Conformational studies of RGDechi peptide by natural‐abundance NMR spectroscopy

Integrins are heterodimeric cell‐surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin‐inhibitor drugs have been developed for integrin‐based medical applications, the structural determinants modulating integrin‐ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for α_vβ3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of α_vβ₃ by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of α_vβ₃ integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance ¹⁵N and ¹³C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.     

Fate and Transport of 2,4,6-Trinitrotoluene in Loams at a Former Explosives Factory

Natural attenuation processes affecting 2,4,6-trinitrotoluene (TNT) were determined within loams for two study areas at the former Explosives Factory Maribyrnong, Australia. TNT fate and transport was investigated through spectrophotometric/High Performance Liquid Chromatography (HPLC) analyses of soil and groundwater, adsorption and microcosm testwork. A five tonne crystalline TNT source zone delineated within near surface soils at the base of a TNT process waste lagoon was found to be supplying aqueous TNT loading (7 ppm) to subsurface soils and groundwater. The resultant plume was localized within the loam aquitard due to a combination of natural attenuation processes and hydrogeological constraints, including low hydraulic conductivity and upward hydraulic gradients. Freundlich described sorptive partitioning was the main TNT sink (K_F = 29 mL/g), while transformation rates were moderate (1.01 × 10^-4 h^-1) under the aerobic conditions. Increasing 2-amino-4,6-dinitrotoluene predominance over 4-amino-2,6-dinitrotoluene was discovered with depth (in situ) and time (microcosms). Simplified dissolution rate calculations indicate that without mitigation of the TNT source, contaminant persistence within the vadose zone may approach 2000 years, while ATRANS20 simulations demonstrate that the TNT plume propagates very slowly along the flow path within the aquitard.     

Specific MALDI imaging and profiling for biomarker hunting and validation: fragment of the 11S proteasome activator complex, Reg alpha fragment, is a new potential ovary cancer biomarker

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.     

Post-transcriptional regulation of P-glycoprotein expression in cancer cell lines

The present study of inhibitors shows that the histone deacetylase-induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5' end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5' untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA.     

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.     

Plant proteome analysis: a 2006 update

This 2006 'Plant Proteomics Update' is a continuation of the two previously published in 'Proteomics' by 2004 (Canovas et al., Proteomics 2004, 4, 285-298) and 2006 (Rossignol et al., Proteomics 2006, 6, 5529-5548) and it aims to bring up-to-date the contribution of proteomics to plant biology on the basis of the original research papers published throughout 2006, with references to those appearing last year. According to the published papers and topics addressed, we can conclude that, as observed for the three previous years, there has been a quantitative, but not qualitative leap in plant proteomics. The full potential of proteomics is far from being exploited in plant biology research, especially if compared to other organisms, mainly yeast and humans, and a number of challenges, mainly technological, remain to be tackled. The original papers published last year numbered nearly 100 and deal with the proteome of at least 26 plant species, with a high percentage for Arabidopsis thaliana (28) and rice (11). Scientific objectives ranged from proteomic analysis of organs/tissues/cell suspensions (57) or subcellular fractions (29), to the study of plant development (12), the effect of hormones and signalling molecules (8) and response to symbionts (4) and stresses (27). A small number of contributions have covered PTMs (8) and protein interactions (4). 2-DE (specifically IEF-SDS-PAGE) coupled to MS still constitutes the almost unique platform utilized in plant proteome analysis. The application of gel-free protein separation methods and 'second generation' proteomic techniques such as multidimensional protein identification technology (MudPIT), and those for quantitative proteomics including DIGE, isotope-coded affinity tags (ICAT), iTRAQ and stable isotope labelling by amino acids in cell culture (SILAC) still remains anecdotal. This review is divided into seven sections: Introduction, Methodology, Subcellular proteomes, Development, Responses to biotic and abiotic stresses, PTMs and Protein interactions. Section 8 summarizes the major pitfalls and challenges of plant proteomics.     

Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein-Barr virus replication and the simple tetracycline repressor

We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100:1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24:1 and 34:1, respectively. With enhanced green fluorescent protein, a 23:1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.