clarify every step of CFTR transport. Perhaps insight into how CFTR is transported could eventually lead to the ability to effectively increase or direct transport of CFTR to appropriate locations within the body. Maybe some mutations in CF lead to decreased recognition by transporter proteins, leading to failure of appropriate transport. Or possibly, more effective transport of CFTR could lead to a decreased phenotype, because the greater amount of CFTR would offset the imbalanced chloride ion gradient.
Lastly, it is known that a defective CFTR protein, if recognized, will be ubiquinated and subsequently destroyed by the proteasome. But it is unknown by which mechanisms the mutant CFTR protein is recognized and where it is recognized. I recommend that more research be focused on the degradation of CFTR protein within cells. It should be determined when is CFTR degraded, during translation or transport or within the membrane. It should also be determined if mutant CFTR has the same lifetime as a wild type CFTR protein. Current research is focused on “rescue proteins” (such as vasoactive intestinal peptide) which save functional CFTR proteins. Maybe some CF patients do not have a mutation within the CFTR gene, but within the gene that codes for the degradation protein targeted for CFTR. Maybe a drug can be found which acts as an antagonist to this degradation protein, turning it off an allowing the CFTR to be transported successfully. Perhaps researchers could create a drug that resembles some sort of heat shock protein or other molecular chaperone to protect it from degradation as it is transported. The preservation of CFTR is essential, increased degradation would lead to a more severe phenotype and increased rates of fatality; therefore it follows that increased preservation would lead to decreased symptoms.
I believe that these areas of research would lead to the discovery of many drug targets that would decrease the severity of the CF phenotype and lead to a better quality of life for many CF patients. Although, these treatments would only remedy the symptoms of CF, not completely cure it. I believe that in order to cure CF, gene therapy must be utilized to correct the genetic defect that causes CF. Much has been anticipated in terms of gene therapy as a cure for CF. Although the technology is still in its infancy, it has been used with some success. However, it has still not been proven to be a cure.
All these avenues of research have yet to be fully investigated, and there are many more areas and types of research that have yet to be discovered as relevant to CF and CFTR. Those afflicted with CF should remain hopeful that someday soon, a better drug or even a cure will be discovered to treat this devastating disease.
Nearly all homology and phylogeny analyses for both the mRNA and amino acid sequence of CFTR gave the same results. Pan troglodytes has a nearly identical sequence and is evolutionarily the closest to Homo sapiens, thereforethese results were expected.
One finding that was somewhat unexpected was that the homologs of both Saccharomyces cerevisiae and C. elegans had more than 50 extra amino acids at the N-termini and shortened C-termini. No other homologs displayed such great modifications. Although, the fact that these homologs even existed in such distant relatives of Homo sapiens shows a great deal of evolutionary conservation of the CFTR gene.
I also used gene ontology (GO) to gain a better understanding of purpose and localization of the CFTR protein in well-differentiated cells. The GO results were expected, given its classification as an ABC transporter. Most of the GO terms confirmed CFTR's involvement in transmembrane ion transport, lung development, and ATP binding; and CFTR’s localization to the apical plasma membrane and endosome.
The research into protein domains supported CFTR’s GO terms. CFTR has an ATP-binding cassette and a six-helix transmembrane domain and when correctly folded, is transported to the plasma membrane as a chloride channel. Many mutations within the CFTR gene can lead to a misfolded protein. Thousands have been documented, but the most common mutation (fΔ508) and the majority of others are found in the most highly conserved domain, the ATP-binding cassette. A defect here leads to the most severe phenotypes of CF, even something as small as a single amino acid deletion (such as fΔ508).
Because misfolded CFTR proteins do not allow for the passage of chloride, I wanted to further explore the process for correct folding of CFTR. I found that the CFTR protein has a regulatory domain situated between the two ABC transporter domains. The regulatory domain must be phosphorylated by PKA or PKC (which are stimulated by cAMP) in order to promote correct folding of the entire protein. After researching the regulatory domain of CFTR specifically, I found that it has over 20 serine residues which are phosphorylated primarily by PKA and enhanced by PKC. For my hypothesis to resolve CF symptoms, I postulated that the upregulation of PKA and PKC in CFTR -/- organisms would increase the functionality of mutant CFTR proteins, thereby reducing the severity of the phenotype.
The main conclusions that can be drawn from this project are that CFTR's role in CF is through loss of its function as a chloride ion transporter, caused by the misfolding of the protein. While researching, I noticed this became the principal focus due to its role as the primary cause of the CF phenotype and its predominance in the literature. CFTR, when mutated, leads to a well-defined and well-known set of symptoms unique from any other disease. CFTR is a highly researched protein and therefore has become well-understood. Although there has yet to be a cure discovered for this debilitating and often fatal genetic disease.
I believe that even though CFTR is well-studied, that several areas of research should be focused on to further examine the role of CFTR and several promising drug targets. The exact mechanisms of CFTR folding, transport, and destruction are unknown. Through knowledge of these detailed mechanisms, I believe several drug targets could expose themselves, leading to a substantial increase in the standard of living for severely affected CF patients.
First, it is not well-understood how the folding of CFTR occurs or exactly how phosphorylation affects CFTR. The correct folding of CFTR is necessary for the protein to function as a transporter. Misfolded CFTR does not allow chloride to pass through, leading to an imbalance of water in mucus. It is known that the regulatory domain of CFTR is necessary for correct folding, via phosphorylation of its serine residues by PKA and PKC. But it has not been found how exactly PKA and PKC work together (or separately?) to phosphorylate the correct amount and exact serine residues. I propose that further studies examine the roles of PKA and PKC in CFTR phosphorylation to determine if 1) phosphorylation by PKA and PKC reaches mutant CFTR protein, 2) PKA and PKC could be supplemented to increase phosphorylation of CFTR, and 3) if this increase in PKA and PKC leads to increased phosphorylation of mutant, not just wild type CFTR. If this is possible, it would result in increased functionality of mutant CFTR proteins in CF patients, leading to a decrease in the thickness and stickiness of their mucus.
Secondly, the transport of CFTR beginning from nascent polypeptide and ending in apical membrane localization is not well known. The transport of CFTR is critical if CFTR is to be of use. A perfect CFTR protein is unable to perform its job of ion transport across membranes if it never becomes localized to the membrane. I suggest that more research be done to clarify every step of CFTR transport. Perhaps insight into how CFTR is transported could eventually lead to the ability to effectively increase or direct transport of CFTR to appropriate locations within the body. Maybe some mutations in CF lead to decreased recognition by transporter proteins, leading to failure of appropriate transport. Or possibly, more effective transport of CFTR could lead to a decreased phenotype, because the greater amount of CFTR would offset the imbalanced chloride ion gradient.
Lastly, it is known that a defective CFTR protein, if recognized, will be ubiquinated and subsequently destroyed by the proteasome. But it is unknown by which mechanisms the mutant CFTR protein is recognized and where it is recognized. I recommend that more research be focused on the degradation of CFTR protein within cells. It should be determined when is CFTR degraded, during translation or transport or within the membrane. It should also be determined if mutant CFTR has the same lifetime as a wild type CFTR protein. Current research is focused on “rescue proteins” (such as vasoactive intestinal peptide) which save functional CFTR proteins. Maybe some CF patients do not have a mutation within the CFTR gene, but within the gene that codes for the degradation protein targeted for CFTR. Maybe a drug can be found which acts as an antagonist to this degradation protein, turning it off an allowing the CFTR to be transported successfully. Perhaps researchers could create a drug that resembles some sort of heat shock protein or other molecular chaperone to protect it from degradation as it is transported. The preservation of CFTR is essential, increased degradation would lead to a more severe phenotype and increased rates of fatality; therefore it follows that increased preservation would lead to decreased symptoms.
I believe that these areas of research would lead to the discovery of many drug targets that would decrease the severity of the CF phenotype and lead to a better quality of life for many CF patients. Although, these treatments would only remedy the symptoms of CF, not completely cure it. I believe that in order to cure CF, gene therapy must be utilized to correct the genetic defect that causes CF. Much has been anticipated in terms of gene therapy as a cure for CF. Although the technology is still in its infancy, it has been used with some success. However, it has still not been proven to be a cure.
All these avenues of research have yet to be fully investigated, and there are many more areas and types of research that have yet to be discovered as relevant to CF and CFTR. Those afflicted with CF should remain hopeful that someday soon, a better drug or even a cure will be discovered to treat this devastating disease.
_______________
References
1. Rafferty S, Alcolado N, Norez C, Chappe F, Pelzer S, Becq F, Chappe V. Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada. Rescue of functional F508del cystic fibrosis transmembrane conductance regulator by vasoactive intestinal peptide in the human nasal epithelial cell line JME/CF15. J Pharmacol Exp Ther. 2009 Oct;331(1):2-13. Epub 2009 Jul 7.
2. Seavilleklein G, Amer N, Evagelidis A, Chappe F, Irvine T, Hanrahan JW, Chappe V. Dept. of Physiology and Biophysics, 5850 College St., Halifax, NS, Canada B3H 1X5. PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR. Am J Physiol Cell Physiol. 2008 Nov;295(5):C1366-75. Epub 2008 Sep 17.
3. OLAFUR BALDURSSON, HERBERT A. BERGER, AND MICHAEL J. WELSH. Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia. Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41.
4. David Dahan, Alexandra Evagelidis, John W. Hanrahan, Deborah A. R. Hinkson, Yanlin Jia, Jiexin Luo, Tang Zhu. Department of Physiology, McGill University, Montréal, Québec, Canada. Regulation of the CFTR channel by phosphorylation. Pflügers Arch - Eur J Physiol (2001) 443 [Suppl1]: S92–S96 .
References
1. Rafferty S, Alcolado N, Norez C, Chappe F, Pelzer S, Becq F, Chappe V. Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada. Rescue of functional F508del cystic fibrosis transmembrane conductance regulator by vasoactive intestinal peptide in the human nasal epithelial cell line JME/CF15. J Pharmacol Exp Ther. 2009 Oct;331(1):2-13. Epub 2009 Jul 7.
2. Seavilleklein G, Amer N, Evagelidis A, Chappe F, Irvine T, Hanrahan JW, Chappe V. Dept. of Physiology and Biophysics, 5850 College St., Halifax, NS, Canada B3H 1X5. PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR. Am J Physiol Cell Physiol. 2008 Nov;295(5):C1366-75. Epub 2008 Sep 17.
3. OLAFUR BALDURSSON, HERBERT A. BERGER, AND MICHAEL J. WELSH. Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia. Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41.
4. David Dahan, Alexandra Evagelidis, John W. Hanrahan, Deborah A. R. Hinkson, Yanlin Jia, Jiexin Luo, Tang Zhu. Department of Physiology, McGill University, Montréal, Québec, Canada. Regulation of the CFTR channel by phosphorylation. Pflügers Arch - Eur J Physiol (2001) 443 [Suppl1]: S92–S96 .
Alexandra Reynolds
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