Lianwu Fu

The silent codon change I507-ATC→ATT contributes to the severity of the ΔF508 CFTR channel dysfunction

The most common disease‐causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out‐of‐frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine‐508 (ΔF508) and a silent codon change (SCC) for isoleucine‐507 (I507‐ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum‐associated degradation (ERAD). We have demonstrated that the I507‐ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507‐ATT and I507‐ATC ΔF508 CFTR, we establish that the I507‐ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR We demonstrate that the I507‐ATC ΔF508 CFTR is less susceptible to the ER quality‐control machinery during translation than the I507‐ATT, although 27°C correction is necessary for sufficient cell‐surface expression. Whole‐cell patch‐clamp recordings indicate sustained, thermally stable cAMP‐activated Cl– transport through I507‐ATC and unstable function of the I507‐ATT ΔF508 CFTR Single‐channel recordings reveal improved gating properties of the I507‐ATC compared to I507‐ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507‐ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.—Lazrak, A., Fu, L., Bali, V., Bartoszewski, R., Rab, A., Havasi, V., Keiles, S., Kappes, J., Kumar, R., Lefkowitz, E., Sorscher, E. J., Matalon, S., Collawn, J. F., Bebok, Z. The silent codon change I507‐ATC→ATT contributes to the severity of the ΔF508 CFTR FASEB J. 27, 4630–4645 (2013). www.fasebj.org

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (∼10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.

ARFGAP1 promotes AP-2-dependent endocytosis

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.COPI (coat protein I) and the clathrin–AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.

CFTR expression regulation by the unfolded protein response.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel and key regulator of epithelial functions. Mutations in the CFTR gene lead to reduced or dysfunctional CFTR protein and cause cystic fibrosis (CF), a generalized exocrinopathy affecting multiple organs. In the airways, loss of CFTR function leads to thickened mucus, reduced mucociliary clearance, chronic infections, and respiratory failure. Common airway disorders such as bronchitis and chronic obstructive pulmonary disease (COPD) also present CF-like symptoms such as mucus congestion and chronic inflammation without mutations in CFTR. The primary risk factors for COPD and chronic bronchitis include environmental stress insults such as pollutants and infections that often result in hypoxic conditions. Furthermore, environmental factors such as cigarette smoke and reactive oxygen species have been implicated in reduced CFTR function. Activation of cellular stress responses by these factors promotes differential, stress-associated gene expression regulation. During our investigations on the mechanisms of CFTR expression regulation, we have shown that the ER stress response, the unfolded protein response (UPR), decreases CFTR expression at the transcriptional, translational, and maturational levels. Here, we provide a detailed description of the methods we employ to study CFTR expression regulation by the UPR. Similar approaches are applicable in studies on other genes and how they are affected by the UPR.

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload

BACKGROUND Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymases role in autophagy induction. CONCLUSION In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.

Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

The therapeutic potential of CFTR modulators for COPD and other airway diseases

HighlightsCommon airway diseases including COPD, asthma and non‐CF bronchiectasis (NCFBE) share the common pathophysiologic feature of mucus obstruction.Acquired CFTR dysfunction is a potential overlapping mechanism to explain abnormal mucus clearance in these diseases.CFTR modulators have shown promise in CF and thus should be tested in diseases with acquired CFTR dysfunction.Early studies indicate safety and promise efficacy for the CFTR potentiator ivacaftor in COPD with chronic bronchitis. &NA; Airways diseases, especially chronic obstructive pulmonary disease (COPD) and asthma, are common causes of morbidity and mortality worldwide. There is an ongoing unmet need for novel and effective therapies. There is an established pathophysiological link and phenotypic similarity between the chronic bronchitis phenotype of COPD and cystic fibrosis (CF). New evidence suggests that CFTR dysfunction may play a role in other common airways diseases such as COPD, non‐atopic asthma and non‐CF bronchiectasis. Newly approved and investigational drugs that target both mutant and wild‐type CFTR channels have provided a new treatment opportunity addressing the mucus defect in pulmonary diseases that share the same pathophysiology with CF.

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator

Synonymous mutations, such as I507‐ATCÅATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease‐associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small‐molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX‐809), on I507‐ATT and I507‐ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507‐ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX‐809 biochemically corrected ~10% of both of the variants, leading to stable, forskolin+3‐isobutyl‐1‐methylxanthine (IBMX)‐activated whole‐cell currents in the presence of the corrector. Omitting VX‐809 during whole‐cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX‐809. Treatment of cells with the C4+VX‐809 combination resulted in enhanced rescue and 2‐fold higher forskolin+IBMX‐activated currents of both I507‐ATT and I507‐ATC ΔF508 CFTR, compared with VX‐809 treatment alone. The lack of an effect of C4 on I507‐ATC ΔF508 CFTR, but its additive effect in combination with VX‐809, implies that C4 acted on VX‐809‐modified I507‐ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX‐809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.—Bali, V., Lazrak, A., Guroji, P., Fu, L., Matalon, S., Bebok, Z. A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator. FASEB J. 30, 201‐213 (2016). www.fasebj.org

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes.