Radial nerve cord protein phosphorylation dynamics during starfish arm tip wound healing events.

Echinoderms, as invertebrate deuterostomes, have amazing neuronal intrinsic growth aptitude triggered at any time point during the animal lifespan leading to successful functional tissue regrowth. This trait is known to be in opposition to their mammal close phylogenic relatives that have lost the ability to regenerate their central nervous system. Despite the promising nature of this intrinsic echinoderm trait, it was only recently that this complex biological event started to be unveiled. In the present study, a 2DE gel-based phosphoproteomics approach was used to investigate changes in starfish neuronal protein phosphorylation states at two different wound healing time-graded events following arm tip amputation, 48 h and 13 days. Among the resolved protein spots in 3.0-5.6 NL pH IEF strips, 190, 142, and 124 had a phosphoprotein signal in the control and the two injury experimental groups, respectively. Gel image analysis, highlighted 129 spots with an injury-related protein phosphorylation dynamics, several being exclusively phosphorylated in controls (72 spots), injured nerves (8 spots) or, showing significantly different phosphorylation ratios (37 spots). Within these, a total of 43 proteins were identified with MALDI-TOF/TOF. Altogether, several intervening proteins of important injury-signaling pathways that seem to be modulated through phosphorylation, were identified for the first time in starfish radial nerve cord early regeneration events. These include cytoskeleton re-organization toward the formation of the neuronal growth cones; cell membrane rearrangements, actin filaments, and microtubules dynamics; mRNA binding and transport; lipid signaling; Notch pathway; and neuropeptide processing.

Proteomic analyses of Ehrlichia ruminantium highlight differential expression of MAP1-family proteins.

The Rickettsiales Ehrlichia ruminantium (ER) is the causative agent of heartwater, a fatal tick-borne disease of livestock in sub-Saharan Africa and in the Caribbean, posing strong economical constraints to livestock production. In an attempt to identify the most prominent proteins expressed by this bacterium, especially those encoded by the major antigenic protein 1 (map1) multigene family, a proteome map of ER cultivated in endothelial cells was constructed by using two dimensional gel electrophoresis combined with mass spectrometry. Among the sixty-four spots detected, we could identify only four proteins from the MAP1-family; the other proteins detected were mainly related to energy, amino acid and general metabolism (26%), to protein turnover, chaperones and survival (21%) and to information processes (14%) or classified as hypothetical proteins (23%). Additional studies on MAP1-family protein using immunochemical labeling also revealed that these proteins are differentially expressed along the bacterium life cycle, presenting different structural organization. Interestingly, when infectious elementary bodies (EBs) are released from host cells, MAP1 appears to be organized in SDS and heat-resistant dimers and trimers stabilized by disulfide bridges. Overall, the results presented herein not only reveal the first partial proteome map of ER but provide new insights on the expression ER MAP1-family proteins in host endothelial cells.

Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms.

Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1-D SDS-PAGE followed by nanoLC and 2-D SDS-PAGE) and MALDI-TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm-predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself.

Monitoring virus-like particle and viral protein production by intact cell MALDI-TOF mass spectrometry.

A new application of intact cell MALDI-TOF MS (ICM-MS) methodology is described for monitoring the production of viral proteins and viral like particles using the baculovirus/insect cells expression system. Various MALDI matrices, cell preparation methods, cell/matrix volume ratio and MALDI target application procedures were tested in order to obtain the highest intensity and reproducibility of intact insect cell spectra. The web interface, SPECLUST (http://bioinfo.thep.lu.se/speclust.html), was used to construct dendograms based on MALDI-TOF MS data for evaluation of fingerprint changes. We demonstrate that insect cell mass spectrum fingerprints are characteristic of each viral protein/particle production. Their changes along the time for each production experiment correlate with the intracellular viral protein content determined by Western blot. This work shows that this simple, fast and low cost assay, which requires low sample volume, is a powerful analytical tool that complements the most common analytical methods used for monitoring bioprocesses and has potential application in the biotechnological industry namely, in the production of recombinant proteins.

Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.