CFTR inhibitory factor
Encyclopedia
The CFTR inhibitory factor (Cif) is a protein virulence factor
secreted by the Gram-negative bacterium Pseudomonas aeruginosa
. Discovered at Dartmouth Medical School
, Cif is able to alter the trafficking of select ABC transporters in eukaryotic epithelial cells, such as the cystic fibrosis transmembrane conductance regulator
(CFTR), and P-glycoprotein
by interfering with the host deubiquitinating machinery. By promoting the ubiquitin
-mediated degredation of CFTR, Cif is able to phenocopy cystic fibrosis
at the cellular level. Cif is transcribed as part of a 3 gene operon
, whose expression is negatively regulated by CifR, a TetR
family repressor.
. Cif is secreted by P. aeruginosa PA14 as soluble protein as well as packaged into outer membrane vesicles (OMV). Cif is far more potent when applied in OMVs, likely due to efficiency of delivery. Purified, recombinant Cif protein can be applied to polarized monolayers of mammalian cells and promote the removal of CFTR and P-glycoprotein from the apical membrane. Cif accomplishes this by interfering with the host deubiquitylation system.
(EH) with unique substrate selectivity. Cif is the first example of an EH serving as a virulence factor. Based on structural comparison, it appears that the enzyme utilizes a catalytic triad of residues Asp129, Glu153 and His297, with accessory residues His177 and Tyr239 coordinating the epoxide oxygen during ring opening. Cif is also the first example of an EH utilizing a His-Tyr pair to coordinate an epoxide substrate, rather than the canonical Tyr-Tyr pair. In the proposed enzyme mechanism, Asp129 nucleophilically attacks a carbon of the epoxide moiety of a substrate, forming an ester linked enzyme-acyl intermediate. The preference for which carbon is attacked varies depending upon the substrate. In the second step of the reaction, a water molecule is activated by the charge-relay His297-Glu153 pair, and undergoes nucleophilic attack on the Cγ of Asp129. This hydrolyzes the ester group, liberating the hydrolysis product as a vicinal diol.
of proteins. Its structure was determined by X-ray crystallography and consists of the canonical α/β hydrolase fold with a cap domain, which it uses to constitutively homo-dimerize in solution. The active site is burred in the interior of the protein at the interface between the α/β hydrolase core and the cap.
Virulence factor
Virulence factors are molecules expressed and secreted by pathogens that enable them to achieve the following:* colonization of a niche in the host...
secreted by the Gram-negative bacterium Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many...
. Discovered at Dartmouth Medical School
Dartmouth Medical School
Dartmouth Medical School is the medical school of Dartmouth College, located in Hanover, New Hampshire, in the United States. The fourth-oldest medical school in the United States, Dartmouth Medical School was founded in 1797 by New England physician Nathan Smith and grew steadily over the course...
, Cif is able to alter the trafficking of select ABC transporters in eukaryotic epithelial cells, such as the cystic fibrosis transmembrane conductance regulator
Cystic fibrosis transmembrane conductance regulator
Cystic fibrosis transmembrane conductance regulator is a protein that in humans is encoded by the CFTR gene.CFTR is a ABC transporter-class ion channel that transports chloride and thiocyanate ions across epithelial cell membranes...
(CFTR), and P-glycoprotein
P-glycoprotein
P-glycoprotein 1 also known as multidrug resistance protein 1 or ATP-binding cassette sub-family B member 1 or cluster of differentiation 243 is a glycoprotein that in humans is encoded by the ABCB1 gene...
by interfering with the host deubiquitinating machinery. By promoting the ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
-mediated degredation of CFTR, Cif is able to phenocopy cystic fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...
at the cellular level. Cif is transcribed as part of a 3 gene operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
, whose expression is negatively regulated by CifR, a TetR
TetR
Tetracycline is a broad family of antibiotics to which bacteria have evolved resistance.The expression of tc resistance genes is regulated by Tet Repressor Protein, called TetR....
family repressor.
Cellular mechanism of action
Cif was first discovered by co-culturing P. aeruginosa with human airway epithelial cells and monitoring the resulting effect on chloride ion efflux across a polarized monolayer. After co-culture, the CFTR specific chloride ion efflux was found to be drastically reduced. This was determined to be caused by reduced levels of CFTR at the apical surface of these cells. This effect was later found to be the result of a single secreted protein produced by P. aeruginosa, which was named the CFTR inhibitory factor for this initial phenotypePhenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
. Cif is secreted by P. aeruginosa PA14 as soluble protein as well as packaged into outer membrane vesicles (OMV). Cif is far more potent when applied in OMVs, likely due to efficiency of delivery. Purified, recombinant Cif protein can be applied to polarized monolayers of mammalian cells and promote the removal of CFTR and P-glycoprotein from the apical membrane. Cif accomplishes this by interfering with the host deubiquitylation system.
Epoxide hydrolase enzyme mechanism
Cif is an epoxide hydrolaseEpoxide hydrolase
Epoxide hydrolase functions in detoxication during drug metabolism. It converts epoxides to trans-dihydrodiols, which can be conjugated and excreted from the body. Epoxides result from the degradation of aromatic compounds...
(EH) with unique substrate selectivity. Cif is the first example of an EH serving as a virulence factor. Based on structural comparison, it appears that the enzyme utilizes a catalytic triad of residues Asp129, Glu153 and His297, with accessory residues His177 and Tyr239 coordinating the epoxide oxygen during ring opening. Cif is also the first example of an EH utilizing a His-Tyr pair to coordinate an epoxide substrate, rather than the canonical Tyr-Tyr pair. In the proposed enzyme mechanism, Asp129 nucleophilically attacks a carbon of the epoxide moiety of a substrate, forming an ester linked enzyme-acyl intermediate. The preference for which carbon is attacked varies depending upon the substrate. In the second step of the reaction, a water molecule is activated by the charge-relay His297-Glu153 pair, and undergoes nucleophilic attack on the Cγ of Asp129. This hydrolyzes the ester group, liberating the hydrolysis product as a vicinal diol.
Structure
Cif belongs to the α/β hydrolase familyAlpha/beta hydrolase fold
In molecular biology, the alpha/beta hydrolase fold is common to a number of hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is an alpha/beta-sheet , containing 8 strands connected byhelices...
of proteins. Its structure was determined by X-ray crystallography and consists of the canonical α/β hydrolase fold with a cap domain, which it uses to constitutively homo-dimerize in solution. The active site is burred in the interior of the protein at the interface between the α/β hydrolase core and the cap.