Cystic fibrosis lecture 1 Flashcards
CF is one of the most common lethal genetic disorders affecting Caucasian populations. Currently life expectancy is 32 years of age, but for babies born in the 1990s it is 40 years of age. In the 1950s life expectancy was less than 5 years of age, and life expectancy is therefore increasing, which is the result of improved therapy, but it is still a lethal disease for which there is no cure.
It is most common in the Western World. There is very little CF in India or the Far East, for example. The reason for this is that a mutation in the CF gene arose spontaneously 50,000 years ago in the region of Turkey, and as nomadic tribes moved west, the mutation was carried through Europe, with relatively little movement eastwards. Some racial groups are affected in the middle east, and CF is found in Africa, but it is much ………. common than in ……………… populations. Overall CF affects 80-100,000 people world wide, around 30,000 in the US and around 11,000 in the UK.
1:25 of us are carriers of the genetic defect, however you will not know it because genetic screening for CF in new born babies was not carried out routinely until very recently. Because CF is inherited as a recessive characteristic, then babies with CF are born to parents who are ………… carriers of the genetic defect.
CF is one of the most common lethal genetic disorders affecting Caucasian populations. Currently life expectancy is 32 years of age, but for babies born in the 1990s it is 40 years of age. In the 1950s life expectancy was less than 5 years of age, and life expectancy is therefore increasing, which is the result of improved therapy, but it is still a lethal disease for which there is no cure.
It is most common in the Western World. There is very little CF in India or the Far East, for example. The reason for this is that a mutation in the CF gene arose spontaneously 50,000 years ago in the region of Turkey, and as nomadic tribes moved west, the mutation was carried through Europe, with relatively little movement eastwards. Some racial groups are affected in the middle east, and CF is found in Africa, but it is much less common than in caucasian populations. Overall CF affects 80-100,000 people world wide, around 30,000 in the US and around 11,000 in the UK.
1:25 of us are carriers of the genetic defect, however you will not know it because genetic screening for CF in new born babies was not carried out routinely until very recently. Because CF is inherited as a recessive characteristic, then babies with CF are born to parents who are BOTH carriers of the genetic defect.
The genetic defect in cystic fibrosis is a mutation in the gene coding for a protein that acts as a chl……….. ion channel in ep……… cells This chloride channel protein is called the cystic fibrosis ……………………… conductance regulator (CFTR). It is expressed in ……… epithelial cells and cystic fibrosis is therefore a ……….-organ disease, affecting all organs lined with epithelial cells, such as the lu……., the g….., the repro…………… tract, pan………., liver and sweat glands.
There has been great progress in our understanding of the basic genetic defect in CF since the defect was first localised to the long arm of chromosome 7 in 1985, and the gene was cloned and sequenced.
Since then, more than 2000 different mutations in the gene coding for CFTR have been de………… CF is in…………. as an autosomal recessive disease, which means that TWO defective CF g……….. must be inherited, one from each parent, and the inherited mutations need not be the same. However 90% of European patients carry at least one copy of the most common defect, DF………..
This refers to a de………… (D) of a single phen…………… (F) residue at position 508 in the amino acid sequence of CFTR, ie the deletion of 3 base pairs coding for a single amino acid is responsible for most CF disease.
The genetic defect in cystic fibrosis is a mutation in the gene coding for a protein that acts as a chloride ion channel in epithelial cells This chloride channel protein is called the cystic fibrosis transmembrane conductance regulator (CFTR). It is expressed in ALL epithelial cells and cystic fibrosis is therefore a multi-organ disease, affecting all organs lined with epithelial cells, such as the lungs, the gut, the reproductive tract, pancreas, liver and sweat glands.
There has been great progress in our understanding of the basic genetic defect in CF since the defect was first localised to the long arm of chromosome 7 in 1985, and the gene was cloned and sequenced.
Since then, more than 2000 different mutations in the gene coding for CFTR have been described. CF is inherited as an autosomal recessive disease, which means that TWO defective CF genes must be inherited, one from each parent, and the inherited mutations need not be the same. However 90% of European patients carry at least one copy of the most common defect, DF508.
This refers to a deletion (D) of a single phenylalanine (F) residue at position 508 in the amino acid sequence of CFTR, ie the deletion of 3 base pairs coding for a single amino acid is responsible for most CF disease.
In CF the genetic defect has an …………… recessive pattern of inheritance.
Carriers have a single copy of the defective gene (small g), and it may be one of any of the 1000 that have been described. In the carriers the defective gene is masked by the normal …………….. gene (big G) and carriers have no symptoms of the disease.
The offspring of two carriers of a mutation in the CF gene may be;
Ho……………….. normal (GG)
He…………… carriers (Gg)
Homozygous for CF (gg) inheriting two defective genes and expressing the symptoms of CF.
Inheritance is autosomal ie it is not sex-l…………. and equal numbers of both boys and girls may have ……. Each child, male or female has a 25% chance of having CF when both parents are carriers.
In CF the genetic defect has an autosomal recessive pattern of inheritance. Carriers have a single copy of the defective gene (small g), and it may be one of any of the 1000 that have been described. In the carriers the defective gene is masked by the normal dominant gene (big G) and carriers have no symptoms of the disease. The offspring of two carriers of a mutation in the CF gene may be; Homozygous normal (GG) Heterozygous carriers (Gg) Homozygous for CF (gg) inheriting two defective genes and expressing the symptoms of CF. Inheritance is autosomal ie it is not sex-linked and equal numbers of both boys and girls may have CF. Each child, male or female has a 25% chance of having CF when both parents are carriers.
Processing of the normal CFTR
The CF gene is transcribed in the …………… and the normal protein is synthesised on the …… and transported to the cell membrane in ……… vesicles. During the transport process the protein undergoes post-…………….l modification. That is to say the protein becomes ……………… by the addition of sugar residues. These sugar residues are essential for normal trafficking of CFTR to the cell ………………….
The protein finally becomes an integral part of the cell surface membrane, where its activity as a chloride channel is further regulated by two distinct mechanisms. Firstly the protein is activated by ………………… which allows the channel to open and chloride to diffuse passively through the opening, in either direction. Conversely, depho…………… closes the channel. Secondly, the binding of ……. and its hyd………….., regulates activity of the channel and the rate of ch…………… conductance.
Since the CFTR protein is expressed in epithelial cells of the pan………., int…………, sweat gland bile ……….., vas defe……….., s………. glands and sub…………….. glands of the airways it is clear that a defect in this protein will have wide ranging physiological consequences.
Processing of the normal CFTR
The CF gene is transcribed in the nucleus and the normal protein is synthesised on the ER and transported to the cell membrane in golgi vesicles. During the transport process the protein undergoes post-translational modification. That is to say the protein becomes glycosylated by the addition of sugar residues. These sugar residues are essential for normal trafficking of CFTR to the cell membrane.
The protein finally becomes an integral part of the cell surface membrane, where its activity as a chloride channel is further regulated by two distinct mechanisms. Firstly the protein is activated by phosphorylation which allows the channel to open and chloride to diffuse passively through the opening, in either direction. Conversely, dephosphorylation closes the channel. Secondly, the binding of ATP and its hydrolysis, regulates activity of the channel and the rate of chloride conductance.
Since the CFTR protein is expressed in epithelial cells of the pancreas, intestine, sweat gland bile ducts, vas deferens, salivary glands and submucosal glands of the airways it is clear that a defect in this protein will have wide ranging physiological consequences.
Regulation of CFTR chloride channel activity
The structure of CFTR has been predicted from the amino acid sequence. CFTR consists of two transmembrane domains (T….. and T….), two nu…………… binding domains (NBD1 and NBD2), and a unique i…………… h……………. reg………… (R ) domain. Each membrane spanning domain has s…. membrane spanning segments that make up the pore through which ch………. ions move and determine the ion-selectivity of the channel.
Act……………. of CFTR by pho…………… is catalysed by the cyclic AMP dep………….. protein kinase, protein kinase A. The R domain is ph……………… at multiple sites by protein kinase A, which causes a confor…………… change in the protein and the channel to open.
The channel is ATP-g…………… Binding and hydr…………. of A…….. on NBD1 and NBD2 forms a d……….. that activates chloride ……………, whereas ATP hydrolysis at NBD2 closes the channel and inh……….. chloride co……………
Dep………………. of the R domain ensures the channel remains ……………
The extracellular loops in TM2 contain the sites for gly………….. of the protein.
Regulation of CFTR chloride channel activity
The structure of CFTR has been predicted from the amino acid sequence. CFTR consists of two transmembrane domains (TM1 and TM2), two nucleotide binding domains (NBD1 and NBD2), and a unique intracellular hydrophilic regulatory (R ) domain. Each membrane spanning domain has six membrane spanning segments that make up the pore through which chloride ions move and determine the ion-selectivity of the channel.
Activation of CFTR by phosphorylation is catalysed by the cyclic AMP dependent protein kinase, protein kinase A. The R domain is phosphorylated at multiple sites by protein kinase A, which causes a conformational change in the protein and the channel to open.
The channel is ATP-gated. Binding and hydrolysis of ATP on NBD1 and NBD2 forms a dimer that activates chloride conductance, whereas ATP hydrolysis at NBD2 closes the channel and inhibits chloride conductance.
Dephosphorylation of the R domain ensures the channel remains closed
The extracellular loops in TM2 contain the sites for glycosylation of the protein.
Activation of CFTR chloride channel activity by
2-adrenergic receptors
Agents that inc…………. intracellular c…….. in airway epithelial cells include the ……-agonists such as salb…………, that act on …….. adrenergic receptors. Activation of β2 adrenergic receptors sti………….. the stimulatory …..-protein (Gs) that activates a………….. cyclase, which is the enzyme responsible for the formation of c………… This pool of cAMP is highly com……………… and the local increase in cAMP activation leads to an increase in protein kinase ……. activity, and ph………………… and activation of CFTR.
The reason behind this com……………… response is that CFTR and the β2 adrenergic receptors …..-localise in the cell me……………. of airway epithelial cells, and both CFTR and β2 adrenergic receptors bind to the …………. binding protein ……….. (EBP50). These proteins form a ………-molecular complex (CFTR-EBP50- β2 adrenergic receptor). Ezrin is a cyto……………. protein that also binds protein kinase A, ensuring that protein kinase A is in close proximity to CFTR. Following channel activation, the complex …………. apart and the signal is turned off.
These protei-protein interactions (ma………………. complex assembly) are essential for full activation of the channel by the β2-agonist rec……….. pathway. These inte………….. may be critical for a rapid and specific signal tran………….. from the receptor to the channel in a compartmentalised fashion.
It also indicates that defective forms of CFTR may lead to …………….. CFTR function in response to receptor based signalling.
Activation of CFTR chloride channel activity by
2-adrenergic receptors
Agents that increase intracellular cAMP in airway epithelial cells include the β2-agonists such as salbutamol, that act on β2 adrenergic receptors. Activation of β2 adrenergic receptors stimulates the stimulatory G-protein (Gs) that activates adenyl cyclase, which is the enzyme responsible for the formation of cAMP This pool of cAMP is highly compartmentalised and the local increase in cAMP activation leads to an increase in protein kinase A activity, and phosphorylation and activation of CFTR.
The reason behind this compartmentalised response is that CFTR and the β2 adrenergic receptors co-localise in the cell membrane of airway epithelial cells, and both CFTR and β2 adrenergic receptors bind to the ezrin binding protein 50 (EBP50). These proteins form a tri-molecular complex (CFTR-EBP50- β2 adrenergic receptor). Ezrin is a cytoskeletal protein that also binds protein kinase A, ensuring that protein kinase A is in close proximity to CFTR. Following channel activation, the complex falls apart and the signal is turned off.
These protei-protein interactions (macromolecular complex assembly) are essential for full activation of the channel by the β2-agonist receptor pathway. These interactions may be critical for a rapid and specific signal transduction from the receptor to the channel in a compartmentalised fashion.
It also indicates that defective forms of CFTR may lead to abnormal CFTR function in response to receptor based signalling.
Syntaxin 1A inhibits CFTR activity via the cytoplasmic N-terminal tail
CFTR is …………….. regulated by another membrane protein, syntaxin ……, which binds at the …………. (N) terminus of CFTR and negatively regulates channel opening time and also CFTR trafficking to the cell membrane.
Therefore protein networks are important both ………. pos………… (β2-AR) and ne…………. (syntaxin 1A) reg………….. of CFTR.
Protein-protein interactions are also important in the way that CFTR regulates other ion channels such as the epi…………. sodium channel.
Syntaxin 1A inhibits CFTR activity via the cytoplasmic N-terminal tail
CFTR is NEGATIVELY regulated by another membrane protein, syntaxin 1A, which binds at the amino (N) terminus of CFTR and negatively regulates channel opening time and also CFTR trafficking to the cell membrane.
Therefore protein networks are important both for positive (β2-AR) and negative (syntaxin 1A) regulation of CFTR.
Protein-protein interactions are also important in the way that CFTR regulates other ion channels such as the epithelial sodium channel.
The cytoplasmic C-terminal tail of CFTR interacts with regulatory networks
Protein-protein interactions are also important in the way that CFTR reg………….. other ion channels such as the epithelial sodium channel (ENaC), ie in the way that CFTR acts as a con……………. regulator, in the airways
The C terminus of CFTR interacts with proteins that link CFTR to a complex network of reg…………. proteins, through the pro……. bi………… do…………… (PDZ) of NH…………. (previously called EBP50), to the epithelial sodium channel (E………).
In this case, CFTR is NORMALLY assembled as a complex with e………., protein kinase A (PK-A), and the YAP and cYES proteins. As before, the function is to tether PK-…. near to CFTR and to link CFTR to the cytos………… and localise it to the plasma membrane.
NHERF1/EBP50 interacts with Y…….. and this recruits cYES to the complex, which acts to IN………… ENaC activity. Since CFTR is defective in CF we can speculate that this complex is disrupted in CF, so there is both d…………. chloride channel activity and increased sodium channel activity in CF.
This inh…………… effect of CFTR on ENac has been demonstrated in the airways, but not in the sweat gland where the channels work together to regulate Na….. transport, indicating tissue-specific regulatory interactions between CFTR and ENaC.
Understanding these interactions suggests new pharmacological approaches to the regulation of CFTR activity.
The cytoplasmic C-terminal tail of CFTR interacts with regulatory networks
Protein-protein interactions are also important in the way that CFTR regulates other ion channels such as the epithelial sodium channel (ENaC), ie in the way that CFTR acts as a conductance regulator, in the airways
The C terminus of CFTR interacts with proteins that link CFTR to a complex network of regulatory proteins, through the protein binding domains (PDZ) of NHERF1 (previously called EBP50), to the epithelial sodium channel (ENaC).
In this case, CFTR is NORMALLY assembled as a complex with ezrin, protein kinase A (PK-A), and the YAP and cYES proteins. As before, the function is to tether PK-A near to CFTR and to link CFTR to the cytoskeleton and localise it to the plasma membrane.
NHERF1/EBP50 interacts with YAP and this recruits cYES to the complex, which acts to INHIBIT ENaC activity. Since CFTR is defective in CF we can speculate that this complex is disrupted in CF, so there is both decreased chloride channel activity and increased sodium channel activity in CF.
This inhibitory effect of CFTR on ENac has been demonstrated in the airways, but not in the sweat gland where the channels work together to regulate NaCl transport, indicating tissue-specific regulatory interactions between CFTR and ENaC.
Understanding these interactions suggests new pharmacological approaches to the regulation of CFTR activity.
Processing of the deltaF508 CFTR
At present there are over 1000 known mutations in the CFTR gene, but the most common and the one accounting for 70% of CF chromosomes worldwide is the delta F 508 mutation. Delta F 508 is derived from D for de………….., F is from the one letter code for amino acids and stands for phenyl-alanine and 508 is the position in the amino acid sequence of the CFTR protein which is actually made up of 1460 a…………. acids in total. In other words the deletion of t……….. base pairs in the CF gene, coding for a single amino acid in the final product, is responsible for the CF phenotype.
The missing phe…………….. is critically responsible for post-tran…………….. defects in the CFTR protein. Immature forms of the protein are synt……………., but they do not become glyc…………, do not traffick through the g………. system and are not inserted into the cell membrane. Because of the missing amino acid in the tertiary structure, the protein does not fold properly and it is taken up and quickly destroyed in the prote……………. The net defect is that the CFTR is not delivered to the cell membrane and this chl……….. channel is missing, leading to a wide range of phys………….. defects in those epithelial cells where it should normally be expressed.
Processing of the F508 CFTR
At present there are over 1000 known mutations in the CFTR gene, but the most common and the one accounting for 70% of CF chromosomes worldwide is the delta F 508 mutation. Delta F 508 is derived from D for deletion, F is from the one letter code for amino acids and stands for phenyl-alanine and 508 is the position in the amino acid sequence of the CFTR protein which is actually made up of 1460 amino acids in total. In other words the deletion of three base pairs in the CF gene, coding for a single amino acid in the final product, is responsible for the CF phenotype.
The missing phenylalanine is critically responsible for post-translational defects in the CFTR protein. Immature forms of the protein are synthesised, but they do not become glycosylated, do not traffick through the golgi system and are not inserted into the cell membrane. Because of the missing amino acid in the tertiary structure, the protein does not fold properly and it is taken up and quickly destroyed in the proteosomes. The net defect is that the CFTR is not delivered to the cell membrane and this chloride channel is missing, leading to a wide range of physiological defects in those epithelial cells where it should normally be expressed.
Classes of CFTR mutations.
More than 2000 gene variants have been described, most of which (…..%) have been associated with disease ……………… Mutations can have effects on the synt…………, matur……….., sta………. and function of the C……….. protein.
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be divided into six classes.
Class I mutations result in …….. ……… ……….
Class II mutations (including the most prevalent, Phe508del, which accounts for 70% of all mutations) cause ret…………. of a misf………… protein at the endoplasmic reticulum, and subsequent deg………….. in the prot…………… An immature protein is formed but deletion of phenylalanine results in misfolding, degradation and lack of trafficking through the go……..
Class III mutations affect ch…………… re……………….., impairing channel o……………. (eg, Gly551Asp). These are channel gating mutations.
Class IV mutants show reduced con………… —ie, decreased flow of ………….. (eg, Arg117His).
Class V mutations cause substantial reduction in m………. or protein, or both,
Class VI mutations cause substantial pl……….. membrane ins…………. and include Phe508del when rescued by most cor………….. (rPhe508del).
Diagnosis of CF is usually made early in life following newborn screening and D……… analysis for CFTR mutations.
Classes of CFTR mutations.
More than 2000 gene variants have been described, most of which (85%) have been associated with disease causation. Mutations can have effects on the synthesis, maturation, stability and function of the CFTR protein.
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be divided into six classes.
Class I mutations result in no protein production.
Class II mutations (including the most prevalent, Phe508del, which accounts for 70% of all mutations) cause retention of a misfolded protein at the endoplasmic reticulum, and subsequent degradation in the proteasome. An immature protein is formed but deletion of phenylalanine results in misfolding, degradation and lack of trafficking through the golgi.
Class III mutations affect channel regulation, impairing channel opening (eg, Gly551Asp). These are channel gating mutations.
Class IV mutants show reduced conduction—ie, decreased flow of ions (eg, Arg117His).
Class V mutations cause substantial reduction in mRNA or protein, or both,
Class VI mutations cause substantial plasma membrane instability and include Phe508del when rescued by most correctors (rPhe508del).
Diagnosis of CF is usually made early in life following newborn screening and DNA analysis for CFTR mutations.
Chloride ions move through a p…………. that ………….. when CFTR is activated by ………..and by phosp……………. of a reg………… domain
Chloride ions move through a pore that opens when CFTR is activated by ATP and by phosphorylation of a regulatory domain
