Protein quality control in lung disease: it's all about cloud networking.

Protein quality control involves the comprehensive management of protein function in the cell and is called “proteostasis” [1]. It ranges from translation and chaperone-assisted three-dimensional folding, interaction with protein partners, signal-induced post-translational modifications to disposal by the proteasome or autophagy pathways. Dysfunctional protein quality control is emerging as a key pathogenic mechanism for chronic lung diseases. Two major hereditary conformational disorders of the lung, cystic fibrosis and α1-antitrypsin (α1-AT) deficiency, and some familial forms of idiopathic pulmonary fibrosis (IPF) are caused by the expression of mutant and misfolded proteins that disrupt protein homeostasis and drive the onset of pulmonary diseases [2, 3]. Disturbed proteostasis also causes sporadic respiratory diseases [1, 4]. Cigarette smoke-induced protein misfolding, aberrant proteasomal protein degradation and induction of autophagy have been observed in chronic obstructive pulmonary disease (COPD) patients and smoke-exposed mice [4, 5]. Dysregulation of autophagy and endoplasmic reticulum (ER) stress have also been implicated in cystic fibrosis, pulmonary arterial hypertension, IPF and other lung diseases [6, 7]. Impairment of protein quality control pathways exacerbates the detrimental effects of environmentally induced protein damage in lung pathogenesis [1]. The European Respiratory Society (ERS) research seminar Protein Quality Control in Lung Disease, held on March 1–2, 2014, at Lake Starnberg in Germany, brought together international experts to develop a comprehensive view of protein quality control in general and in the lung in particular. Understanding the complex interplay of protein misfolding, ER homeostasis and protein degradation as interrelated components of adaptive proteostasis will identify novel therapeutic targets for treatment of pulmonary diseases, as outlined here.</p

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events

Toxicity and inhibitory profile of bortezomib and oprozomib in alveolar epithelial cells.

<p>MTT assay after 72 hours of treatment with (A) BZ or (B) OZ (Data represent mean ± SEM. <i>n</i> = 3 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Proteasome activity 24 hours after treatment with BZ or (D) OZ (Data represent mean ± SEM. <i>n</i> = 3 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (E) and (F) MTT assay of primary murine ATII cells after 52 hours of treatment with OZ or BZ (Data represent mean ± SEM. <i>n</i> = 4 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test).</p

Inhibition profile of oprozomib in primary mouse lung fibroblasts.

<p>(A) Proteasome activity and (B) Luciferase activity of ODD-Luc FVB-LF 24 hours after treatment with OZ (Data represent mean ± SEM. <i>n</i> = 3 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Native gel of ODD-Luc FVB-LF 24 hours after OZ treatment.</p

Dose response to local pulmonary application of 0.5, 1, and 5 mg/kg OZ or Pluronic F-127 0.1% solvent control after 24 hours or 96 hours.

<p>(A) Treatment scheme: local pulmonary application of OZ, (B) CT-L proteasome activity after 24 hours, (C) percent of PMNs to total BAL count after 24 hours, (D) CT-L proteasome activity after 96 hours, and (E) percent of PMNs to total BAL count after 96 hours (Data represent mean ± SEM. <i>n</i> = 5 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test).</p

Antifibrotic effects of oprozomib in primary mouse lung fibroblasts.

<p>(A) Immunofluorescence staining for Coll-I (green), F-Actin (red) and nuclei (blue) after 72 hours of treatment with OZ. (B) BrdU proliferation assay of primary lung fibroblasts treated with OZ for 72 hours (Data represent mean ± SEM. <i>n</i> = 4 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Immunofluorescence staining for Coll-I (green), F-Actin (red) and nuclei (blue) after treatment with TGF-β and OZ. (D) and (E) RT-qPCR analysis of mRNA expression of Coll-I and αSMA after treatment with TGF-β and OZ (Data represent mean ± SEM. <i>n</i> = 3 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. Paired t-Test).</p

Oral application of oprozomib does not reduce proteasome activity in fibrotic lungs and is not well tolerated in bleomycin challenged animals.

<p>(A) Treatment scheme: repeated oral application of OZ. (B) CT-L proteasome activity (Data represent mean ± SEM. <i>n</i> = 5–6 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. Mann Whitney t-test) and (B) weight loss of animals at different time points (Data represent mean ± SEM. <i>n</i> = 5–6 per group. *<i>P</i> ≤ 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. 1way ANOVA Bonferroni‘s Multiple Comparison Test).</p

Protease-Mediated Release of Chemotherapeutics from Mesoporous Silica Nanoparticles to <i>ex Vivo</i> Human and Mouse Lung Tumors

Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules <i>via</i> MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of <i>ex vivo</i> 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of K<i>ras</i> mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors