Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo\"id cell lines nuclear proteomes

One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta 2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of observed changes are due to post-translational modifications, thereby a exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications

Conformational analysis of some pyridinium phenolates and synthetic precursors based on X-Ray and IR characterisations

7 pagesInternational audienceThis manuscript reports X-Ray and IR characterizations of representative pyridinium phenolates, model compounds for nonlinear optics. These analyses reveal the close dependence existing between molecular structure and the contribution of quinone and zwitterionic limiting forms. The bond length alternation (BLA) values, the well-known parameter correlated to hyperpolarisability β, are also discussed and compared with literature data

About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis.

web publisher www.interscience.wiley.comInternational audienceThe influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels

Zinc adaptation and resistance to cadmium toxicity in mammalian cells. Molecular insight by proteomic analysis

To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium- resistant human epithelial cell line (HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included co-chaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity. Among endoplasmic reticulum stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to cadmium toxicity more acutely than tunicamycin, implying that these cells heavily relied on proper intracellular calcium distribution. The similar sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as MG-132 or lactacystin, excluded improved proteasome activity as a mechanism associated with zinc adaptation of HZR cells. The enzyme 4-hydroxyphenylpyruvate dioxygenase was overproduced in HZR cells as compared to HeLa cells. It transforms 4-hydroxyphenylpyruvate to homogentisate in the second step of tyrosine catabolism. Inhibition of 4-hydroxyphenylpyruvate dioxygenase decreased the resistance of HZR cells against cadmium, but not that of HeLa cells, suggesting that adaptation to zinc overload and increased 4-hydroxyphenylpyruvate removal are linked in HZR cellsComment: in press in Proteomic

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations

Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents

Progress in the definition of a reference human mitochondrial proteome

Owing to the complexity of higher eukaryotic cells, a complete proteome is likely to be very difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting. The optimization steps in two-dimensional electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.Comment: website publisher http://www.interscience.wiley.co

High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.Comment: cpyright: American Society of Biochemistry and Molecular Biolog