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Mechanism of disease

PKA (protein kinase A) or PKC (protein kinase C). It interacts with different regions of the protein simultaneously and enhances ATP binding to the NBDs, which in turn will lead to opening of the channel. Subsequent hydrolysis of ATP then returns the channel to the basal, closed conductance state. ATP-dependent conformational changes of the NBDs are transmitted to the MSDs through interdomain interactions of NBD1 and NBD2 with interfacing CL4/CL1 and CL2/ CL3, respectively.

This complex protein structure is proposed to be responsible for the limited folding efficiency of the wild-type (WT) CFTR. It has been found in nonpolarised cells (heterologous systems) that only 20–40% of the nascent chain achieves its folded conformation, while the remaining 60–80% of molecules are targeted for endoplasmic reticulum-associated degradation (ERAD), especially via the ubiquitin-proteasome proteolytic pathway. CFTR folding and maturation is a complex and hierarchical process, which takes place in multiple cellular compartments, and involves several folding machineries. Experimental data suggest cooperative domain folding/ assembly model, where all the domains, like elements of a puzzle, are required to attain the fully assembled mature protein. CFTR folds during its synthesis in a modular manner, domain by domain, with insertion of transmembrane segments into the ER membrane.3 Post-translational folding allows interdomain interactions needed to form a physiologically stable and functional structure. The compactly folded, native tertiary structure with NBDs–MSDs interfaces is assisted by prolonged interaction with molecular chaperones. Formation of complexes with chaperones protects the channel from aggregation and facilitates folding, as well as the degradation of non-native conformers. Misfolded CFTR activates the unfolded protein response (UPR) or is recognised by protein quality control system, ubiquitinated and directed to the ERAD. Folded and core-glycosylated CFTR is transported out of the ER to the Golgi apparatus via common cytosolic budding machinery, and is then modified in the trans-Golgi network to produce a complex glycosylated protein by N-glycosylation of two sites located in MSD2. The trafficking of native CFTR from the Golgi to the plasma membrane (PM) is modulated by dynamic interactions of its C-terminal

peptide (DTRL) with PDZ-domain- containing proteins. Ten percent of the plasma membrane CFTR is constitutively internalised each minute by endocytosis through clathrin-coated vesicles.4


-14–18 h). Conversely, non-native CFTR that has escaped ERAD is rapidly eliminated from the plasma membrane by lysosomal degradation.

mature CFTR is very stable and recycles by default back to the cell surface with high efficiency that is necessary to maintain the long residence time of CFTR (t½

CFTR protein mutations The CFTR gene is localised on the seventh chromosome (7q31.2) and is composed of 27 exons spreading over 190 kb, which encodes a 6.5 kb mRNA transcript. To date, over 1900 naturally occurring sequence variations have been identified

is generated but is degraded rapidly in the cytoplasm.

2. Class II mutations alter the cellular maturation of the protein. This leads to the synthesis of the protein that cannot be correctly folded and thus transported to its site of action at the apical plasma membrane. The misfolded protein is retained in the ER, retrotranslocated in the cytoplasm to be degraded by the ubiquitin/ proteasome pathway. In this way, the protein is either absent from the plasma membrane or present in a very small quantity (for example, F508del, I507del, N1303K).

3. Class III mutations alter the channel regulation. The mutated protein is properly trafficked to the plasma membrane but cannot be activated or regulated as a chloride channel. These

“CFTR folding and maturation is a complex and hierarchical process, which takes place in multiple cellular compartments and involves several folding machineries”

in the CFTR gene. In order to catalogue this growing number of mutations, the International Consortium for the Genetic Analysis of CF (www.genet.sickkids.on. ca/cftr/) was created by the discoverers of the gene. A new project, called Clinical and Functional Translation of CFTR (CFTR2) (, has also been initiated to determine the relationships between specific mutations and clinical phenotype of CF. The most common and first identified mutation is F508del, which accounts for 70% of CF alleles in Caucasian patients, with a decreasing prevalence from northwestern to southeastern Europe. The overall frequency of non-F508del mutations is low, occurring at a worldwide frequency of less than 0.1%, except for some rare alleles that segregate with a specific ethnic group.5

Mutations in the CFTR

gene can be classified into five classes (Figure 2): 1. Class I mutations interfere with protein synthesis. This class includes the nonsense, frameshift or severe splicing mutation, that result with a premature termination codon (PTC) (for example, G542X, W1282X). As a consequence, the Nonsense-Mediated mRNA Decay (NMD) pathway eliminates the abnormal mRNA containing PTC, or a truncated protein

mutations are missenses frequently situated in the region of the ATP binding domain (NBDs) (for example, G551D, which abolishes ATP- dependent gating).

4. Class IV are mutations affecting the Cl-

channel conductance. Like Class III, these mutations produce a protein properly trafficked and localised to the cell membrane; however, such a mutated channel generates reduced Cl-

flow and has a modified selectivity. A majority of mutations in this class are missenses located within the membrane-spanning domains forming the pore of the channel, such as R117H or R334W.

5. Class V mutations reduce the level of normally functioning CFTR at the apical membrane. These mutations, either partially aberrant splicing mutations or inefficient trafficking missenses, affect the pre-mRNA splicing and alter the stability of mRNA, which produce a low level of normal CFTR (for example, 3272- 26A>G, 3849+10kbC>T).

Interestingly, specific mutations may have the characteristics of more than one class; therefore, a mutation can combine two defects. For example, the F508del, the prototype of a Class II mutation, can also be classified as a Class III mutation.


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