ABSTRACT
BACKGROUND: Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. MATERIALS AND METHODS: The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. RESULTS: Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. CONCLUSION: The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.
Subject(s)
Epidermal Growth Factor , Neuregulins , Humans , Epidermal Growth Factor/metabolism , Neuregulins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , HeLa Cells , Disulfides/chemistry , Disulfides/metabolismABSTRACT
Phenotypic modulation of NAFLD-severity by molecules derived from white (adipokines) and brown (batokines) adipose tissue may be important in inducing or protecting against the progression of the disease. Adipose tissue-derived factors can promote the progression of NAFLD towards severe histological stages (NASH-fibrosis and NASHcirrhosis). This effect can be modulated by the release of adipokines or batokines that directly trigger an inflammatory response in the liver tissue or indirectly modulate related phenotypes, such as insulin resistance. Metabolically dysfunctional adipose tissue, which is often infiltrated by macrophages and crown-like histological structures, may also show impaired production of anti-inflammatory cytokines, which may favor NAFLD progression into aggressive phenotypes by preventing its protective effects on the liver tissue.
Subject(s)
Adipocytes, Brown/metabolism , Adipocytes, White/metabolism , Adipokines/metabolism , Energy Metabolism , Liver Cirrhosis/etiology , Liver/metabolism , Non-alcoholic Fatty Liver Disease/complications , Adipocytes, Brown/pathology , Adipocytes, White/pathology , Animals , Cytokines/metabolism , Disease Progression , Humans , Inflammation Mediators/metabolism , Liver/pathology , Liver/physiopathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Cirrhosis/physiopathology , Neuregulins/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/physiopathology , Phenotype , Severity of Illness Index , Signal TransductionABSTRACT
Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.