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Topic Name: Using Computer Simulations Researchers Identified a Key Molecular Mechanism May Help to Understanding the Development of Cystic Fibrosis

Category: Genetic Engineering

Research persons: Nikolay Dokholyan

Location: University of North Carolina, Chapel Hill, United States

Details

Researchers from the University of North Carolina at Chapel Hill have identified a key molecular mechanism that may account for the development of cystic fibrosis, which about 1 in 3000 children are born with in the US every year. The findings, published February 29 in the open-access journal PLoS Computational Biology, add new knowledge to understanding the development of this disease and may also point the way to new corrective treatments.

Cystic fibrosis (CF) is a fatal disease caused by a defective gene that produces a misshapen form of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. People with cystic fibrosis do not have enough CFTR for their cells to work normally because their bodies quickly destroy the mutant protein. The deletion of this protein specifically occurs in a major domain of CFTR called NBD1. Earlier experimental studies have shown that the mutant NBD1 has an increased tendency to misfold, resulting in the premature degradation of CFTR.

In CF, the molecular basis of this increased misfolding tendency has remained elusive, said team leader Nikolay Dokholyan.

“Understanding molecular etiology of the disease is a key step to developing pharmaceutical strategies to fight this disease,” Dokholyan said.

Using molecular dynamics simulations, the researchers performed extensive simulations of how normal and mutant NBD1 folded. Molecular dynamics simulation is akin to a “virtual experiment” wherein atoms and molecules are allowed to evolve according to known physical laws. Using computers, this virtual experiment allows researchers to view how atoms actually move. These simulations, when applied to the NBD1 protein, showed that the disease-causing mutant exhibits a higher misfolding tendency.

More importantly, by comparing the structures of the normal and the mutant NBD1 domains as they fold, the authors were able to determine critical pairs of amino acid residues that must come together for NBD1 to fold correctly. These interactions are modulators of CFTR folding, and hence, they are potential modulators of CF.

“Computer simulations approximate our understanding of natural phenomena. That our simulations correlated with known experimental studies is remarkable,” Dokholyan said. “More importantly, the molecular details of aberrant NBD1 folding provides guidance for the design of small molecule drugs to correct the most prevalent and pathogenic mutation in CFTR.”

Note for Cystic Fibrosis
Cystic fibrosis (CF), mucoviscoidosis, or mucoviscidosis, is a hereditary disease that affects mainly the lungs and digestive system, causing progressive disability.
Thick mucus production, as well as a less competent immune system, results in frequent lung infections. Diminished secretion of pancreatic enzymes is the main cause of poor growth, fatty diarrhea and deficiency in fat-soluble vitamins. Males can be infertile due to the condition congenital bilateral absence of the vas deferens. Often, symptoms of CF appear in infancy and childhood. Meconium ileus is a typical finding in newborn babies with CF.
Individuals with cystic fibrosis can be diagnosed prior to birth by genetic testing. Newborn screening tests are increasingly common and effective. The diagnosis of CF may be confirmed if high levels of salt are found during a sweat test. Some false positives may occur.
There is no cure for CF, and most individuals with cystic fibrosis die young: many in their 20s and 30s from lung failure. However, with the continuous introduction of many new treatments, the life expectancy of a person with CF is increasing. Lung transplantation is often necessary as CF worsens.
Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. In the United States, 1 in 3900 children are born with CF. It is most common among Europeans and Ashkenazi Jews; one in twenty-two people of European descent carry one gene for CF, making it the most common genetic disease in these populations.
CF is caused by a mutation in a gene called the cystic fibrosis transmembrane conductance regulator (CFTR). The product of this gene is a chloride ion channel important in creating sweat, digestive juices, and mucus. Although most people without CF have two working copies of the CFTR gene, only one is needed to prevent cystic fibrosis. CF develops when neither gene works normally. Therefore, CF is considered an autosomal recessive disease.
Cystic fibrosis may be diagnosed by many different categories of testing including those such as, newborn screening, sweat testing, or genetic testing. As of 2006 in the United States, 10percent of cases are diagnosed shortly after birth as part of newborn screening programs. The newborn screen initially measures for raised blood concentration of immunoreactive trypsinogen. However, most states and countries do not screen for CF routinely at birth. Therefore, most individuals are diagnosed after symptoms prompt an evaluation for cystic fibrosis. The most commonly-used form of testing is the sweat test. Sweat-testing involves application of a medication that stimulates sweating (pilocarpine) to one electrode of an apparatus and running electric current to a separate electrode on the skin. This process, called iontophoresis, causes sweating; the sweat is then collected on filter paper or in a capillary tube and analyzed for abnormal amounts of sodium and chloride. People with CF have increased amounts of sodium and chloride in their sweat. CF can also be diagnosed by identification of mutations in the CFTR gene.
A multitude of tests is used to identify complications of CF and to monitor disease progression. X-rays and CAT scans are used to examine the lungs for signs of damage or infection. Examination of the sputum under a microscope is used to identify which bacteria are causing infection so that effective antibiotics can be given. Pulmonary function tests measure how well the lungs are functioning, and are used to measure the need for and response to antibiotic therapy. Blood tests can identify liver problems, vitamin deficiencies, and the onset of diabetes. DEXA scans can screen for osteoporosis and testing for fecal elastase can help diagnose insufficient digestive enzymes.

Note for Cystic Fibrosis Transmembrane Conductance Regulator
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC transporter-class protein and ion channel that transports chloride ions across epithelial cell membranes. Mutations of the CFTR gene affect functioning of the chloride ion channels in these cell membranes, leading to cystic fibrosis and congenital absence of the vas deferens.
The gene that encodes for CFTR is found on the human chromosome 7, on the long arm at position q31.2. It contains about 170,000 base pairs. The encoded CFTR is a glycoprotein with 1480 amino acids. The protein consists of five domains. There are two transmembrane domains, each with six spans of alpha helices. These are each connected to a nucleotide binding fold (NBF) in the cytoplasm. These two nucleotide binding folds are linked to a single regulatory R-domain that is a unique feature belonging only to this type of ABC protein. The ion channel only opens when its R-domain has been phosphorylated by PKA and two molecules of ATP have bound to the two NBFs. The carboxyl terminal of the protein is anchored to the cytoskeleton by a PDZ domain interaction.
The CFTR is found in the epithelial cells of many organs including the lung, liver, pancreas, digestive tract, reproductive tract, and skin. Normally, the protein moves chloride ions (with a negative charge) out of an epithelial cell to the covering mucus. This results in an electrical gradient being formed and in the movement of (positively charged) sodium ions in the opposite direction via the ENaC. Due to this movement, the water potential of the mucus is reduced, resulting in the movement of water here by osmosis and a more fluid mucus.

Note for Amino Acid
In chemistry, an amino acid is a molecule that contains both amine and carboxyl functional groups. In biochemistry, this term refers to alpha-amino acids with the general formula H2NCHRCOOH, where R is an organic substituent. In the alpha amino acids, the amino and carboxylate groups are attached to the same carbon, which is called the α–carbon. The various alpha amino acids differ in which side chain (R group) is attached to their alpha carbon. They can vary in size from just a hydrogen atom in glycine through a methyl group in alanine to a large heterocyclic group in tryptophan.
Beyond the amino acids that are found in all forms of life, many non-natural amino acids have vital roles in technology and industry. For example, the chelating agents EDTA and nitriloacetic acid are alpha amino acids that are important in the chemical industry.
Alpha-amino acids are the building blocks of proteins. A protein forms via the condensation of amino acids to form a chain of amino acid "residues" linked by peptide bonds. Proteins are defined by their unique sequence of amino acid residues; this sequence is the primary structure of the protein. Just as the letters of the alphabet can be combined to form an almost endless variety of words, amino acids can be linked in varying sequences to form a huge variety of proteins.
Twenty standard amino acids are used by cells in protein biosynthesis, and these are specified by the general genetic code. These 20 amino acids are biosynthesized from other molecules, but organisms differ in which ones they can synthesize and which ones must be provided in their diet. The ones that cannot be synthesized by an organism are called essential amino acids.
Amino acids are the basic structural building units of proteins. They form short polymer chains called peptides or longer chains called either polypeptides or proteins. The process of such formation from an mRNA template is known as translation, which is part of protein biosynthesis. Twenty amino acids are encoded by the standard genetic code and are called proteinogenic or standard amino acids. Other amino acids contained in proteins are usually formed by post-translational modification, which is modification after translation in protein synthesis. These modifications are often essential for the function or regulation of a protein; for example, the carboxylation of glutamate allows for better binding of calcium cations, and the hydroxylation of proline is critical for maintaining connective tissues and responding to oxygen starvation. Such modifications can also determine the localization of the protein, e.g., the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.

The first author of the study is Adrian Serohijos, a graduate student in the department of Physics and Astronomy at UNC and in the Molecular and Cellular Biophysics Program. Other co-authors in the study include John Riordan, Ph.D., co-discoverer of the CFTR gene and professor of biochemistry and biophysics, and Tamas Hegedus, Ph.D. of the UNC Cystic Fibrosis Research Center.

This study was supported in part by grants from the Cystic Fibrosis Foundation, the National Institutes of Health, and the American Heart Association.

In figure 1, Cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)

In figure 2, CFTR protein - Molecular structure of the CFTR protein