Solution Structure of Human p8 MTCP1 , a Cysteine-rich Protein Encoded by the MTCP1 Oncogene, Reveals a New a a a-Helical Assembly Motif

International audienceMature-T-Cell Proliferation) is the ®rst gene unequivocally identi®ed in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8 MTCP1 protein encoded by the MTCP1 oncogene was determined by homonuc-lear proton two-dimensional NMR methods at 600 MHz. After sequence speci®c assignments, a total of 931 distance restraints and 57 dihedral restraints were collected. The location of the three previously unassigned disul®de bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of p8 MTCP1 is presented as a set of 30 DIANA structures, further re®ned by restrained molecular dynamics using a simulated annealing protocol with the AMBER force ®eld. The r.m.s.d. values with respect to the mean structure for the backbone and all heavy atoms for a family of 30 structures are 0.73(AE0.28) and 1.17(AE0.23) A Ê , when the structured core of the protein (residues 5 to 63) is considered. The solution structure of p8 MTCP1 reveals an original scaffold consisting of three a helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disul®de bridges, forming an a-hairpin which resembles an antiparallel coiled-coil. The third helix is oriented roughly parallel to the plane de®ned by the a-antiparallel motif and its axis forms an angle of %60 with respect to the main axis of this motif

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

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

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

Ground-Based Optical Measurements at European Flux Sites: A Review of Methods, Instruments and Current Controversies

This paper reviews the currently available optical sensors, their limitations and opportunities for deployment at Eddy Covariance (EC) sites in Europe. This review is based on the results obtained from an online survey designed and disseminated by the Co-cooperation in Science and Technology (COST) Action ESO903—“Spectral Sampling Tools for Vegetation Biophysical Parameters and Flux Measurements in Europe” that provided a complete view on spectral sampling activities carried out within the different research teams in European countries. The results have highlighted that a wide variety of optical sensors are in use at flux sites across Europe, and responses further demonstrated that users were not always fully aware of the key issues underpinning repeatability and the reproducibility of their spectral measurements. The key findings of this survey point towards the need for greater awareness of the need for standardisation and development of a common protocol of optical sampling at the European EC sites

Reception Test of Petals for the End Cap TEC+ of the CMS Silicon Strip Tracker

The silicon strip tracker of the CMS experiment has been completed and was inserted into the CMS detector in late 2007. The largest sub system of the tracker are its end caps, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted onto the TEC support structures. Each end cap consists of 144 such petals, which were built and fully qualified by several institutes across Europe. Fro

Integration of the End Cap TEC+ of the CMS Silicon Strip Tracker

The silicon strip tracker of the CMS experiment has been completed and inserted into the CMS detector in late 2007. The largest sub-system of the tracker is its end cap system, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted into the TEC support structures. Each end cap consists of 144 petals, and the insertion of these petals into the end cap structure is referred to as TEC integration. The two end caps were integrated independently in Aachen (TEC+) and at CERN (TEC--). This note deals with the integration of TEC+, describing procedures for end cap integration and for quality control during testing of integrated sections of the end cap and presenting results from the testing

Validation practices for satellite based earth observation data across communities

Assessing the inherent uncertainties in satellite data products is a challenging task. Different technical approaches have been developed in the Earth Observation (EO) communities to address the validation problem which results in a large variety of methods as well as terminology. This paper reviews state-of-the-art methods of satellite validation and documents their similarities and differences. First the overall validation objectives and terminologies are specified, followed by a generic mathematical formulation of the validation problem. Metrics currently used as well as more advanced EO validation approaches are introduced thereafter. An outlook on the applicability and requirements of current EO validation approaches and targets is given

Selenomethionine and Selenocysteine Double Labeling Strategy for Crystallographic Phasing

International audienc