The activity of aminoacyl-tRNA synthetase-interacting multi-functional protein 1 (AIMP1) on endothelial cells is mediated by the assembly of a cytoskeletal protein complex.

AIMP1 was first found as a factor associated with the aminoacyl-tRNA synthetase (ARS) complex. However, it is also secreted and acts on different target cells such as endothelial cells, macrophages, and fibroblasts as an extracellular regulator, respectively, of angiogenesis, inflammatory responses and dermal regeneration. AIMP1 has also been reported to suppress in vivo tumor growth. In this study, we investigated the signaling pathways activated by exogenous AIMP1 in an in vitro endothelial model. AIMP1 decreases EC viability through an α5ß1 integrin-dependent mechanism and inhibits cell adhesion, is internalized and shows an asymmetric pattern of distribution and accumulation in cell protrusions. Experiments of affinity purification, pull down, and co-immunoprecipitation showed that AIMP1 interacts with four cytoskeletal proteins (filamin-A, α-tubulin, vinculin, and cingulin). α-Tubulin also gets phosphorylated upon cell treatment with AIMP1 and colocalization between AIMP1 and filamin-A as well as between AIMP1 and cingulin was observed through immunofluorescence assays. In this work, we propose that AIMP1 effect on EC adhesion is mediated by the assembly of a cytoskeletal protein complex on the cytosolic face of the cell membrane which could regulate cellular architecture maintenance and remodeling. Moreover, this activity is able to indirectly influence cell viability.

Acute-phase proteins investigation based on lectins affinity capture prior to 2-DE separation: application to serum from multiple sclerosis patients.

Plasma acute-phase proteins (APPs) glyco-isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco-isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate-binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2-DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up-regulated and 7 down-regulated in MS samples. ESI LC-Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N-linked sugar structures is well known. Performing galectin-3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco-isoforms of ß-haptoglobin and MS. In conclusion, although the patho-physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease-specific marker identification approaches.

Lymphocyte proteomics of Parkinson's disease patients reveals cytoskeletal protein dysregulation and oxidative stress.

AIMS: There is increasing evidence of biochemical alterations in peripheral blood lymphocytes of Parkinson's disease (PD) patients. In this work, we describe the changes in protein levels in peripheral lymphocytes of PD patients in order to identify potential peripheral biomarkers. MATERIALS & METHODS: By means of 2D electrophoresis and mass spectrometry protein identification, we compared patients under L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, patients under subthalamic nucleus deep-brain stimulation and healthy controls. RESULTS: Statistical analysis of the results demonstrated that cofilin-1, tropomyosin, and a specific actin isoform vary significantly in patients, regardless of the therapy. Two different isoforms of gamma-fibrinogen either correlate with the disease state or with the disease duration. Eventually, specific changes associated with the different therapies allowed to highlight oxidative stress conditions in lymphocytes in patients treated with higher doses of L-DOPA. CONCLUSIONS: As a whole, peripheral blood lymphocytes are sensitive reporters of PD over inter-individual variability, and allow the identification of specific alterations that could be further exploited for diagnostic purposes.

Effects of malachite green (MG) and its major metabolite, leucomalachite green (LMG), in two human cell lines.

Malachite green (MG) is still illegally used as a fungicide in aquaculture. In fish it is absorbed and metabolised to its major reduced metabolite, leucomalachite green (LMG). This latter represents the main residue found in fish tissues and may persist for several months. Since MG, suspected to act as a tumour promoter in vitro and in vivo, might be also present as a residue in fish, the present study was undertaken to ascertain the in vitro toxicity of both compounds in two human tumour cell lines (Caco-2 and HEp-2). After 24h incubation with MG, significant decreases of cell viability, measured by neutral red uptake (NRU) or total protein content (TPC) as well as proliferation capability (colony-forming ability, CFA) were noticed in HEp-2 cells; the mean IC(50) value was about 2 microM. As regards the differentiated Caco-2 cells, MG caused a dose-related significant cytotoxicity, measured either by MTT test, the LDH leakage or NRU, with a mean IC(50) value of about 15 microM. By contrast, LMG disclosed, in both cell lines, a lower cytotoxicity compared to MG. These results also show that HEp-2 cells are more sensitive than intestinal cells to the toxic action of both compounds.