Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

<p>Abstract</p> <p>Background</p> <p>The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.</p> <p>Results</p> <p>We analysed the proteome of PBs induced in <it>Nicotiana benthamiana </it>leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.</p> <p>Conclusions</p> <p>This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.</p

Excreted/Secreted Proteins from Trypanosome Procyclic Strains

Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families) because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission

Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions

Idea generation in the fuzzy front-end of small entrepreneurial projects

The success of the new product development (NPD) process is vital for the survival of projects. Especially for small entrepreneurial projects that try to survive in a very competitive environment, affected by constant market and technical changes. Previous literature emphasize the importance of the generation of new ideas, to improve the product concept during the pre-development stage, but it did not pay a lot of attention to the sources of ideas. For this reason, this study aims to develop the knowledge concerning the sources of ideas of small entrepreneurial projects, during the fuzzy front-end (FFE). The literature review of this study presents the previous researches that relate to the sources of ideas, during the idea generation stage of the FFE. The structure of the existing model, integrating the sources of ideas, is based on three categories: environmental scanning, innovative organizational culture and joint research. An inductive study and a research, based on the analysis of one small high-tech entrepreneurial project, were conducted to cover the literature gap. This choice of methodology reflects the experimental purpose of this research. The empirical data are only primary data, collected by analyzing the diaries of the two entrepreneurs involved in the project. Our findings reveal that contact with lead user, entrepreneur experience, customer involvement, brainstorming sessions, competitor analysis, resource constraints and prototype conception represent the main sources of ideas of small entrepreneurial projects in the FFE. The practical purpose of this study is to offer some advices to entrepreneurs of small entrepreneurial projects for accessing to sources of ideas, during the idea generation stage of the FFE, and manage their impact on the development of the product concept. The theoretical implications contribute to the identification of new sources of ideas and a proposition of framework of their impact

Visible and Fluorescent Staining of Two-Dimensional Gels

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Regulation in primary metabolism of transgenic maize endosperm after metabolic engineering of astaxanthin biosynthesis highlighted by a label-free quantitative proteomics strategy

Regulation in primary metabolism of transgenic maize endosperm after metabolic engineering of astaxanthin biosynthesis highlighted by a label-free quantitative proteomics strategy. 33. Congrès de la Société Française d'Electrophorèse et d'Analyse Protéomique (SFEAP

Proteomic characterization of arabidopsis ecotypes under phosphate starvation

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