Risultati finali
We have developed a database for the project. Our aims were to provide storage space for large amount of information coming from experimental data, to improve the information flow between participants, thus helping the coordination process of the whole project, and finally to help in the analysis of the data and in drawing conclusions based on several different types of information (mass-spectrometry data, Multi Photon Detection, 2D electrophoresis) obtained during the project (data mining).
The major steps in database development were as follows:
- Building the hardware setup and operating system,
- Building the database structure, and linking to other existing biological resources,
- Creating user interfaces to the database.
- Creating helper applications for data analysis.
The user friendly software for 2d-gel analysis. A software for the analysis of images obtained from classical 2D electrophoresis and Multi Photon Detection has been created. It was written in Borland Delphi and can be run on Microsoft Windows operating system, however using Borland Kylix as a compiler enables our application to run on under linux as well. It allows for selecting, enlarging and analyzing parts of the image processed and allows comparison of 2D gels. One of its most valuable features is the creation of image of theoretical gel based on sequences of proteins. The software contains a database of post-translational modifications and is linked to the PROSITE database.
The post-translational modification status of the protein we want to see on the image can be changed on demand. In this way one can observe influence of attaching many different chemical groups to protein on its physical-chemical properties. Images created by the software can be merged with experimentally obtained ones. It permits easy identification of proteins (and possible posttranslational modifications) in the sample analyzed. Analysis of the existing, publicly available, well resolved 2D gels allowed us to revise the pK values used by software packages for similar tasks. We have also undertaken molecular modeling studies on NBDs of the CFTR protein.
The most common mutation of the CFTR gene, the delta F508 prevents the protein from maturing correctly and reaching the apical membrane to exert its functions, which accelerates its intracellular degradation. A direct strategy is to investigate the differences in protein profiles between cells expressing the normal and the mutated channel. This is the rationale of a study performed within the context of the EUROPROCF project that scrutinizes the protein expression patterns in a cell system (HeLa) stably transfected with either the mutated or the wild-type CFTR. In the study, the 2-D gel screening showed five spots of greater intensity in the mutation-bearing line, identified as type II keratin 8 and type I keratin 18. It can be argued that interaction of mutated CFTR with these proteins could be one of the mechanisms responsible for proteasomal degradation and prevention of protein addressing to the apical membrane. This was reversed by low temperature, and confirmed by keratin 18 siRNA silencing. Because this study was conducted on undifferentiated HeLa cells, these exciting conclusions would be reinforced after re-investigation in a more physiological CF model.
Primary cultures of nasal polyps from normal individuals can be used as an alternative to cell lines of identical genetic back ground and compared with those of patients bearing the deltaF508 mutation. This cell model has been used in a preliminary study aiming to determine the proteins that are not changed in spite of differences in the genetic background of donors. 2-D gels in the pH range 5-8 revealed 24 unchanged proteins, including cytokeratins 6,7,8 and 19, HSP 27 and 70, and elongation factors ER-Tu and ER-2. In addition, 20 protein isoforms were found only in patients (i.e. keratins 4 and 18), and 13 only in controls (i.e., annexin-2, keratin 19). Some of them were identified in both CF and control cells (i.e. chaperonin GroEL precursor, keratins 4 and 17), but showing a migration displacement that could again be attributed to post-translational modifications. Despite the limitations of the study in terms of number of subjects and cell model, it seems to confirm the potential of cytoskeletal proteins as participants in the pathogenesis of CF.
Although multiple functions have been attributed to CFTR, the mature protein located at the apical membrane of epithelial cells is believed to act as a way out for chloride ions and water. The use of CFTR-null (cftr -/-) or CFTR-dysfunctinal mouse models is the most consequent procedure to analyze the effects of channel dysfunction. However, one of the limiting factors of proteomics is the difficulty to examine the membrane proteome, which is mainly composed of hydrophobic and basic proteins that cannot be properly resolved by the conventional IEF-based 2-D technology. A study performed within the scope of the EUROPROCF project successfully overcame this obstacle by a blue native-based electrophoresis approach (BN-PAGE). BN consists of a non-denaturing electrophoresis of protein samples solubilized in a non-ionic detergent that preserves native conformation and interactions, allowing the separation of membrane proteins and complexes. It can be combined with a classic SDS-PAGE to dissociated the separated complexes and resolve their constitutive subunits in a second dimension.
This way, not only the membrane proteome can be addressed and trans-membrane domain-containing proteins identified, but also protein-protein interactions may be unmasked. The study by Brouillard et al. (Mol. Cell. Proteomics, 4, 1762-1775, 2005) showed that in colonic crypts and lung epithelium from cftr -/- mice, the expression of the calcium (sensitive chloride channel ClCA3 is decreased. This channel, which has been suggested to compensate for the lack of functional CFTR in the human intestine, could account, when dysfunctional in the mouse, for the digestive pathology in this animal model. It is proposed that ClCA and most likely mClCA3, which is localized in goblet cells, is involved in this mucus secretory response. In turn, underexpression of mClCA3 protein in cftr -/- mice would participate in the defect of fluid and mucus transport in the colon. In addition, MS analysis per se of mClCA3 pointed to the existence of novel isoforms in mouse colonic epithelial cells that probably results from proteolytic cleavage. The following step is to investigate the expression and function of ClCA proteins in human patients.
One of the most intriguing pathological features of CF is the abnormal inflammatory response in several organs. In a study using a global proteomic approach on colonic crypts from the cftr -/- mouse model, the anti-inflammatory protein annexin-I was identified and found to be totally absent. A similar pattern was found in the pancreas and lung of the same model. Interestingly, the non-targeted organs showed a normal level of annexin-I expression.
ProteoSys AG developed ProteoTope, which provides a direct differential display of two or more samples for proteomic analysis. Samples are labelled with different radioactive isotopes of iodine, such as I-125 and I-131, under chemically identical conditions, then the samples are mixed and separated together by two dimensional polyacrylamide gel electrophoresis (2D-PAGE).
ProteoTope can separate the radioactive signals coming from each radioisotope, and hence from proteins from each sample, and the differential ratio of intensities or signals from each of the samples can be determined. ProteoTope is part of the proprietary technology platform of ProteoSys AG, a small biotechnology company with 25 employees.
P3 has been unable to find a buyer for the commercial rights to ProteoTope despite efforts to sell the technology. At the moment, ProteoTope is the exclusive proprietary property of ProteoSys AG. ProteoSys also patented the stable isotope mass spectrometry method analysis of relative isotopologue abundances (ARIA). This patent application has been discontimued since no suitable commercial vendor could be identified.
ProteoTope is an advertised part of the commercial technology platform of ProteoSys AG (P3) on the company website, which helps maintain the employment of a staff of 25.
Further development of MPD for ultra-high sensitivity analysis of proteins.
It was decided to develop further the MultiPhotonDetection technology to perform ultra-sensitive analyses of differentially displayed phosphoproteins from HeLa cells expressing either WT-CFTR or ?F508-CFTR and, at the same time, test the new back-to-back HT-MPD imager.
By using standard proteins that were trace labelled with 125I (specific activity of 0.5 Ci/mmole tyrosine residues = 1 labelled tyrosine residue in 12000 available ones), then fractionated on 1D gels and measures for 12 hrs on the MPD imager, we reached a sensitivity limit of protein detection of 90 attomoles. Serial dilution curves showed that the MPD measurement gave excellent linear quantitation over the full concentration range. The method used for protein radiolabelling can provide specific activities of about 200 Ci/mmole tyrosine residues, which means that the sensitivity limit with our current settings is at 200 zeptomoles.
To compare MPD with phosphor imager detection, 60 ng of 125I iodinated HeLa phosphoproteins (specific activity 5 Ci/mmole tyrosine residues) were separated on narrow pH unit 2D gels (pH 5.5 - 6.7), measured for 3 days on a phosphor imager and subsequently for 12 hrs on the MPD imager. In the MPD analysis a very conservative background cut-off filter was used. The MPD measurement showed approximately 10 times as many protein spots as the phosphor imager analysis.
We were also able to set up a first multicolour MPD analysis. Total proteins isolated from HeLa pTracer cells, labelled with 131I and phosphoproteins from the same cells, labelled with 125I, were separated in the same narrow pH unit 2D gel (pH 5.5 - 6.7). The 125I-labelled phosphoproteins and the 131I-labelled total proteins were then individually detected by multicolour MPD without any cross-talk problems between the two different isotopes.
The major achievement of Partner 2 was obtained surprisingly with glycoproteins in saliva of CF patients, a study not envisaged at the drafting of the project and therefore not included. A combination of lectin column chromatography and electrophoretic separation showed significant and distinct increases in CF patient saliva glycoproteins, as reported in the third progress report and verified and further detailed during the extension period. P2 has planned a detailed project with Danish partners to collect saliva samples, to investigate the glycoprotein changes and to identify the particular glycoprotein components undergoing changes, i.e. increases in amounts and character, indicating a possible diagnostic approach of non-invasive character. The laboratory tools involved are simple and non-expensive and P2 is led to believe that the potential diagnostic value of the indications will be further substantiated by the planned activities with the focus on development of a simple, economical, and patient-friendly non-invasive diagnostic tool. The significance for the patient being that here may be a method to assess the disease without any harm or troubles for the patient, except to spit in a tray.
To study cystic fibrosis, we used Human 1A Oligo Microarray chips from Agilent. The microarray represents 20,173 60-mer oligonucleotide probes which span conserved exons across the transcripts of the targeted full length genes. A total of 18716 well known human genes is present on the chips. Coupled with Agilent's probe selection process, the design method of each probe prevents redundancy in gene coverage. Virtually all of the genes and corresponding probes have been mapped to the human genome DNA backbone and are experimentally validated.
Agilent�s chips were chosen on three main criteria :
- The chips present an almost full coverage of human genome;
- The proper use of the chips is totally suitable to our transcriptome platform which has been set up in order to make it s own microarrays as well as using commercialy available arrays;
- Agilent�s amplification and labeling kits enable the use of very low amounts of mRNA and can theoretically reveal low abundant species differentially displayed.IB3 cell lines and C38 cell lines were studied with Agilent�s Human 1A Oligo Microarray chips .
IB3-1cells are immortalized pseudotetraploid CF bronchial epithelial cell lines established from a patient with Cystic Fibrosis bearing the two mutations delta F508 and W1282X. C38 cell lines are the corrected IB3 cells for the CFTR gene, meaning that both mutated alleles are replaced with normal CFTR alleles. C38 cell lines can be considered as the patient cell lines after the definitive treatment from his disease. CF is known to be associated with a chronic inflammatory syndrome and pulmonary infections.
CF cells are overeactive to bacterial stimuli like lipopolysaccharide (LPS) compared to normal cells. In order to test LPS effects, which are partly mediated by the NFK-B pathway, we have compared the level of mRNA extracted respectively from IB3 cells treated with IFN? and TNF?, which enhance NKF-B pathway, and from C38 cells treated with IFN? and TNF?. To validate the model, 4 slides were used with 2 dye-swap labelings : IB3 treated cells labeled with Cy3 versus C38 treated cells labeled with Cy5 and IB3 treated cells labeled with Cy5 versus C38 treated cells labeled with Cy3. Our results from IB3 cells treated with TNF? and IFN??versus treated C38 cells, using pan genomic Agilent human slides, has shown 478 up or down regulated genes. Quality control tests have been applied to each step of the process to insure the accuracy and reproducibility of the results.
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