Cigarette smoke induces significant changes in oxidant defense responses; some of these are further amplified, but not in a linear fashion, in individuals who develop COPD.

Transforming growth factor-␤ 1 (TGF-␤ 1 ) induces ␣-smooth muscle actin (␣-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased ␣-SMA expression. Because TGF-␤ 1 is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-␤ 1 alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [ 35 S]methionine, followed by protein separation on twodimensional gel electrophoresis, displayed ϳ2500 proteins in the pI interval of 3-10. Treatment of TGF-␤ 1 led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-␤ 1 , and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-␤ 1 induces not only ␣-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of ␣-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium-activated chloride channels (CLCAs), appear in fact to be membrane anchored metal-dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well-conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N-terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three-dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP-11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full-length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full-length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel-activating and/or channel-accessory roles.

Chronic obstructive pulmonary disease (COPD) is a disease primarily caused by cigarette smoking, which in turn has been shown to affect the susceptibility to and progression of airway infections. The question addressed in this study was how components from cigarette smoke could affect the defence mechanisms of T-cells and epithelial cells, and thereby contribute to the development of the COPD pathology. T-cells and monocytes were isolated from buffycoats from healthy donors and T-cell responses studied in response to cigarette smoke extract (CSE). Activation level (CD25 expression), proliferation (BrdU incorporation) and intracellular expression of the cytotoxic markers granzyme-b and TIA-1 were determined using flowcytometry. Normal human bronchial epithelial cells were obtained from Cambrex and differentiated in air-liquid interface cultures. After exposure to CSE barrier function (trans-epithelial electric resistance, TEER), MUC5AC and interleukin-8 production were measured. T-cell activation, proliferation and expression of the cytotoxic proteins granzyme-b and TIA-1 were significantly reduced in response to 0.5-1% of CSE. The epithelial cells were more resistant to CSE and responded at doses 20 times higher than T-cells. The expression of interleukin-8 and MUC5AC was significantly increased after exposure to 15% and 30% CSE and TEER was largely unaffected at 30% CSE but clearly reduced at 40% CSE. This study shows that mechanisms, in both T-cells and airway epithelial cells, involved in the defence against infectious agents are modulated by CSE.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular

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BackgroundDuring wound healing processes fibroblasts account for wound closure by adopting a contractile phenotype. One disease manifestation of COPD is emphysema which is characterized by destruction of alveolar walls and our hypothesis is that fibroblasts in the COPD lungs differentiate into a more contractile phenotype as a response to the deteriorating environment.MethodsBronchial (central) and parenchymal (distal) fibroblasts were isolated from lung explants from COPD patients (n = 9) (GOLD stage IV) and from biopsies from control subjects and from donor lungs (n = 12). Tissue-derived fibroblasts were assessed for expression of proteins involved in fibroblast contraction by western blotting whereas contraction capacity was measured in three-dimensional collagen gels.ResultsThe basal expression of rho-associated coiled-coil protein kinase 1 (ROCK1) was increased in both centrally and distally derived fibroblasts from COPD patients compared to fibroblasts from control subjects (p < 0.001) and (p < 0.01), respectively. Distally derived fibroblasts from COPD patients had increased contractile capacity compared to control fibroblasts (p < 0.01). The contraction was dependent on ROCK1 activity as the ROCK inhibitor Y27632 dose-dependently blocked contraction in fibroblasts from COPD patients. ROCK1-positive fibroblasts were also identified by immunohistochemistry in the alveolar parenchyma in lung tissue sections from COPD patients.ConclusionsDistally derived fibroblasts from COPD patients have an enhanced contractile phenotype that is dependent on ROCK1 activity. This feature may be of importance for the elastic dynamics of small airways and the parenchyma in late stages of COPD.