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.
Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.
Proteins and peptides present within clinical samples represent a valuable library of information regarding the ongoing processes within cells and tissues in health and disease. We have developed and validated novel technology applications that can be used to characterize the patterns of global protein expression in tissue and biofluids in either gel-based systems or by automated multidimensional nanocapillary liquid chromatography. Mass spectrophotometry platforms using MALDI MS and MS/MS or LTQ ion trap MS were capable of delivering sensitive and accurate identifications of hundreds of proteins contained in individual samples including individual forms of processing intermediates such as phospho peptides. The Systems Biology approach of integrating protein expression data with clinical data such as histopathology, clinical functional measurements, medical imaging scores, patient demographics, and clinical outcome provides a powerful tool for linking biomarker expression with biological processes that can be segmented and linked to disease presentation.
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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