RICERCA ITALIANA     

Signalling Proteomics (Signalomics): differential studies by novel strategies and technologies

Università degli Studi di Napoli "Federico II"

Abstract

The project focuses on differential studies of proteomes or protein complexes (complexomes) involved in the different steps of signal transduction. A major goal of the project is the development of novel experimental strategies and advanced technologies required by the proposed studies.
Cell survival relies on multiple regulated metabolic processes, prompted by the ability to react to external stimuli. Cellular activity can be viewed as the integration of activation processes, stabilization of activated molecular complexes and degradation. A characterization of the bulk of proteins activated in vivo via phosphorylation (phosphoproteome) is essential for understanding regulatory mechanisms of the signalling pathways. The first aim of the proposal, besides the identification of proteins involved in a given signalling pathway, is the identification of residues recognised by kinase-phosphatase enzymatic systems in various phases of the cell cycle. The second goal of the proposal is directed to understanding the systemic response to CA2+-dependent stimulation due to formation of isopeptidic bonds catalysed by transglutaminase. The goal is the differential analysis of the proteins and amino acid residues involved (transglutamome). Conjugation to ubiquitin is an important metabolic step for termination of the signalling process; a description of the proteome involved in the ubiquitin pathway (ubiquitinome) will be the third goal of the project. Finally identification of the protein subunits of ancestral anionic receptors and nuclear orphan receptors, identification of their endogenous ligands and of the complementary protein machinery (gephyrin, ubiquitin, catenins, cadherins: receptor complexomes) for differential analysis purposes represents the fourth goal of the proposal. Thus the general aim of the project is the identification of basic molecular complexes operating in different cellular systems, as a first step in the process of unravelling proteome complexity, which can be predicted to be two or even three orders of magnitude higher than that of the expressed genome, owing to the different post-synthetic modifications.
Attainment of such challenging goals will require adequate methodologies for the studies to be undertaken. Mass Spectrometry has proven to be the methodological key to proteomics studies. Proteins, either native or derived by post-translational modifications, are separated by bidimentional electrophoresis and subjected to hydrolysis. MALDI-TOF mass spectrometry of these peptidic maps allows the identification of the proteins of interest. This approach still requires solving many problems of separation/purification and sensitivity of the available techniques. An essential part of the present proposal is devoted to setting up new multi-dimensional electrophorestic and chromatographic techniques essentially based on affinity purification. In order to identify protein complexes, high molecular weight proteins or proteins poorly representated in bidimensional maps a new methodology for spectrometric analysis will be developed, based on the physical properties of novel detectors. A prototype mass spectrometer equipped with cryogenic detectors is available to one of the teams participating to the project, that will provide the development of adequate methodologies. Exploitation of this technical advancement will be complementary to MALDI-TOF spectrometry, in which another team of the project has a long-standing expertise.
In brief, the proposal aims at studies of functional proteomics by advanced technlogies in different enzymatic systems in vivo and in vitro; phage or synthetic peptidic libraries; transmembrane or nuclear receptors and associated protein complexes; different model systems (yeast, hydra, rodent) and cell systems (macrophages, neuroectodermal cell lines). <<<

Principal Investigator

GENNARO MARINO Universita' degli Studi di NAPOLI "Federico II"

Research Goal

There is an essential need to improve present proteomics methodologies towards an extensive sensitivity coupled with affordable and confident quantitation.
The final aim of this project is to make significant advances in differential proteomics toward this end. To face with this challenge the project is focused on differentially displayed proteomics in connection with the major events of signal transduction, a key issue in functional genomics. Since mass spectrometry and multidimensional separations are the core methods in proteomic studies, the most experienced Italian groups in biomolecular mass spectrometry and in separation methodologies will merge with an outstanding research group, specialised in low-temperature physics, who have developed a prototype of an extremely sensitive MALDI mass spectrometer. To focus on the specific biological issue of signal transduction proteomics, several research groups, all in the forefront of the international cellular and molecular biology research, will be involved in the project with the aim both to integrate their expertise and to make significant advancement in their fields by making use of the proteomics strategies and technologies made already available or to be developed.
The use of a novel mass spectrometer, equipped with a cryogenic detector, in proteomics may be considered a result per se and may pave the way to the discovery of so far unknown proteins modulating fine cellular signals. These expression products and/or their modifications have to be transient species and, as such, present only in very few copies in the cell and may have escaped to presently available methodologies.The specific aims of this part of the project are: 1) to increase the sensitivity and accuracy of large molecules identification and characterisation by improving and making accessible a novel detector technology into mass spectrometry. The key technology on which this proposal is based are cryodetectors. These can detect single molecules (up to m>150 kDa) with a high signal-to-noise ratio and an efficiency of 100%, which is independent from molecular mass. By comparison with today's ionising detectors, a three order of magnitude improvement in intrinsic detection efficiency for molecules with mass beyond 150 kDa can be achieved. These properties will be particularly useful when very small quantities of analyte are available or in case of analysis of species in trace amounts. 2) to apply with success the novel instrumentation for the identification and investigation of the various proteins of interest in the project. 3) to produce validation of performances of the novel instrumentation and evaluation of the advantages offered by this new technology when facing the complex analytical problems of the present research project. Furthermore by combining the extreme sensitivity of the machine with a new generation of Isotope Coded Affinity Tags one can expect to report the target modifications resulting from signal transduction event(s) in strictly quantitative terms. Finally, development of new protocols for multidimensional pre-fractionation of peptide mixtures from biological extracts will be the essential complement of of these technological advancements and will be the main concern of Verona RU.
The expected results are described in detail in the individual Work Packages and basically concern the discovery of differentially displayed substrates in the phosphorylation/dephosphorylation cycles, in the ubiquitination pathway and in the transglutamination events, and in receptor proteins or multimolecular receptor complexes.
In brief, the research efforts will be directed and coordinated as follows:
Identification of phosphorylation sites and phosphoproteome changes will be performed in collaboration with the Firenze and Padova RU by a multisteps approach based on the ICAT strategy to i) transform Ser-P and Thr-P into dehydroalanine or dehydro-amino2-butyrric acid respectively by beta-elimination; ii) introduce deuterated/non deuterated ICAT reagent through Michael addition of the free SH group onto the vynilic bond of DSer and/or DThr; iii) isolate modified peptides by affinity chromatography with streptavidin; iv) quantitate peptides by mass spectrometry. The relative abundance of different protein components will be determined by the ratio between peptides labelled with the heavy and light forms of the reagent which differ by 4/8 Da. Protein mixtures from cells grown under different conditions will be treated with these reagents and enzymatically digested. Modified peptides will be fished out by affinity chromatography with streptavidin and characterised by mass spectrometry leading to the identification of the specific protein substrates.
Identification, within the yeast phosphoproteome, of sub-sets of phosphorylated proteins which are targeted by CK2 and piD261, respectively will be pursued by Padua RU. Moreover, the analysis of the Golgi apparatus phosphorylated proteome, facilitated by the limited number of protein kinases present in this cellular compartment, will be exploited for getting a thorough insight into the endogenous substrates of another protein kinase, partially characterized, the "Golgi casein kinase (G-CK)", a ubiquitous and pleiotropic protein kinase, whose physiological roles, besides that of phosphorylating caseins in the mammary gland, are still poorly understood. Identification of physiological docking proteins (both substrates and other molecular partners) for LMWPTP and ACP, and the study of the effect of extracellular stimuli, particularly oxidative stimuli, on the cellular action and localisation of both enzymes will be carried out by Florence RU.
Covalent ligation of ubiquitin to specific target proteins within the cell represents a critical regulatory mechanism involved in in a number of key biological processes from the clearance of damaged proteins to the control of the population of GABA receptors on the cell surface. Analogously, TGase-mediated reactions constitute Ca2+ modulated events involved in different physio-pathological states and in various stages of cell life. Identification of the ubiquitin specific targets and TGase substrates will be carried out by RU Marino in collaboration with the Roma RU using biotynilated ubiquitin and synthetic TGase peptide substrates. The specific aims are: 1. Identification of TG substrates that are used in normal (epidermal differentiation, apoptosis) and pathological (neurodegenerative and Celiac diseases) conditions. 2. Identification of specific sites of postranslational modification targeted by TGs in different biological systems. 3. Identification of possible regulatory domains (e.g. BH3) on TGs that control their enzymatic activity and their involvement in various biological processes.
The integration of laboratories dealing with transduction signals may add a further value to the project since multiple post-translational events on the same expression product may be discovered and related to still unknown physio-/pathological statuses. This may be indeed the case of cell surface receptors whose number and stability are likely regulated by more than a single modification. The specific aims of this part of the project, carried out by RU CNR IC-IIGB in collaboration with RU Naples and RU CNR IC, are summarised as follows:
Molecular characterisation of inhibitory ionotropic receptor subunits (GABA and glycine receptors) in a primitive nervous system (Hydra vulgaris, Cnidaria, Hydrozoa); transcriptional and functional analysis. Description of the identified protein complexes. The aim is to achieve a better understanding of the phylogenesis of mammalian ionotropic receptors. Identification and characterisation of anchoring proteins involved in clustering and targeting of these receptors in Hydra, (gephyrin, GABARAP, Plic-1). Identification of endogenous ligand(s) for the orphan nuclear receptor (Nurr1) in primary dopaminergic cultures and rodent cell lines. Characterisation of the molecular machinery involved in transcriptional regulation of Nurr1 target genes.
Expected results: Development of new protocols and application of novel Mass Spectrometry technologies (MALDI-TOF, cryogenic spectrometry, eventually multidimensional chromatography) to the analysis of crude extracts of eucariotic membranes (Hydra) and nuclear extracts of primary cultures and rodent cell lines. Differential analysis of protein extracts in control and treated conditions. <<<

Timescale

36 months