Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Hybrid Structure of the RagA/C-Ragulator mTORC1 Activation Complex.

The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity.

Effect of complicated, untreated and uncontrolled diabetes and pre-diabetes on treatment outcome among patients with pulmonary tuberculosis.

BACKGROUND AND OBJECTIVE: Patients with tuberculosis and diabetes have a higher risk of unfavourable anti-tuberculosis treatment outcomes. In the present study, we aimed to evaluate the effects of various diabetes statuses on the outcomes of patients with pulmonary tuberculosis. METHODS: Among the patients with pulmonary tuberculosis enrolled in the Korea Tuberculosis Cohort (KTBC) registry and the multicentre prospective cohort study of pulmonary tuberculosis (COSMOTB), those with diabetes and complicated diabetes were identified. The primary and secondary outcomes were unfavourable outcomes and mortality, respectively. The effect of diabetes and complicated diabetes on the outcomes was assessed using multivariable logistic regression analysis. Using COSMOTB, subgroup analyses were performed to assess the association between various diabetes statuses and outcomes. RESULTS: In the KTBC, diabetes (adjusted odds ratio [aOR] = 1.93, 95% CI = 1.64-2.26) and complicated diabetes (aOR = 1.96, 95% CI = 1.67-2.30) were significantly associated with unfavourable outcomes, consistent with the COSMOTB data analysis. Based on subgroup analysis, untreated diabetes at baseline was an independent risk factor for unfavourable outcomes (aOR = 2.72, 95% CI = 1.26-5.61). Prediabetes and uncontrolled diabetes increased unfavourable outcomes and mortality without statistical significance. CONCLUSION: Untreated and complicated diabetes at the time of tuberculosis diagnosis increases the risk of unfavourable outcomes and mortality.

IL-37 Attenuates Lung Fibrosis by Inducing Autophagy and Regulating TGF-ß1 Production in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive and destructive lung disease with a poor prognosis resulting in a high mortality rate. IL-37 is an anti-inflammatory cytokine that inhibits innate and adaptive immunity by downregulating proinflammatory mediators and pathways. However, the exact role of IL-37 in lung fibrosis is unclear. In this study, we found that the IL-37 protein was expressed in alveolar epithelial cells (AECs) and alveolar macrophages in healthy controls but significantly reduced in patients with IPF. IL-37 significantly inhibited oxidative stress-induced primary mouse AEC death in a dose-dependent manner, and knockdown of IL-37 significantly potentiated human lung cancer-derived AEC (A549 cells) death. IL-37 attenuated constitutive mRNA and protein expression of fibronectin and collagen I in primary human lung fibroblasts. IL-37 inhibited TGF-ß1-induced lung fibroblast proliferation and downregulated the TGF-ß1 signaling pathway. Moreover, IL-37 enhanced beclin-1-dependent autophagy and autophagy modulators in IPF fibroblasts. IL-37 significantly decreased inflammation and collagen deposition in bleomycin-exposed mouse lungs, which was reversed by treatment with the autophagy inhibitor 3-methyladenine. Our findings suggested that a decrease in IL-37 may be involved in the progression of IPF and that IL-37 inhibited TGF-ß1 signaling and enhancement of autophagy in IPF fibroblasts. Given its antifibrotic activity, IL-37 could be a therapeutic target in fibrotic lung diseases, including IPF.

Impact of capsaicin concentration evoking coughs on clinical variables in patients with asthma.

BACKGROUND: Inhaled capsaicin (8-methyl-N-vanillyl-6-nonenamide) has been used to induce cough in a safe and dose-dependent manner. Chronic cough is associated with an increased sensitivity to inhaled capsaicin in patients with asthma. The aim of this study was to evaluate clinical impact of capsaicin provocation test for chronic cough, and to find relationship between capsaicin concentration producing coughs and clinical variables in patients with asthma. Methods: 385 patients with chronic cough [capsaicin provocation test (+, n = 152)] vs. [capsaicin provocation test (-, n = 233)] who has done with capsaicin provocation test recruited and evaluated by asthma diagnosis and clinical variables. Asthma diagnoses were based on the Global Initiative for Asthma guidelines. Results: Capsaicin positivity was more prevalent in patient with asthma diagnosis than in patients without asthma diagnosis (129/304 vs. 24/81, p = 0.037). Capsaicin positivity was more prevalent in female patients than in male patients (123/271 = 45.4% vs. 30/114 = 26.3%, p = 0.001). Capsaicin concentration producing coughs correlated with smoke amount (r = 0.126, p = 0.014). Capsaicin positivity was more prevalent in nonsmoker patients than in smoker patients (133/295 = 45.1% vs. 20/90 = 22.2%, p = 0.001). Capsaicin concentration producing coughs negatively correlated with methacholine PC20 (4 mg mL-1, p = 0.037), (16 mg mL-1, p = 0.069) and (20 mg mL-1, p = 0.045). Capsaicin concentration producing coughs correlated with BMI (r = 0.120, p = 0.019). Capsaicin concentration producing coughs negatively correlated with FEV1/FVC % pred. (r = -0.137, p = 0.007). There was no relationship between capsaicin concentration producing coughs and age, IgE, and atopy. Conclusions: Capsaicin test for asthma diagnosis should be considered for variable clinical factors. Key message Cough in asthmatic patients is not only common and troublesome but also predicts disease severity and poor prognosis. The capsaicin cough challenge test is a simple and reproducible provocation method for assessing cough susceptibility in patients with cough. Capsaicin test for asthma diagnosis should be considered for variable clinical factors.

Validation of Previous Spirometric Reference Equations and New Equations.

BACKGROUND: Pulmonary functions are interpreted using predicted values from reference equations that vary with ethnicity, gender, age, height, and weight. The universally used Choi's reference equations are not validated for Korean populations, and the purpose of this study was to validate them and develop new reference equations. METHODS: Subjects with normal spirometry and chest radiographs, no co-morbidities, and non-smokers, from the Korean National Health and National Examination Survey (KNHANES)-VI were enrolled (n = 117). Intraclass correlation coefficient (ICC) was assessed for reliability of reference equations. New reference equations were developed using linear regression analysis. Differences between observed and predicted values were assessed to compare the reference equations from Choi's, Global Lung Function Initiative 2012, KNHANES-IV, and newly developed equations. RESULTS: The ICC of Choi's reference equations was 0.854 (P < 0.001). The new reference equations for men were: forced vital capacity (FVC) (L) = - 4.38775 - 0.01184 × age + 0.05547 × height, forced expiratory volume - 1 second (FEV1) (L) = - 2.40147 - 0.02134 × age + 0.04103 × height; and for women: FVC (L) = - 3.09063 + 0.003904 × age + 0.038694 × height; FEV1 (L) = - 1.32933 - 0.00872 × age + 0.02762 × height. The differences between the predicted and observed means were largest in Choi's equations, but lowest in the new equations with highest goodness of fit. CONCLUSION: Because Choi's reference equations presented larger differences from the observed values, despite reliability, and the new reference equations showed better goodness of fit, we suggest the latter for Korean populations.

Involvement of Alveolar Epithelial Cell Necroptosis in Idiopathic Pulmonary Fibrosis Pathogenesis.

Alveolar epithelial cell (AEC) injury leading to cell death is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF). Among regulated/programmed cell death, the excessive apoptosis of AECs has been widely implicated in IPF pathogenesis. Necroptosis is a type of regulated/programmed necrosis. A multiprotein complex composed of receptor-interacting protein kinase (RIPK)-1 and -3 plays a key regulatory role in initiating necroptosis. Although necroptosis participates in disease pathogeneses through the release of damage-associated molecular patterns, its association with IPF progression remains elusive. In this study, we attempted to illuminate the involvement of RIPK3-regulated necroptosis in IPF pathogenesis. IPF lung tissues were used to detect necroptosis, and the role of RIPK3 was determined using cell culturing models of AECs. Lung fibrosis models of bleomycin (BLM) treatment were also used. RIPK3 expression levels were increased in IPF lungs, and both apoptosis and necroptosis were detected mainly in AECs. Necrostatin-1 and RIPK3 knockout experiments in AECs revealed the participation of necroptosis in BLM and hydrogen peroxide-induced cell death. BLM treatment induced RIPK3 expression in AECs and increased high-mobility group box 1 and IL-1ß levels in mouse lungs. The efficient attenuation of BLM-induced lung inflammation and fibrosis was determined in RIPK3 knockout mice and by necrostatin-1 with a concomitant reduction in high-mobility group box 1 and IL-1ß. RIPK3-regulated necroptosis in AECs is involved in the mechanism of lung fibrosis development through the release of damage-associated molecular patterns as part of the pathogenic sequence of IPF.

Annexin A1 in plasma from patients with bronchial asthma: its association with lung function.

BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = - 0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.

Efficacy and safety of aprepitant for the prevention of chemotherapy-induced nausea and vomiting during the first cycle of moderately emetogenic chemotherapy in Korean patients with a broad range of tumor types.

PURPOSE: This study evaluated the efficacy and safety of a 3-day aprepitant regimen for the prevention of chemotherapy-induced nausea and vomiting (CINV) during the first cycle of non-anthracycline plus cyclophosphamide (AC)-based moderately emetogenic chemotherapy (MEC) based on government guidelines in Korean patients. METHODS: This multicenter, randomized, double-blind, phase IV trial (NCT01636947) enrolled adult South Korean patients with a broad range of tumor types who were scheduled to receive a single dose of ≥1 MEC agent. Patients were randomized to a 3-day regimen of aprepitant (aprepitant regimen) or placebo (control regimen) on top of ondansetron plus dexamethasone. The primary and key secondary efficacy endpoints were the proportions of subjects who achieved no vomiting and complete response (CR) during the overall phase. RESULTS: Of the 494 randomized subjects, 480 were included in the modified intent-to-treat population. Response rates for no vomiting and CR in the overall phase were numerically higher for the aprepitant regimen compared with the control regimen groups, but failed to reach statistical significance (no vomiting 77.2 vs 72.0%; p = 0.191; CR 73.4 vs 70.4%; p = 0.458). Both the aprepitant and control regimens were generally well tolerated. CONCLUSION: A 3-day aprepitant regimen was numerically better but not statistically superior to a control regimen with respect to the achievement of no vomiting or CR during the overall phase in a non-AC MEC Korean population based on government reimbursement guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT01636947 ( https://clinicaltrials.Gov/ct2/show/NCT01636947 ).

Assembly and dynamics of the autophagy-initiating Atg1 complex.

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17-Atg31-Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen-deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼ 100-nM affinity. The Atg1-Atg13 complex in turn binds as a unit to the Atg17-Atg31-Atg29 scaffold with ∼ 10-µM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.

Attenuation of Cigarette Smoke-Induced Emphysema in Mice by Apolipoprotein A-1 Overexpression.

Chronic inflammation, oxidative stress, and proteolysis participate primarily in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. COPD is a highly prevalent smoking-related disease for which no effective therapy exists to improve the disease course. Although apolipoprotein A-1 (ApoA1) has antiinflammatory and antioxidant properties as well as cholesterol efflux potential, its role in cigarette smoke (CS)-induced emphysema has not been determined. Therefore, we investigated whether human ApoA1 transgenic (TG) mice, with conditionally induced alveolar epithelium to overexpress ApoA1, are protected against the CS-induced lung inflammatory response and development of emphysema. In this study, ApoA1 levels were significantly decreased in the lungs of patients with COPD and in the lungs of mice exposed to CS. ApoA1 TG mice did not develop emphysema when chronically exposed to CS. Compared with the control TG mice, ApoA1 overexpression attenuated lung inflammation, oxidative stress, metalloprotease activation, and apoptosis in CS-exposed mouse lungs. To explore a plausible mechanism of antiapoptotic activity of ApoA1, alveolar epithelial cells (A549) were treated with CS extract (CSE). ApoA1 prevented CSE-induced translocation of Fas and downstream death-inducing signaling complex into lipid rafts, thereby inhibiting Fas-mediated apoptosis. Taken together, the data showed that ApoA1 overexpression attenuated CS-induced lung inflammation and emphysema in mice. Augmentation of ApoA1 in the lung may have therapeutic potential in preventing smoking-related COPD/emphysema.

Metabolic Profiling Regarding Pathogenesis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, eventually fatal disease characterized by fibrosis of the lung parenchyma and loss of lung function. IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair process including uncontrolled proliferation of lung (myo) fibroblasts and excessive deposition of extracellular matrix proteins in the interstitial space; however, the pathogenic pathways involved in IPF have not been fully elucidated. In this study, we attempted to characterize metabolic changes of lung tissues involved in the pathogenesis of IPF using gas chromatography-mass spectrometry-based metabolic profiling. Partial least-squares discriminant analysis (PLS-DA) model generated from metabolite data was able to discriminate between the control subjects and IPF patients (R(2)X = 0.37, R(2)Y = 0.613 and Q(2) (cumulative) = 0.54, receiver operator characteristic AUC > 0.9). We discovered 25 metabolite signatures of IPF using both univariate and multivariate statistical analyses (FDR < 0.05 and VIP score of PLS-DA > 1). These metabolite signatures indicated alteration in metabolic pathways: adenosine triphosphate degradation pathway, glycolysis pathway, glutathione biosynthesis pathway, and ornithine aminotransferase pathway. The results could provide additional insight into understanding the disease and potential for developing biomarkers.

Circulating angiopoietin-1 and -2 in patients with stable and exacerbated asthma.

BACKGROUND: Angiopoietin (Ang)-1 and -2 are involved in the pathogenesis of asthma and have been identified as markers of asthma severity. OBJECTIVE: To determine the relation between circulating angiopoietins and clinical variables of patients with asthma. METHODS: Fifty patients with bronchial asthma and 25 healthy controls were enrolled. Ang1 and Ang2 plasma levels were analyzed in patients with stable and exacerbated asthma. RESULTS: Plasma Ang1 levels were 28.4 ± 4.01 pg/mg in patients with bronchial asthma and 21.2 ± 5.21 pg/mg in healthy controls. Plasma Ang2 levels were 23.96 ± 1.38 pg/mg in patients with bronchial asthma compared with 36.8 ± 4.46 pg/mg in healthy controls (P = .010). The ratio of Ang2 to Ang1 was lower in patients with asthma than in control subjects. Plasma Ang1 concentrations were correlated with the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC), and plasma Ang2 levels were correlated with FEV1 percentage of predicted, FEV1/FVC, and total immunoglobulin E values. The ratio of Ang2 to Ang1 was correlated with FEV1 percentage of predicted and FEV1/FVC. Although plasma Ang1 levels tended to be lower in the exacerbated state than in the stable state in patients with asthma, Ang2 levels were higher in the exacerbated state than in the stable state in patients with asthma (P = .001). Plasma Ang2 levels were correlated with initial eosinophil proportions and initial neutrophil proportions. Plasma Ang2 levels and the ratio of Ang2 to Ang1 were correlated with blood eosinophil proportions in the exacerbated state. CONCLUSION: These results indicate that circulating angiopoietins could be a useful marker of asthma exacerbation.

Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from Arabidopsis thaliana in complex with AMP.

Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/ß overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members.

Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N(ε)-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N(α)-acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.

Crystal structure of JHP933 from Helicobacter pylori J99 shows two-domain architecture with a DUF1814 family nucleotidyltransferase domain and a helical bundle domain.

The jhp0933 gene in the plasticity region of Helicobacter pylori J99 encodes a hypothetical protein (JHP933), which may play some roles in pathogenesis. Here, we have determined the crystal structure of JHP933 at 2.17 Å. It represents the first crystal structure of the DUF1814 protein family. JHP933 consists of two domains: an N-terminal domain of the nucleotidyltransferase (NTase) fold and a C-terminal helix bundle domain. A highly positively charged surface patch exists adjacent to the putative NTP binding site. Structural similarity of JHP933 to known NTases is very remote, suggesting that it may function as a novel NTase.

Association analysis of melanocortin 3 receptor polymorphisms with the risk of pulmonary tuberculosis.

PURPOSE: Melanocortin 3 Receptor (MC3R) is one of the families of seven-transmembrane G-protein-coupled receptors, and a recent study showed that MCR3 promoter polymorphism was significantly associated with the susceptibility of tuberculosis (TB) in South African population. METHODS: We analyzed six MC3R polymorphisms to examine the genetic effects on the risk of pulmonary TB in Korean subjects by using TaqMan assays and case-control analyses. RESULTS: Using statistical analyses, one common promoter polymorphism (MC3R rs11575886 T > C) was found to be associated with an increased risk of pulmonary TB. The frequency of the C-bearing genotype of rs11575886 was higher in pulmonary TB patients than in normal controls (p = 0.03, OR = 1.46) although the significance was not retained after correction. In silico analysis for the difference of transcription binding factor (TF), motif between C and T allele demonstrated that the TF motif and its threshold scores of C allele were lower than those of T allele. CONCLUSIONS: The C allele of rs11575886 could be a risk allele for the pulmonary TB by affecting the binding of TF. Our findings suggest that polymorphisms in MC3R might be one of genetic factors for the risk of pulmonary TB development in Korean subjects.

Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-ß1 activity.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling and the excessive accumulation of extracellular matrix (ECM) proteins. In a previous study, we found that the levels of ornithine aminotransferase (OAT), a principal enzyme in the proline metabolism pathway, were increased in the lungs of patients with IPF. However, the precise role played by OAT in the pathogenesis of IPF is not yet clear. The mechanism by which OAT affects fibrogenesis was assessed in vitro using OAT-overexpressing and OAT-knockdown lung fibroblasts. The therapeutic effects of OAT inhibition were assessed in the lungs of bleomycin-treated mice. OAT expression was increased in fibrotic areas, principally in interstitial fibroblasts, of lungs affected by IPF. OAT levels in the bronchoalveolar lavage fluid of IPF patients were inversely correlated with lung function. The survival rate was significantly lower in the group with an OAT level >75.659 ng/mL than in the group with an OAT level ≤75.659 ng/mL (HR, 29.53; p = 0.0008). OAT overexpression and knockdown increased and decreased ECM component production by lung fibroblasts, respectively. OAT knockdown also inhibited transforming growth factor-ß1 (TGF)-ß1 activity and TGF-ß1 pathway signaling. OAT overexpression increased the generation of mitochondrial reactive oxygen species (ROS) by activating proline dehydrogenase. The OAT inhibitor L-canaline significantly attenuated bleomycin-induced lung injury and fibrosis. In conclusion, increased OAT levels in lungs affected by IPF contribute to the progression of fibrosis by promoting excessive mitochondrial ROS production, which in turn activates TGF-ß1 signaling. OAT may be a useful target for treating patients with fibrotic lung diseases, including IPF.

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ-like exonuclease domain-containing protein with homohexameric assembly.

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ-like 3'-5' exonuclease domain-containing protein (AtDECP). The DnaQ-like 3'-5' exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed ß-sheet flanked by α-helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring-shaped structure and comparison with WRN-exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N-terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ-like 3'-5' exonuclease and its substrate nucleic acids.