17 results on '"Diane E. Grove"'
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2. Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice
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Laura A. Hancock, Corinne E. Hennessy, George M. Solomon, Evgenia Dobrinskikh, Alani Estrella, Naoko Hara, David B. Hill, William J. Kissner, Matthew R. Markovetz, Diane E. Grove Villalon, Matthew E. Voss, Guillermo J. Tearney, Kate S. Carroll, Yunlong Shi, Marvin I. Schwarz, William R. Thelin, Steven M. Rowe, Ivana V. Yang, Christopher M. Evans, and David A. Schwartz
- Subjects
Science - Abstract
The promoter variant rs35705950 confers a gain of function to the MUC5B gene and is the dominant risk factor for idiopathic pulmonary fibrosis. Here the authors show that mice overexpressing Muc5b in distal airspaces show impaired mucociliary clearance and increased susceptibility to bleomycin-induced lung fibrosis, and that both characteristics are reduced by treatment with a mucolytic agent.
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- 2018
- Full Text
- View/download PDF
3. Disulfide disruption reverses mucus dysfunction in allergic airway disease
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Siddharth K. Shenoy, Corinne E. Hennessy, William J. Janssen, Fernando Holguin, Jung Soo Suk, Chelsea M. Magin, Christopher M. Evans, Ana M. Jaramillo, Vanessa L. Richardson, Naoko Hara, James C. NeeDell, Leslie E. Morgan, Diane E. Grove Villalon, Dorota S Raclawska, David J. Thornton, William R. Thelin, Nkechinyere A. Emezienna, Anna Q. Harder, Justin Hanes, Hassan M. El-Batal, and Gregg A. Duncan
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0301 basic medicine ,Male ,General Physics and Astronomy ,urologic and male genital diseases ,Biochemistry ,Mice ,0302 clinical medicine ,fluids and secretions ,immune system diseases ,Bronchodilator ,Medicine ,030212 general & internal medicine ,Disulfides ,Respiratory system ,Lung ,Expectorants ,0303 health sciences ,Mice, Inbred BALB C ,Multidisciplinary ,Middle Aged ,respiratory system ,3. Good health ,medicine.anatomical_structure ,Female ,medicine.symptom ,medicine.drug ,Adult ,Adolescent ,medicine.drug_class ,Mucociliary clearance ,Science ,Inflammation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Allergic inflammation ,03 medical and health sciences ,In vivo ,Hypersensitivity ,Animals ,Humans ,030304 developmental biology ,Asthma ,Glycoproteins ,business.industry ,Mucin ,General Chemistry ,Translational research ,medicine.disease ,Mucus ,respiratory tract diseases ,Disease Models, Animal ,030104 developmental biology ,030228 respiratory system ,Immunology ,Methacholine ,business - Abstract
Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma., In asthma, mucus plugging is an important cause of airflow obstruction, but it is not targeted by widely used bronchodilator and anti-inflammatory drugs. Here the authors show that reduction of disulfide bonds that hold mucin polymers together reverses asthma-like obstruction in mice.
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- 2021
4. Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice
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Kate S. Carroll, George M. Solomon, Yunlong Shi, Steven M. Rowe, Alani Estrella, Christopher M. Evans, Evgenia Dobrinskikh, Marvin I. Schwarz, Matthew R. Markovetz, Naoko Hara, Ivana V. Yang, Diane E. Grove Villalon, David B. Hill, Corinne E. Hennessy, William J. Kissner, Laura A. Hancock, Guillermo J. Tearney, Matthew E. Voss, David A. Schwartz, and William R. Thelin
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0301 basic medicine ,Male ,General Physics and Astronomy ,Idiopathic pulmonary fibrosis ,Mice ,0302 clinical medicine ,Pulmonary fibrosis ,Medicine ,Promoter Regions, Genetic ,lcsh:Science ,Lung ,Expectorants ,Multidisciplinary ,respiratory system ,Mucin-5B ,Pulmonary Surfactant-Associated Protein C ,3. Good health ,Mucociliary Clearance ,Gain of Function Mutation ,Female ,Cell type ,Mucociliary clearance ,Science ,Transgene ,Mice, Transgenic ,Respiratory Mucosa ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Bleomycin ,Animals ,Humans ,Genetic Predisposition to Disease ,business.industry ,Mucin ,Surfactant protein C ,Epithelial Cells ,General Chemistry ,medicine.disease ,Idiopathic Pulmonary Fibrosis ,respiratory tract diseases ,Respiratory pharmacology ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,030228 respiratory system ,Cancer research ,lcsh:Q ,business - Abstract
The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF., The promoter variant rs35705950 confers a gain of function to the MUC5B gene and is the dominant risk factor for idiopathic pulmonary fibrosis. Here the authors show that mice overexpressing Muc5b in distal airspaces show impaired mucociliary clearance and increased susceptibility to bleomycin-induced lung fibrosis, and that both characteristics are reduced by treatment with a mucolytic agent.
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- 2018
5. An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases
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Nicholas C. Fontana, Takafumi Kato, Barbara R. Grubb, Scott H. Donaldson, Charles R. Esther, William R. Thelin, Camille Ehre, Boya Wang, Richard C. Boucher, M. Delion, Cameron B. Morrison, Matthew R. Markovetz, Alessandra Livraghi-Butrico, Zachary L. Rushton, Lauren N. Hothem, David B. Hill, and Diane E. Grove Villalon
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Pulmonary and Respiratory Medicine ,Male ,Cystic Fibrosis ,Mucociliary clearance ,Respiratory System ,Pulmonary disease ,In Vitro Techniques ,urologic and male genital diseases ,Critical Care and Intensive Care Medicine ,03 medical and health sciences ,Mice ,Pulmonary Disease, Chronic Obstructive ,0302 clinical medicine ,Medicine ,Animals ,Humans ,030212 general & internal medicine ,Lung Diseases, Obstructive ,Respiratory system ,Expectorants ,business.industry ,Mucin ,Original Articles ,respiratory system ,Mucus ,Pathophysiology ,Asthma ,Acetylcysteine ,Disease Models, Animal ,Dithiothreitol ,030228 respiratory system ,Mucociliary Clearance ,Immunology ,Airway ,business ,Obstructive diseases - Abstract
Rationale: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. Objectives: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. Methods: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. Measurements and Main Results: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in βENaC-overexpressing mice, whereas NAC did not (n = 6–24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. Conclusions: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.
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- 2018
6. Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis
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William R. Thelin, Ian Seim, Lidija Turkovic, Bryan Zorn, Richard C. Boucher, David B. Hill, Matthew R. Markovetz, Camille Ehre, Matthew C. Wolfgang, M. Gregory Forest, M. Delion, Charles R. Esther, Kathryn A. Ramsey, Juan R. Sabater, Marianne S. Muhlebach, Mehmet Kesimer, Cameron B. Morrison, Sarath Ranganathan, Ian C. Garbarine, Stephen M. Stick, and Diane E. Grove Villalon
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0301 basic medicine ,Male ,Cystic Fibrosis ,Inflammation ,Cystic fibrosis ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Respiratory system ,Child ,Lung ,Sheep ,medicine.diagnostic_test ,business.industry ,Microbiota ,Mucin ,Infant ,General Medicine ,respiratory system ,medicine.disease ,Mucus ,Pathophysiology ,respiratory tract diseases ,030104 developmental biology ,Bronchoalveolar lavage ,medicine.anatomical_structure ,030228 respiratory system ,Case-Control Studies ,Child, Preschool ,Immunology ,Female ,medicine.symptom ,business ,Biomarkers - Abstract
Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography–defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared “permanent” because they did not dissolve in dilute BALF matrix, they could be solubi-lized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.
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- 2018
7. Endoplasmic reticulum stress–induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis
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Douglas M. Cyr, Diane E. Grove, Hong Yu Ren, and Pattarawut Sopha
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0301 basic medicine ,Proteasome Endopeptidase Complex ,Carcinoma, Hepatocellular ,Recombinant Fusion Proteins ,Antineoplastic Agents ,Apoptosis ,Caspase 3 ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Chlorocebus aethiops ,Animals ,Humans ,Molecular Biology ,Caspase ,biology ,Protein Stability ,Endoplasmic reticulum ,Liver Neoplasms ,Cell Biology ,HSP40 Heat-Shock Proteins ,Endoplasmic Reticulum Stress ,Neoplasm Proteins ,Cell biology ,Receptors, Autocrine Motility Factor ,Thiazoles ,HEK293 Cells ,030104 developmental biology ,Proteostasis ,Amino Acid Substitution ,Proto-Oncogene Proteins c-bcl-2 ,Proteasome ,Protein Synthesis and Degradation ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Chaperone (protein) ,COS Cells ,Mutation ,Proteolysis ,Cancer cell ,biology.protein ,Unfolded protein response ,RNA Interference - Abstract
DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.
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- 2017
- Full Text
- View/download PDF
8. VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1
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Diane E. Grove, Pattarawut Sopha, Oxana Adolfovna de la Rosa, Hong Yu Ren, Douglas M. Cyr, Beth Jennifer Hoffman, Fredrick Van Goor, and Scott A. Houck
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congenital, hereditary, and neonatal diseases and abnormalities ,Protein Folding ,Cystic Fibrosis ,Protein Conformation ,Regulator ,Mutation, Missense ,Aminopyridines ,Cystic Fibrosis Transmembrane Conductance Regulator ,medicine.disease_cause ,Cystic fibrosis ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,medicine ,Humans ,Benzodioxoles ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Mutation ,biology ,Lumacaftor ,Cell Biology ,Articles ,respiratory system ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,Transmembrane protein ,digestive system diseases ,3. Good health ,Cell biology ,respiratory tract diseases ,Protein Structure, Tertiary ,Biochemistry ,chemistry ,Cell Biology of Disease ,030220 oncology & carcinogenesis ,biology.protein ,Protein folding ,Signal Transduction - Abstract
Misfolding of cystic fibrosis transmembrane conductance regulator protein (CFTR) causes the fatal lung disease cystic fibrosis. VX-809 was developed to suppress disease-related folding defects in CFTR. VX-809 suppresses folding defects in CFTR by modulating the conformation of membrane-spanning domain 1. VX-808 is thereby able to partially restore function to F508del-CFTR and other disease-related mutants., Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.
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- 2013
9. The endoplasmic reticulum–associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3–dependent degradation of nascent CFTRΔF508
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Diane E. Grove, Hong Yu Ren, Douglas M. Cyr, and Chun-Yang Fan
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congenital, hereditary, and neonatal diseases and abnormalities ,Protein Folding ,HSC70 Heat-Shock Proteins ,Ubiquitin-Protein Ligases ,Biosynthesis and Biodegradation ,Cystic Fibrosis Transmembrane Conductance Regulator ,Endoplasmic Reticulum ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Molecular Biology ,030304 developmental biology ,Suppressor mutation ,0303 health sciences ,biology ,Endoplasmic reticulum ,HEK 293 cells ,Cell Biology ,Articles ,HSP40 Heat-Shock Proteins ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Ubiquitin ligase ,HEK293 Cells ,biology.protein ,Protein folding ,030217 neurology & neurosurgery ,Biogenesis - Abstract
A specialized Hsp40 protein, DNAJB12, was found to function on the cytoplasmic face of the ER with the RMA1 E3 ligase to regulate the folding efficiency of CFTR., Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRΔF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRΔF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRΔF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRΔF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRΔF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.
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- 2011
10. Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508
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Diane E. Grove, Meredith F.N. Rosser, Hong Yu Ren, Anjaparavanda P. Naren, and Douglas M. Cyr
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Protein Folding ,Cystic Fibrosis ,Protein Conformation ,Cystic Fibrosis Transmembrane Conductance Regulator ,Endoplasmic Reticulum ,medicine.disease_cause ,Cell Line ,Suppression, Genetic ,Protein structure ,Ubiquitin ,medicine ,Humans ,RNA, Small Interfering ,ΔF508 ,Molecular Biology ,Genetics ,Mutation ,biology ,Protein Stability ,Endoplasmic reticulum ,Articles ,Cell Biology ,Protein Structure, Tertiary ,Ubiquitin ligase ,Cell biology ,Folding (chemistry) ,Thiazoles ,Gene Knockdown Techniques ,biology.protein ,Protein folding - Abstract
Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.
- Published
- 2009
11. The Use of Small Molecules to Correct Defects in CFTR Folding, Maturation, and Channel Activity
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Meredith F.N. Rosser, Douglas M. Cyr, and Diane E. Grove
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congenital, hereditary, and neonatal diseases and abnormalities ,biology ,Chemistry ,Mutant ,Biochemistry (medical) ,Clinical Biochemistry ,Gating ,Apical membrane ,Potentiator ,medicine.disease ,Cystic fibrosis ,Small molecule ,Biochemistry ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,medicine ,biology.protein ,Molecular Biology ,Biogenesis - Abstract
Cystic Fibrosis, one of the most common inherited lethal disease among Caucasians, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR protein acts as a gated Cl - channel at the apical membrane of epithelial cells, thereby facilitating proper hydration of mucosal linings. Disease causing muta- tions in the CFTR protein can affect a variety of steps in the biogenesis of a functional protein including the folding and trafficking of CFTR as well as the channel activity of plasma membrane-localized protein. Therefore, current research is focused on the use of small molecules to not only correct folding defects but also to enhance channel activity of mutant CFTR proteins. This review discusses the current knowledge of the folding, trafficking, and gating defects caused by CFTR mutations, the manner by which these defects are monitored by the cell, as well as the strategies which are cur- rently being utilized to develop and screen for small molecule therapeutics.
- Published
- 2009
12. Effect of Mg2+ on the DNA Binding Modes of the Streptococcus pneumoniae SsbA and SsbB Proteins
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Diane E. Grove and Floyd R. Bryant
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Recombinant Fusion Proteins ,Molecular Sequence Data ,DNA, Single-Stranded ,Cooperativity ,Plasma protein binding ,Biology ,Biochemistry ,Single-stranded binding protein ,chemistry.chemical_compound ,Protein structure ,Tetramer ,Escherichia coli ,Magnesium ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Polyacrylamide gel electrophoresis ,Binding Sites ,Sequence Homology, Amino Acid ,Escherichia coli Proteins ,DNA ,Cell Biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,stomatognathic diseases ,Streptococcus pneumoniae ,chemistry ,biology.protein ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,Protein Binding - Abstract
The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dT(n) oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of Mg2+, the results indicated that the SsbEc protein was able to bind to the dT(n) oligomers in the SSB(35) mode, with only two of the four subunits of the tetramer interacting with the dT(n) oligomers. In the presence of Mg2+, however, the results indicated that the SsbEc protein was bound to the dT(n) oligomers in the SSB(65) mode, with all four subunits of the tetramer interacting with the dT(n) oligomers. The SsbA protein behaved similarly to the SsbEc protein under all conditions, indicating that it undergoes Mg2+ -dependent changes in its DNA binding modes that are analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind to the dT(n) oligomers in an SSB(65)-like mode in either the presence or the absence of Mg2+, suggesting that it may not exhibit the pronounced negative intrasubunit cooperativity in the absence of Mg2+ that is required for the formation of the SSB(35) mode. Additional experiments with a chimeric SsbA/B protein indicated that the structural determinants that govern the transitions between the different DNA binding modes may be contained within the N-terminal domains of the SSB proteins.
- Published
- 2006
13. Expression and purification of the SsbB protein from Streptococcus pneumoniae
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Scott E. Steffen, Diane E. Grove, Floyd R. Bryant, and Mohammad Hedayati
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Expression vector ,Biology ,medicine.disease_cause ,DNA-binding protein ,Molecular biology ,Recombinant Proteins ,law.invention ,Single-stranded binding protein ,DNA-Binding Proteins ,chemistry.chemical_compound ,Streptococcus pneumoniae ,Bacterial Proteins ,chemistry ,law ,Escherichia coli ,medicine ,Recombinant DNA ,biology.protein ,bacteria ,Gene ,Genome, Bacterial ,DNA ,Biotechnology - Abstract
The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.
- Published
- 2005
14. Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein
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Diane E. Grove, Geetha Anne, Mohammad Hedayati, and Floyd R. Bryant
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Biophysics ,medicine.disease_cause ,Biochemistry ,DNA-binding protein ,Cofactor ,Article ,Single-stranded binding protein ,law.invention ,chemistry.chemical_compound ,Bacterial Proteins ,law ,ATP hydrolysis ,Streptococcus pneumoniae ,medicine ,Nucleotide ,Molecular Biology ,chemistry.chemical_classification ,Recombination, Genetic ,biology ,Cell Biology ,DNA Transformation Competence ,DNA-Binding Proteins ,Rec A Recombinases ,chemistry ,biology.protein ,Recombinant DNA ,DNA - Abstract
The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.
- Published
- 2012
15. Analysis of CFTR folding and degradation in transiently transfected cells
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Richard L Watkins, Diane E. Grove, Meredith F.N. Rosser, and Douglas M. Cyr
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Quality Control ,congenital, hereditary, and neonatal diseases and abnormalities ,Protein Folding ,Time Factors ,Ubiquitin-Protein Ligases ,Blotting, Western ,Cystic Fibrosis Transmembrane Conductance Regulator ,Cell Separation ,Endoplasmic Reticulum ,Transfection ,Article ,Humans ,Immunoprecipitation ,RNA, Small Interfering ,Cell Proliferation ,Sequence Deletion ,biology ,Base Sequence ,Endoplasmic reticulum ,HEK 293 cells ,Molecular biology ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Folding (chemistry) ,Blot ,Kinetics ,HEK293 Cells ,Gene Knockdown Techniques ,biology.protein ,Protein folding ,Biogenesis - Abstract
Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.
- Published
- 2011
16. Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2
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Liling Chen, Meredith F.N. Rosser, Diane E. Grove, and Douglas M. Cyr
- Subjects
Models, Molecular ,Protein Folding ,congenital, hereditary, and neonatal diseases and abnormalities ,Calnexin ,Cystic Fibrosis Transmembrane Conductance Regulator ,Biology ,Cell Line ,Protein structure ,Heat shock protein ,Humans ,HSP70 Heat-Shock Proteins ,Molecular Biology ,Sequence Deletion ,Endoplasmic reticulum ,Indolizines ,Cell Biology ,Articles ,respiratory system ,Cystic fibrosis transmembrane conductance regulator ,digestive system diseases ,Cell biology ,Protein Structure, Tertiary ,respiratory tract diseases ,Membrane protein ,Biochemistry ,Ubiquitin ligase complex ,biology.protein ,Protein folding ,Mutant Proteins ,Protein Processing, Post-Translational ,Protein Binding - Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl−channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.
- Published
- 2008
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17. Differential single-stranded DNA binding properties of the paralogous SsbA and SsbB proteins from Streptococcus pneumoniae
- Author
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Diane E. Grove, Floyd R. Bryant, Smaranda Willcox, and Jack D. Griffith
- Subjects
Molecular Sequence Data ,DNA, Single-Stranded ,medicine.disease_cause ,Biochemistry ,Single-stranded binding protein ,chemistry.chemical_compound ,Bacterial Proteins ,Streptococcus pneumoniae ,medicine ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,Peptide sequence ,biology ,Cell Biology ,biology.organism_classification ,Molecular biology ,DNA-Binding Proteins ,Transformation (genetics) ,chemistry ,DNA, Viral ,biology.protein ,Agarose ,DNA ,Bacteria ,Bacteriophage phi X 174 ,Bacteriophage M13 - Abstract
The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB. The SsbA protein is similar in size to the well characterized SSB protein from Escherichia coli (SsbEc). The SsbB protein, in contrast, is a smaller protein that is specifically induced during natural transformation and has no counterpart in E. coli. In this report, the single-stranded DNA (ssDNA) binding properties of the SsbA and SsbB proteins were examined and compared with those of the SsbEc protein. The ssDNA binding characteristics of the SsbA protein were similar to those of the SsbEc protein in every ssDNA binding assay used in this study. The SsbB protein differed from the SsbA and SsbEc proteins, however, both in its binding to short homopolymeric dT(n) oligomers (as judged by polyacrylamide gel-shift assays) and in its binding to the longer naturally occurring X and M13 ssDNAs (as judged by agarose gel-shift assays and electron microscopic analysis). The results indicate that an individual SsbB protein binds to ssDNA with an affinity that is similar or higher than that of the SsbA and SsbEc proteins. However, the manner in which multiple SsbB proteins assemble onto a ssDNA molecule differs from that observed with the SsbA and SsbEc proteins. These results represent the first analysis of paralogous SSB proteins from any bacterial species and provide a foundation for further investigations into the biological roles of these proteins.
- Published
- 2005
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