ABSTRACT
The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid ß-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid ß-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.
Subject(s)
Idiopathic Pulmonary Fibrosis , Animals , Apoptosis , Bleomycin , Fibrosis , Humans , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/metabolism , Macrophages, Alveolar , MiceABSTRACT
Pulmonary fibrosis is a progressive lung disease often occurring secondary to environmental exposure. Asbestos exposure is an important environmental mediator of lung fibrosis and remains a significant cause of disease despite strict regulations to limit exposure. Lung macrophages play an integral role in the pathogenesis of fibrosis induced by asbestos (asbestosis), in part by generating reactive oxygen species (ROS) and promoting resistance to apoptosis. However, the mechanism by which macrophages acquire apoptosis resistance is not known. Here, we confirm that macrophages isolated from asbestosis subjects are resistant to apoptosis and show they are associated with enhanced mitochondrial content of NADPH oxidase 4 (NOX4), which generates mitochondrial ROS generation. Similar results were seen in chrysotile-exposed WT mice, while macrophages from Nox4-/- mice showed increased apoptosis. NOX4 regulated apoptosis resistance by activating Akt1-mediated Bcl-2-associated death phosphorylation. Demonstrating the importance of NOX4-mediated apoptosis resistance in fibrotic remodeling, mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages exhibited increased apoptosis and were protected from pulmonary fibrosis. Moreover, resolution occurred when Nox4 was deleted in monocyte-derived macrophages in mice with established fibrosis. These observations suggest that NOX4 regulates apoptosis resistance in monocyte-derived macrophages and contributes to the pathogenesis of pulmonary fibrosis. Targeting NOX4-mediated apoptosis resistance in monocyte-derived macrophages may provide a novel therapeutic target to protect against the development and/or progression of pulmonary fibrosis.
Subject(s)
Apoptosis , Disease Progression , Idiopathic Pulmonary Fibrosis/enzymology , Idiopathic Pulmonary Fibrosis/pathology , Macrophages/enzymology , Macrophages/pathology , NADPH Oxidase 4/metabolism , Animals , Cell Line , Female , Lung/pathology , Male , Mice, Inbred C57BL , Mitochondria/metabolism , Models, Biological , Monocytes/metabolism , Phosphorylation , Reactive Oxygen Species/metabolism , bcl-Associated Death Protein/metabolismABSTRACT
A functional polyurethane based on the heterocyclic group was synthesized and its self-healing and mechanical properties were examined. To synthesize a heterocyclic polyurethane, a polyol and a heterocyclic compound with di-hydroxyl groups at both ends were blended and the blended solution was reacted with a crosslinker containing multiple isocyanate groups. The heterocyclic polyurethane demonstrates better self-healing efficiency than the conventional polyurethane with no heterocyclic groups. Furthermore, unlike the conventional self-healing materials, the heterocyclic polyurethane examined in this study shows an outstanding recovery of the mechanical properties after the self-healing process. These results are attributed to the unique supramolecular network resulting from the strong hydrogen bonding interaction between the urethane group and the heterocyclic group in the heterocyclic polyurethane matrix.
ABSTRACT
Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full-length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full-length murine cDNA produces a > 250 kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1-/- cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant-directed therapeutics.
Subject(s)
Genetic Variation/genetics , Neurofibromatosis 1/genetics , Neurofibromin 1/genetics , Structure-Activity Relationship , Animals , Cell Line , DNA, Complementary/genetics , Gene Expression Regulation/genetics , Gene Knockdown Techniques , Genetic Testing , Humans , Mice , Mutation/genetics , Neurofibromatosis 1/diagnosis , Neurofibromatosis 1/pathology , Signal Transduction/geneticsABSTRACT
The genomic RNA of negative-strand RNA viruses, such as vesicular stomatitis virus (VSV), is completely enwrapped by the nucleocapsid protein (N) in every stage of virus infection. During viral transcription/replication, however, the genomic RNA in the nucleocapsid must be accessible by the virus-encoded RNA-dependent RNA polymerase in order to serve as the template for RNA synthesis. With the VSV nucleocapsid and a nucleocapsid-like particle (NLP) produced in Escherichia coli, we have found that the RNA in the VSV nucleocapsid can be removed by RNase A, in contrast to what was previously reported. Removal of the RNA did not disrupt the assembly of the N protein, resulting in an empty capsid. Polyribonucleotides were reencapsidated into the empty NLP, and the crystal structures were determined. The crystal structures revealed variable degrees of association of the N protein with a specific RNA sequence.
Subject(s)
Nucleocapsid/physiology , RNA, Viral/metabolism , Vesiculovirus/physiology , Escherichia coli , Nucleocapsid Proteins/metabolism , Protein Binding , Recombinant Proteins/metabolism , Ribonuclease, Pancreatic/metabolismABSTRACT
The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was coexpressed with the phosphoprotein (P) in Escherichia coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsidlike ring that has a diameter of approximately 20 nm with 13 subunits. There is a piece of single-stranded RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also visible. The MuV NP protein may provide weaker protection of the RNA than the NP protein of some other negative-strand RNA viruses, reflecting the degree of NP protein association.
