59 research outputs found
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
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
Detection of Prion Protein in Urine-Derived Injectable Fertility Products by a Targeted Proteomic Approach
BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD) is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD), a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG). METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG) and highly-purified urinary human menopausal gonadotropin (hMG-HP) products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and young women exposed to prions can be expected to survive an incubation period associated with a minimal inoculum. The risks of urine-derived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products
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