Cystic fibrosis is a very serious genetic disease that affects one in 3600 Canadian children (Canadian Cystic Fibrosis Foundation, 2010). In cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR) protein does not function to its full capacity and the individual suffers form many serious side effects such as weight loss, pulmonary disease, and depression (Goss et al., 2009). Recently, with developments in genetics, cellular biology, and pharmaceuticals, CF is better understood and effective treatments are being developed (Rooman, 2007). This essay will explore the regular function of the protein, what it does for a cell and the body, and how it is normally made. The basic literature knowledge of the genetic basis, misfolding and the direct and indirect effects of these errors will be described. Lastly, possible therapies with cell biology basis will be explored.
CFTR protein is a cAMP-activated chloride ion channel protein composed of approximately 1440 amino acids, found in epithelial cells in the lungs, digestive tract and other such surfaces (Turnbull et al., 2007). It is situated in the apical membrane of these cells and is very important to processes such as chlorine ion transport, water balance, and transport of other ions (such as sodium) (Rubin, 2007). It weighs about 150 kDa and is composed of 5 protein domains. Two of these domains extend across the plasma membrane (Ko and Pedersen, 2001). There are also two nucleotide-binding domains that are believed to play a role in binding of ATP, through a conformational change (Ko and Pedersen, 2001). ATP is necessary for the opening and closing of the channel (Ko and Pedersen, 2001). The 5th domain is a regulatory domain and is phosphorylated in the cAMP process (Ko and Pedersen, 2001). The role of the protein is to transport chloride ions. As chloride ions are transported into or out of a cell, the water molecules will also follow.
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The protein is made in the rough endoplasmic reticulum (Turnbull et al., 2007). The mRNA containing coding information for the CFTR gene is brought to the RER, and synthesis continues so that the newly forming peptide is within the ER (Turnbull et al., 2007). As soon as the entire peptide is synthesized, the chaperone proteins, Hsp70, Hsc70 and Hsp90, assist in folding (Rooman, 2007). Even in unaffected individuals, the rate of incorrect folding is quite high, about 30%, (Kopito, 1999) and many incorrectly folded proteins are degraded (Ko and Pedersen, 2001). The folded protein interacts with calnexin, which determines if the protein is folded correctly (Norez et al. 2006). The approved proteins are transported out of the RER in a COP II vesicle (Ko and Pedersen, 2001). The proteins are taken to the Golgi apparatus where the immature protein is modified through glycolsylation, into its mature form and taken to the apical plasma membrane of the cell, where it serves its function (Turnbull et al., 2007; Ko and Pederson, 2001).
Mutations that will possibly lead to CF are separated into 5 categories (Turnbull, et al., 2007). The first class contains the mutations that originate from errors in the mRNA, such as frame shift or nonsense mutations (Turnbull, et al., 2007). The second of class mutations are the CFTR proteins that are degraded after synthesis because of their inability to fold properly (Turnbull, et al., 2007). Class 3, consists of the proteins that although are folded incorrectly, have been inserted into the apical membrane but cannot respond to ATP, and so do not function (Turnbull, et al., 2007). Class 4 is similar to class 3, except the protein does respond to ATP (Turnbull, et al., 2007). Normal working proteins made at a lower rate are put into class 5 (Turnbull, et al., 2007). The most common mutation in CF patients is delF508 CFTR, which includes a deletion of the amino acid phenylalanine in position 508 of the peptide (Ko and Pedersen, 2001). This causes recognizable misfolding, but functionally only decreases the time the protein channel stays open (Rooman, 2007). When calnexin recognizes an incorrectly folded protein it is marked for degradation by ubiquitin (Norez et al., 2006). The lack of chloride transport proteins affects water balance and leads to the secretion of thick mucus (Rubin, 2007). This mucus is very difficult to clear out of the airways and digestive tract and leads to deadly infections (Rubin, 2007). The CFTR protein is also linked to other ion transport systems, and in CF many of them become unbalanced (Donaldson and Boucher, 2007).
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One possible treatment that is being explored for CF is 4-Phenylbutyrate (Rooman, 2007). An increase in cellular chaperones, such as HSP70, was observed. This enzyme prevents misfolded proteins from being destroyed by ubiquitin and chaperones them to the plasma membrane (Singh et al., 2007). Although the CFTR protein may not function at a high rate, even this small change can make a difference (Rooman, 2007). This drug can help CF patients by increasing the amount of semi-functional CFTR proteins on the membrane.
A research group showed that another drug, miglustat, is effective in transforming cells that contain the delF508 mutation, into cells that behave as if they did not have the deletion (Norez et al., 2009). Miglustat is a glycosidase inhibitor (Norez et al., 2006). Glycosylation allows for the quality control mechanism, calnexin, to check for proper folding (Norez et al., 2006). When the calnexin interaction is inhibited by miglustat, the misfolded CFTR protein is not rejected, but will be incorporated into the apical membrane (Norez, et al., 2006). Basically, the quality control mechanism is limited in the selectivity for functional proteins, and many proteins with the delF508 mutation are allowed to reach the apical membrane and complete some of their function.
Knowledge about the systems that contribute to CF is constantly increasing (Rooman, 2007). New mechanisms of the function, production, and activation of transport proteins give researchers expanding understanding of chloride transport with CFTR and this leads to the development of new treatment for patients.