Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
TL;DR: WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms, and molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation.
Abstract: CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.
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TL;DR: Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.
Abstract: The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.
718 citations
TL;DR: A new mechanistic definition of chronic pancreatitis is proposed that recognizes the complex nature of CP, separates risk factors from disease activity markers and disease endpoints, and allows for a rational approach to early diagnosis, classification and prognosis.
335 citations
Cites background from "Mechanisms of CFTR functional varia..."
...Attempts to use pancreatic duct function as an early diagnostic tool for CP also faces challenges since multiple CFTR gene mutations result in defective bicarbonate conductance that is independent of inflammation or other pancreatic disease [43]....
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TL;DR: The different operational roles of CFTR in epithelial function are illustrated by describing its characteristics in three different tissues: the airways, the pancreas, and the sweat gland.
Abstract: Salt and fluid absorption and secretion are two processes that are fundamental to epithelial function and whole body fluid homeostasis, and as such are tightly regulated in epithelial tissues. The CFTR anion channel plays a major role in regulating both secretion and absorption in a diverse range of epithelial tissues, including the airways, the GI and reproductive tracts, sweat and salivary glands. It is not surprising then that defects in CFTR function are linked to disease, including life-threatening secretory diarrhoeas, such as cholera, as well as the inherited disease, cystic fibrosis (CF), one of the most common life-limiting genetic diseases in Caucasian populations. More recently, CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease (COPD), and the hyper-responsiveness in asthma, underscoring its fundamental role in whole body health and disease. CFTR regulates many mechanisms in epithelial physiology, such as maintaining epithelial surface hydration and regulating luminal pH. Indeed, recent studies have identified luminal pH as an important arbiter of epithelial barrier function and innate defence, particularly in the airways and GI tract. In this chapter, we will illustrate the different operational roles of CFTR in epithelial function by describing its characteristics in three different tissues: the airways, the pancreas, and the sweat gland.
259 citations
TL;DR: Understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling is furthered, and novel roles for this pathway in immune cell function and cell proliferation are uncovered.
168 citations
TL;DR: The genetics, cell biology, and immunology of pancreatitis is reviewed with a focus on protease activation pathways and other early events, and clinical and experimental observations provide compelling evidence that premature intrapancreatic activation of digestive proteases is critical in pancreatitis onset.
151 citations
References
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16,894 citations
TL;DR: The development, current features, and some directions for future development of the Amber package of computer programs, which contains a group of programs embodying a number of powerful tools of modern computational chemistry, focused on molecular dynamics and free energy calculations of proteins, nucleic acids, and carbohydrates.
Abstract: We describe the development, current features, and some directions for future development of the Amber package of computer programs. This package evolved from a program that was constructed in the late 1970s to do Assisted Model Building with Energy Refinement, and now contains a group of programs embodying a number of powerful tools of modern computational chemistry, focused on molecular dynamics and free energy calculations of proteins, nucleic acids, and carbohydrates.
7,672 citations
"Mechanisms of CFTR functional varia..." refers methods in this paper
...Molecular dynamics simulations and elastic network model analysis Molecular dynamics simulations were performed using the AMBER11[74] package (GPU version of the pmemd program), CFTR Bicarbonate Permeation Variants...
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...Molecular dynamics simulations and elastic network model analysis Molecular dynamics simulations were performed using the AMBER11[74] package (GPU version of the pmemd program), CFTR Bicarbonate Permeation Variants PLOS Genetics | www.plosgenetics.org 12 July 2014 | Volume 10 | Issue 7 | e1004376 with the Amber99SB[75] force field and using the TIP3P water model....
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TL;DR: The SWISS-MODEL workspace is a web-based integrated service dedicated to protein structure homology modelling that assists and guides the user in building protein homology models at different levels of complexity.
Abstract: Motivation: Homology models of proteins are of great interest for planning and analysing biological experiments when no experimental three-dimensional structures are available. Building homology models requires specialized programs and up-to-date sequence and structural databases. Integrating all required tools, programs and databases into a single web-based workspace facilitates access to homology modelling from a computer with web connection without the need of downloading and installing large program packages and databases.
Results: SWISS-MODEL workspace is a web-based integrated service dedicated to protein structure homology modelling. It assists and guides the user in building protein homology models at different levels of complexity. A personal working environment is provided for each user where several modelling projects can be carried out in parallel. Protein sequence and structure databases necessary for modelling are accessible from the workspace and are updated in regular intervals. Tools for template selection, model building and structure quality evaluation can be invoked from within the workspace. Workflow and usage of the workspace are illustrated by modelling human Cyclin A1 and human Transmembrane Protease 3.
Availability: The SWISS-MODEL workspace can be accessed freely at http://swissmodel.expasy.org/workspace/
Contact: Torsten.Schwede@unibas.ch
Supplementary information: Supplementary data are available at Bioinformatics online.
7,107 citations
TL;DR: An effort to improve the φ/ψ dihedral terms in the ff99 energy function achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data.
Abstract: The ff94 force field that is commonly associated with the Amber simulation package is one of the most widely used parameter sets for biomolecular simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of alpha-helices, were reported by us and other researchers. This led to a number of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in determining which should be used for a particular application. We show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addition, the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone phi/psi dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. We report here an effort to improve the phi/psi dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mechanical calculations. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which we denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published experimental data for conformational preferences of short alanine peptides and better accord with experimental NMR relaxation data of test protein systems.
6,146 citations
"Mechanisms of CFTR functional varia..." refers methods in this paper
...Molecular dynamics simulations and elastic network model analysis Molecular dynamics simulations were performed using the AMBER11[74] package (GPU version of the pmemd program), CFTR Bicarbonate Permeation Variants PLOS Genetics | www.plosgenetics.org 12 July 2014 | Volume 10 | Issue 7 | e1004376 with the Amber99SB[75] force field and using the TIP3P water model....
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...with the Amber99SB[75] force field and using the TIP3P water model....
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TL;DR: X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site, and that loss of this cleavage site would permit autodigestion resulting in pancreatitis.
Abstract: Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg–His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.
1,471 citations
"Mechanisms of CFTR functional varia..." refers methods in this paper
...Genotyping PRSS1 genotyping was done by DNA sequencing [69]....
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