The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the DeltaF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of 'corrector' drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Acetylharpagide Protects Mice from Staphylococcus Aureus-Induced Acute Lung Injury by Inhibiting NF-κB Signaling Pathway

Staphylococcus aureus (S. aureus)-induced acute lung injury (ALI) is a serious disease that has a high risk of death among infants and teenagers. Acetylharpagide, a natural compound of Ajuga decumbens Thunb. (family Labiatae), has been found to have anti-tumor, anti-inflammatory and anti-viral effects. This study investigates the therapeutic effects of acetylharpagide on S. aureus-induced ALI in mice. Here, we found that acetylharpagide alleviated S. aureus-induced lung pathological morphology damage, protected the pulmonary blood-gas barrier and improved the survival of S. aureus-infected mice. Furthermore, S. aureus-induced myeloperoxidase (MPO) activity of lung homogenate and pro-inflammatory factors in bronchoalveolar lavage (BAL) fluid were suppressed by acetylharpagide. Mechanically, acetylharpagide inhibited the interaction between polyubiquitinated receptor interacting protein 1 (RIP1) and NF-κB essential modulator (NEMO), thereby suppressing NF-κB activity. In summary, these results show that acetylharpagide protects mice from S. aureus-induced ALI by suppressing the NF-κB signaling pathway. Acetylharpagide is expected to become a potential treatment for S. aureus-induced ALI.     

Combined Delivery of a Lipopolysaccharide‐Binding Peptide and the Heme Oxygenase‐1 Gene Using Deoxycholic Acid‐Conjugated Polyethylenimine for the Treatment of Acute Lung Injury

A ternary complex comprising plasmid DNA, lipopolysaccharide‐binding peptide (LBP), and deoxycholic acid‐conjugated polyethylenimine (PEI‐DA) is prepared for combinational therapy of acute lung injury (ALI). The LBP is designed as an anti‐inflammatory peptide based on the lipopolysaccharide (LPS)‐binding domain of HMGB‐1. In vitro cytokine assays show that LBP reduces levels of proinflammatory cytokines by inhibiting LPS. PEI‐DA is synthesized as the gene carrier by conjugation of deoxycholic acid to low‐molecular weight polyethylenimine (2 kDa, PEI2k). PEI‐DA has higher transfection efficiency than high‐molecular weight polyethylenimine (25 kDa, PEI25k). The ternary complex of an HO‐1 plasmid (pHO‐1), PEI‐DA, and LBP is prepared as a combinational system to deliver the therapeutic gene and peptide. The transfection efficiency of the ternary complex is higher than that of the pHO‐1/PEI‐DA binary complex. The ternary complex also reduces TNF‐α secretion in LPS‐activated Raw264.7 macrophage cells. Administration of the ternary complex into the lungs of an animal ALI model by intratracheal injection induces HO‐1 expression and reduces levels of proinflammatory cytokines more efficiently than the pHO‐1/PEI‐DA binary complex or LBP alone. In addition, the ternary complex reduces inflammation in the lungs. Therefore, the pHO‐1/PEI‐DA/LBP ternary complex may be an effective treatment for ALI.

     

Structure-based optimizations of a necroptosis inhibitor (SZM594) as novel protective agents of acute lung injury

Targeting RIPK1 is a promising strategy for the treatment or alleviation of acute lung injury (ALI). SZM594, a benzothiazole compound previously developed by our research group, possessed good dual-targeting receptor-interacting protein kinase 1 (RIPK1) and RIPK3 activity and anti-necroptosis activity as well as acceptable in vivo efficacy. In this study, the cyclopropyl moiety of SZM594 was modified based on a structure-based design strategy. The resulting cyclohexanone-containing analogue 41 improved the selectivity toward RIPK1 over RIPK3 and the anti-necroptosis activity was also increased compared with those of SZM594. More importantly, compound 41 could inhibit the tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-induced peritoneal macrophage cell model, and significantly alleviate LPS-induced ALI in a mouse model. This compound could significantly inhibit the expressions of the phosphorylation of RIPK1 and down-stream RIPK3 and mixed lineage kinase domain-like protein (MLKL). Thus, these cyclohexanone-containing benzothiazole analogues represent promising lead structures for the discovery of novel protective agents of ALI.     

PAK1 as a Potential Therapeutic Target in Male Smokers with EGFR-Mutant Non-Small Cell Lung Cancer

P21-activated kinases (PAKs) are serine/threonine protein kinases that contribute to several cellular processes. Here, we aimed to determine the prognostic value of PAK1 and its correlation with the clinicopathological characteristics and five-year survival rates in patients with non-small cell lung cancer (NSCLC). We evaluated PAK1 mRNA and protein expression in NSCLC cells and resected tumor specimens, as well as in healthy human bronchial epithelial cells and adjacent healthy lung tissues, respectively, for effective comparison. Immunohistochemical tissue microarray analysis of 201 NSCLC specimens showed the correlation of PAK1 expression with clinicopathological characteristics. The mRNA and protein expression of PAK1 were 2.9- and 4.3-fold higher in six of seven NSCLC cell types and human tumors (both, p < 0.001) than in healthy human bronchial epithelial BEAS-2B cells and adjacent healthy lung tissues, respectively. Decreased survival was significantly associated with PAK1 overexpression in the entire cohort (χ² = 8.48, p = 0.0036), men (χ² = 17.1, p < 0.0001), and current and former smokers (χ² = 19.2, p < 0.0001). Notably, epidermal growth factor receptor (EGFR) mutation-positive lung cancer patients with high PAK1 expression showed higher mortality rates than those with low PAK1 expression (91.3% vs. 62.5%, p = 0.02). Therefore, PAK1 overexpression could serve as a molecular target for the treatment of EGFR mutation-positive lung cancer, especially among male patients and current/former smokers.     

Inhibition of <em>Burkholderia multivorans</em> Adhesion to Lung Epithelial Cells by Bivalent Lactosides

Burkholderia cepacia complex (Bcc) is an opportunistic pathogen in cystic fibrosis patients which is inherently resistant to antimicrobial agents. The mechanisms of attachment and pathogenesis of Bcc, a group of 17 species, are poorly understood. The most commonly identified Bcc species in newly colonised patients, Burkholderia multivorans, continues to be acquired from the environment. Development of therapies which can prevent or reduce the risk of colonization on exposure to Bcc in the environment would be a better alternative to antimicrobial agents. Previously, it has been shown that Bcc strains bound to many glycolipid receptors on lung epithelia. Using a real-time PCR method to quantify the levels of binding of B. multivorans to the lung epithelial cells, we have examined glycoconjugate derivatives for their potential to inhibit host cell attachment. Bivalent lactosides previously shown to inhibit galectin binding significantly reduced the attachment of B. multivorans to CF lung epithelial cells at micromolar concentrations. This was in contrast to monosaccharides and lactose, which were only effective in the millimolar range. Development of glycoconjugate therapies such as these, which inhibit attachment to lung epithelial cells, represent an alternative means of preventing infection with inherently antimicrobially resistant pathogens such as B. multivorans.     

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages,HCl/EtOH-induced Gastritis,and LPS-induced Lung Injury via Attenuation of Src and TAK1

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE₂, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.     

8-O-(E-p-methoxycinnamoyl)harpagide Inhibits Influenza A Virus Infection by Suppressing Intracellular Calcium

Calcium (Ca²⁺) dependent signaling circuit plays a critical role in influenza A virus (IAV) infection. The 8-O-(E-p-methoxycinnamoyl)harpagide (MCH) exhibits pharmacological activities that exert neuroprotective, hepatoprotective, anti-inflammatory and other biological effects. However, not have reports of antiviral effects. To investigate the antiviral activity of MCH on IAV-infected human lung cells mediated by calcium regulation. We examined the inhibitory effect of MCH on IAV infections and measured the level of viral proteins upon MCH treatment using Western blotting. We also performed molecular docking simulation with MCH and IAV M2 protein. Finally, we analyzed MCH’s suppression of intracellular calcium and ROS (reactive oxygen species) in IAV-infected human lung cells using a flow cytometer. The results shown that MCH inhibited the infection of IAV and increased the survival of the infected human lung cells. The levels of IAV protein M1, M2, NS1 and PA were inhibited in MCH-treated human lung cells compared to that in infected and untreated cells. Also, docking simulation suggest that MCH interacted with M2 on its hydrophobic wall (L40 and I42) and polar amino acids (D44 and R45), which formed intermolecular contacts and were a crucial part of the channel gate along with W41. Lastly, MCH inhibited IAV infection by reducing intracellular calcium and mitochondrial Ca²⁺/ROS levels in infected human lung cells. Taken together, these data suggest that MCH inhibits IAV infection and increases the survival of infected human lung cells by suppressing calcium levels. These results indicate that MCH is useful for developing IAV treatments.     

Identification of potential lung cancer biomarkers using an in vitro carcinogenesis model

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799) all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.     

The Role of ΔFosB on the Pro-survival Effect of PTHrP in Goat Mammary Epithelial Cells

The mechanism of regulation mammary epithelial cell number in ruminant is not fully understood, but is thought to be dependent on the balance of cell proliferation and cell apoptosis. Parathyroid hormone-related protein (PTHrP) could express in mammary epithelial cells and breast cancer cells, and has been reported to regulate cell survival. Here, we showed that PTHrP induced cell proliferation and increased the expression of CyclinD1 and proliferating cell nuclear antigen (PCNA) in goat mammary epithelial cells (GMEC). PTHrP increased the mRNA levels of anti-apoptosis genes Bcl-2 and Bcl-xl, and protected GMEC from apoptosis. We also found ΔFosB, an alternative splicing of finkel-biskis-jinkins murine osteosarcoma B (fosB), inhibited GMEC apoptosis, and induced cell proliferation with increased Bcl-2/Bax and Bcl-xl/Bax ratios. Interestingly, ΔFosB could further promote the pro-survival effect of PTHrP, and the Bcl-2/Bax and Bcl-xl/Bax ratios showed higher levels. We conclude that the pro-survival role of PTHrP in GMEC may be regulated by ΔFosB.     

PMA-induced up-regulation of MMP-9 is regulated by a PKCalpha-NF-kappaB cascade in human lung epithelial cells

Expression of matrix metalloproteinase-9 (MMP-9) is associated with airway remodeling and tissue injury in asthma. However, little is known about how MMP-9 is up-regulated in airway epithelial cells. In this study, we show that phorbol myristate acetate (PMA) induces MMP-9 expression via a protein kinase Calpha (PKCalpha)-dependent signaling cascade in BEAS-2B human lung epithelial cells. Pretreatment with either GF109203X, a general PKC inhibitor, or Go6976, a PKCalpha/beta isozyme inhibitor, inhibited PMA-induced activation of the MMP-9 promoter, as did transient transfection with PKCalpha antisense oligonuclotides. PMA activated NF-kappaB by phosphorylating IkappaB in these cells and this was also inhibited by GF109203X and Go6976, suggesting that PKCa acts as an upstream regulator of NF-kappaB in PMA-induced MMP-9 induction. Our results indicate that a "PKCalpha-NF- kappaB"-dependent cascade is involved in the signaling leading to PMA-induced MMP-9 expression in the lung epithelium.     

S100A9 in adult asthmatic patients: a biomarker for neutrophilic asthma

The biomarkers and therapeutic targets of neutrophilic asthma (NA) are poorly understood. Although S100 calcium-binding protein A9 (S100A9) has been shown to correlate with neutrophil activation, its role in asthma pathogenesis has not been clarified. This study investigated the mechanism by which S100A9 is involved in neutrophil activation, neutrophil extracellular trap (NET)-induced airway inflammation, and macrophage polarization in NA. The S100A9 levels (by ELISA) in sera/culture supernatant of peripheral blood neutrophils (PBNs) and M0 macrophages from asthmatic patients were measured and compared to those of healthy controls (HCs). The function of S100A9 was evaluated using airway epithelial cells (AECs) and PBNs/M0 macrophages from asthmatic patients, as well as a mouse asthma model. The serum levels of S100A9 were higher in NA patients than in non-NA patients, and there was a positive correlation between serum S100A9 levels and sputum neutrophil counts (r = 0.340, P = 0.005). Asthmatic patients with higher S100A9 levels had lower PC₂₀ methacholine values and a higher prevalence of severe asthma (SA) (P < .050). PBNs/M0 macrophages from SA released more S100A9 than those from non-SA patients. PBNs from asthmatic patients induced S100A9 production by AECs, which further activated AECs via the extracellular signal-regulated kinase (ERK) pathway, stimulated NET formation, and induced M1 macrophage polarization. Higher S100A9 levels in sera, bronchoalveolar lavage fluid, and lung tissues were observed in the mouse model of NA but not in the other mouse models. These results suggest that S100A9 is a potential serum biomarker and therapeutic target for NA.Subject terms: Prognostic markers, Adaptive immunity, Immunological disorders, Diagnostic markers     

Activation of CFTR-mediated Cl- Transport by Magnolin

Magnolin is a herbal compound from Magnolia biondii Pamp. It possesses numerous biological activities. Cystic fibrosis transmembrane conductance regulator（CFTR） is an epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs. In the present study, the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized. In CFTR stably transfected FRT cells, magnolin increases CFTR CI- currents in a concentration-dependent manner. The activation of magnolin on CFTR is rapid, reversible, and cAMP-dependent. Magnolin does not elevate cellular cAMP level, indicating that it activates CFTR by direct binding and interaction with CFTR protein. Magnolin selectively activates wildtype CFTR rather than mutant CFTR. Magnolin may present a novel class of therapeutic lead compound tbr the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca, idiopathic chronic pancreatiti, and chronic constipation.     

Effects of a natural prolyl oligopeptidase inhibitor, rosmarinic acid, on lipopolysaccharide-induced acute lung injury in mice

Rosmarinic acid (RA), a polyphenolic phytochemical, is a natural prolyl oligopeptidase inhibitor. In the present study, we found that RA exerted potent anti-inflammatory effects in in vivo models of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Mice were pretreated with RA one hour before challenge with a dose of 0.5 mg/kg LPS. Twenty-four hours after LPS was given, bronchoalveolar lavage fluid (BALF) was obtained to measure pro-inflammatory mediator and total cell counts. RA significantly decreased the production of LPS-induced TNF-a, IL-6, and IL-1β compare with the LPS group. When pretreated with RA (5, 10, or 20 mg/kg) the lung wet-to-dry weight (W/D) ratio of the lung tissue and the number of total cells, neutrophils and macrophages in the BALF were decreased significantly. Furthermore, RA may enhance oxidase dimutase (SOD) activity during the inflammatory response to LPS-induced ALI. And we further demonstrated that RA exerts anti-inflammation effect in vivo models of ALI through suppresses ERK/MAPK signaling in a dose dependent manner. These studies have important implications for RA administration as a potential treatment for ALI.     

β-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell–cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. β-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether β-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. β-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, β-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.     

Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.