Understanding signal transduction through functional proteomics

The study of signal transduction provides fundamental information regarding the regulation of all biologic processes that support the normal function of life. Functional proteomics, a rapidly emerging discipline that aims to understand the expression, function and regulation of the entire set of proteins in a given cell type, tissue or organism, offers unprecedented opportunity for signal transduction research in terms of understanding cellular behavior and regulation at the systems level. Indeed, swift progress in the area of proteomics has demonstrated the major impact of proteomic approaches on signal transduction and biomedical research. In this review, recent and innovative applications of functional proteomics in determining changes in protein contents, modifications, activities and interactions underpinning signaling transduction pathways are discussed.     

Delineating signal transduction pathways in smooth muscle through focused proteomics

This review will outline examples of the authors' focused proteomics approaches to studying signal transduction pathways in smooth muscle. By focusing the use of traditional proteomics techniques with hypothesis-driven selection methods, this approach efficiently addresses the identification of novel elements in a signal transduction pathway of interest. However, focused proteomics serves only as a starting point in the investigation of novel signaling proteins. While focused proteomics studies can suggest the involvement and general biochemical function of a protein in a signaling pathway, these findings must be further investigated and validated. Through the integrated use of focused proteomics with complementary approaches such as genetics, biochemistry and cell physiology, a complete and detailed mechanism of signal transduction can be determined.     

Understanding type 1 diabetes through proteomics

Introduction: Auto-immunity against pancreatic beta-cells leads to an absolute shortage of the hormone insulin, resulting in hyperglycemia and the onset of type 1 diabetes (T1D). Proteomic approaches have been used to elucidate the mechanisms of beta-cell dysfunction and death.

Areas covered: In the present review, we discuss discoveries in the beta-cell proteome that have contributed to better insights in the role of the beta-cell in T1D. Techniques, such as 2D-DIGE and MALDI imaging, together with new approaches for sample preparation, including laser capture microdissection and immunopeptidomics, have resulted in novel mechanistic insights in the pathogenesis of T1D. We describe how proteomic studies in beta-cell lines as well as isolated islets from animal models and humans have discovered intracellular signaling pathways leading to beta-cell destruction, the generation of neo-antigens through post-translational modifications of beta-cell antigens as well as better biomarkers of disease progression.

Expert commentary: Proteomics has contributed to the discovery of beta-cell neo-autoantigen generation through post-translational modifications, hybrid insulin peptide formation and the generation of defective ribosomal gene products. These concepts are revolutionizing our insights in the pathogenesis of T1D, acknowledging a central role for the beta-cell in its own destruction.     

Functional proteomics to identify critical proteins in signal transduction pathways

Reversible protein phosphorylation plays a crucial role in the regulation of signaling pathways that control various biological responses, such as cell growth, differentiation, invasion, metastasis and apoptosis. Proteomics is a powerful research approach for fully monitoring global molecular responses to the activation of signal transduction pathways. Identification of different phosphoproteins and their phosphorylation sites by functional proteomics provides informational insights into signaling pathways triggered by all kinds of factors. This review summarizes how functional proteomics can be used to answer specific questions related to signal transduction systems of interest. By examining our own example on identifying the novel phosphoproteins in signaling pathways activated by EB virus-encoded latent membrane protein 1 (LMP1), we demonstrated a functional proteomic strategy to elucidate the molecular activity of phosphorylated annexin A2 in LMP1 signaling pathway. Functional profiling of signaling pathways is promising for the identification of novel targets for drug discovery and for the understanding of disease pathogenesis.     

Understanding protein trafficking in plant cells through proteomics

The functions of approximately one-third of the proteins encoded by the Arabidopsis thaliana genome are completely unknown. Moreover, many annotations of the remainder of the genome supply tentative functions, at best. Knowing the ultimate localization of these proteins, as well as the pathways used for getting there, may provide clues as to their functions. The putative localization of most proteins currently relies on in silico-based bioinformatics approaches, which, unfortunately, often result in erroneous predictions. Emerging proteomics techniques coupled with other systems biology approaches now provide researchers with a plethora of methods for elucidating the final location of these proteins on a large scale, as well as the ability to dissect protein-sorting pathways in plants.     

Insulin signal transduction through protein kinase cascades

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.     

Understanding protein trafficking in plant cells through proteomics

The functions of approximately one-third of the proteins encoded by the Arabidopsis thaliana genome are completely unknown. Moreover, many annotations of the remainder of the genome supply tentative functions, at best. Knowing the ultimate localization of these proteins, as well as the pathways used for getting there, may provide clues as to their functions. The putative localization of most proteins currently relies on in silico-based bioinformatics approaches, which, unfortunately, often result in erroneous predictions. Emerging proteomics techniques coupled with other systems biology approaches now provide researchers with a plethora of methods for elucidating the final location of these proteins on a large scale, as well as the ability to dissect protein-sorting pathways in plants.     

Modelling protein functional domains in signal transduction using Maude

Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.     

植物孢子体自交不亲和信号转导功能分子研究进展

孢子体自交不亲和(SSI)是许多植物采取的一种抵制近亲繁殖的重要措施,受S位点复等位基因控制。近年来,参与其信号转导的许多功能分子及它们的编码基因被分离并得到了充分研究：当自花授粉时,SPlI／SCR与SRK特异识别,造成后的Ser／Thr激酶的磷酸化,引发了一系列由SLG、ARC1及水孔蛋白等因子参与的SSI信号转导途径,最终产生自交不亲和的结果。     

Phosphoproteomics strategies for the functional analysis of signal transduction

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.     

Mutants in signal transduction through the T-cell antigen receptor

Mutants of an untransformed helper T-cell clone have been derived by chemical mutagenesis followed by selection for cells incapable of proliferating in response to antigen or anti-CD3. The selection was designed to enrich cells bearing mutations distal to the T-cell antigen receptor. The mutants express normal levels of functional T-cell receptors but are uncoupled from cellular responses, including gene induction, lymphokine secretion, proliferation, and phosphatidylinositol turnover. Responses to phorbol ester plus calcium ionophore and to interleukin-2 are unimpaired. Responses to antigen were restored by fusion with a T-cell receptor-negative thymoma, making the mutants valuable for investigating the mechanisms that couple T-cell receptor stimulation to the induction of second messengers and subsequent physiologic responses.     

Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking

In order to identify integral proteins and peripheral proteins associated with the plasma membrane, highly purified Arabidopsis plasma membranes from green tissue (leaves and petioles) were analyzed by mass spectrometry. Plasma membranes were isolated by aqueous two-phase partitioning, which yields plasma membrane vesicles with a cytoplasmic-side-in orientation and with a purity of 95%. These vesicles were turned inside-out by treatment with Brij 58 to remove soluble contaminating proteins enclosed in the vesicles and to remove loosely bound contaminating proteins. In total, 238 putative plasma membrane proteins were identified, of which 114 are predicted to have transmembrane domains or to be glycosyl phosphatidylinositol anchored. About two-thirds of the identified integral proteins have not previously been shown to be plasma membrane proteins. Of the 238 identified proteins, 76% could be classified according to function. Major classes are proteins involved in transport (17%), signal transduction (16%), membrane trafficking (9%) and stress responses (9%). Almost a quarter of the proteins identified in the present study are functionally unclassified and more than half of these are predicted to be integral.     

Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.     

Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain

Glycosphingolipids (GSLs) and sphingomyelin in animal cells are clustered and organized as membrane microdomains closely associated with various signal transducer molecules such as cSrc, Src family kinases, small G-proteins (e.g., RhoA, Ras), and focal adhesion kinase. GSL clustering in such microdomains causes adhesion to complementary GSLs on the surface of counterpart cells or presented on plastic surfaces, through carbohydrate-to-carbohydrate interaction. GSL-dependent cell adhesion in microdomain causes activation of the signal transducers, leading to cell phenotypic changes. A retrospective of the development of this concept, and current status of our studies, are presented.     

Noise enhanced hormonal signal transduction through intracellular calcium oscillations.

In a wide range of non-linear dynamical systems, noise may enhance the detection of weak deterministic input signals. Here, we demonstrate this phenomenon for transmembrane signaling in a hormonal model system of intracellular Ca(2+) oscillations. Adding Gaussian noise to a subthreshold extracellular pulsatile stimulus increased the sensitivity in the dose-response relation of the Ca(2+) oscillations compared to the same noise signal added as a constant mean level. These findings may have important physiological consequences for the operation of hormonal and other physiological signal transduction systems close to the threshold level.