Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis of total cellular proteins of human neutrophils

Background  

Neutrophils are the most abundant leukocytes in peripheral blood and represent one of the most important elements of innate immunity. Recent subcellular proteomic studies have focused on the identification of human neutrophil proteins in various subcellular membrane and granular fractions. Although there are relatively few studies dealing with the analysis of the total extract of human neutrophils, many biological problems such as the role of chemokines, adhesion molecules, and other activating inputs involved in neutrophil responses and signaling can be approached on the basis of the identification of the total cellular proteins.     

The redoxome: Proteomic analysis of cellular redox networks

Redox-regulated proteins play fundamentally important roles not only during the defense of organisms against oxidative stress conditions but also as targets of cellular signaling events. This realization has spurred the development of proteomic techniques geared towards characterizing the redoxome; proteins with highly reactive cysteine residues, whose thiol oxidation state controls the function of the proteins, and by extension, the pathways they are part of. We will here summarize the most recent advances made in the field of redox proteomic analysis, aimed to elucidate the cellular redox networks that appear to control prokaryotic and eukaryotic organisms.     

Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.     

Proteomic approaches to the analysis of multiprotein signaling complexes

Signal transduction is one of the most active fields in modern biomedical research. Increasing evidence has shown that signaling proteins associate with each other in characteristic ways to form large signaling complexes. These diverse structures operate to boost signaling efficiency, ensure specificity and increase sensitivity of the biochemical circuitry. Traditional methods of protein analysis are inadequate to fully characterize and understand these structures, which are intricate, contain many components and are highly dynamic. Instead, proteomics technologies are currently being applied to investigate the nature and composition of multimeric signaling complexes. This review presents commonly used and potential proteomic methods of analyzing diverse protein complexes along with a discussion and a brief evaluation of alternative approaches. Challenges associated with proteomic analysis of signaling complexes are also discussed.     

Proteomic analysis reveals novel molecules involved in insulin signaling pathway

The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.     

Proteomic analysis of cellular response to microcystin in human amnion FL cells

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.     

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors

The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.     

细胞信号网络功能鲁棒的简并性拓扑特征

细胞信号网络对于外界环境的干扰表现出优良的鲁棒性,但是其维持功能鲁棒的内在机制尚未明确,本文研究了细胞信号网络功能鲁棒性的拓扑特征。选择布尔网络模型模拟细胞网络的动态行为,利用网络节点状态的扰动模拟外界环境干扰。基于演化策略探寻不同网络拓扑的功能并分析其在干扰环境下的鲁棒性,采用埃德尔曼提出的基于信息论的计算方法评估网络拓扑的简并度、冗余度和复杂度等拓扑属性,对比分析它们与功能鲁棒度的相关性及作用机理。结果显示,在网络模型的演化过程中,其拓扑简并度与功能鲁棒度显著正相关,相关性水平高于拓扑冗余度与鲁棒度的相关性。并且,随着鲁棒度的提升,网络的节点数和复杂度也随之升高,同样简并度与网络的节点数和复杂度的相关性高于拓扑冗余度与网络的节点数和复杂度的相关性。这说明增加的网络节点以简并的方式同时提高了网络拓扑的鲁棒度和复杂度。因此,细胞网络功能鲁棒性的拓扑特征是简并而不是冗余,简并为解决生物系统的复杂问题提供了有效手段,为人工系统的可靠性设计提供有益的借鉴。     

Cadherin-mediated cellular signaling

Recent cadherin studies focusing on cellular signaling have shown that several pathways are activated by cadherin-mediated cell-cell contact. Cadherin-mediated contacts activate Rho family GTPases, regulate the availability of beta-catenin to participate in Wnt signaling, and function in receptor tyrosine kinase signaling. Although different classical cadherins bind to the same cytosolic proteins via their cytoplasmic tails, one message that is clear from the recent literature is that downstream signals emanating from cadherin-mediated contacts are both cadherin-specific and cell-context-specific.     

Proteomic analysis

The field of proteomics is becoming increasingly important as genome sequences are being completed and annotated. Recent advances in proteomics include experimental and mathematical proofs of the need to complement microarray analysis with protein analysis, improved sensitivity for mass spectrometric analysis of separated proteins, better informatic tools for gel analysis and protein spot annotation, first steps towards automated experimental procedures, and new technology for quantitation of protein changes.     

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.     

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity

1. Download : Download high-res image (292KB)
2. Download : Download full-size image

     

Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes

Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.     

A network analysis of changes in molecular interactions in cellular signaling

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.     

Spotlight on cellular signaling

On May 21-27, 2004, the fourth in an ongoing series of Dubrovnik Conferences on Cellular Signaling was held at the beautiful Croatian resort of Cavtat, outside the historic town of Dubrovnik. The largest so far in the biannual series attracted an audience of over 300. A series of topics was discussed in depth. Here we concentrate on three areas: receptor tyrosine kinase signaling, actin rearrangements, and endocytosis.     

Bioinformatics and cellular signaling

The understanding of cellular function requires an integrated analysis of context-specific, spatiotemporal data from diverse sources. Recent advances in describing the genomic and proteomic 'parts list' of the cell and deciphering the interrelationship of these parts are described, including genome-wide location analysis, standards for microarray data analysis, and two-hybrid and mass spectrometry approaches. This information is being collected and curated in databases such as the Alliance for Cellular Signaling (AfCS) Molecule Pages, which will serve as vital tools for the reconstruction and analysis of cellular signaling networks.     

Proteomic analysis of proteins associated with cellular senescence by calorie restriction in mesenchymal stem cells

Mesenchymal stem cells (MSCs) hold great promise for use in cell-based therapies because of their multipotency and simple methods for in vitro expansion. However, during in vitro expansion, MSCs will age and lose their multipotency and proliferation capability. Previous studies have reported that calorie restriction (CR) increases proliferation of MSCs and decreases apoptosis. Therefore, in this study, we examined the effect of low glucose (LG) on human bone marrow-derived MSCs. Proliferation under low glucose (LG, 1.4 mM) conditions was compared with that under normal glucose (NG, 5.5 mM) conditions. In addition, comparative studies of population doubling (PD), β-galactosidase (β-GAL) activity, reactive oxygen species (ROS) generation and differentiation capacity (osteocytes and adipocytes) in NG and LG conditions were performed. In addition, protein expression patterns were compared between NG and LG conditions and several proteins were found to be up- or down-regulated under the glucose restriction condition (LG condition). As a result, CR does not seem to have a significant effect on proliferation, ROS generation, glucose consumption concentration, population doublings, and adipogenic differentiation of MSCs. Interestingly, however, the differentiation potential into osteocytes was maintained under CR and a lower senescence-associated β-galactosidase (β-GAL) activity was observed under CR than under the NG condition. In addition, we determined three up-regulated proteins (aldehyde dehydrogenase, neuropolyprptide h3, and prolyl 4-hydroxylase alpha subunit) and seven down-regulated proteins (laminin-binding protein, actin, sec 13 protein, alpha soluble N-ethylmaleimide-sensitive fusion protein (NSF)- attachment protein (SNAP), manganese superoxide dismutase, proteasome alpha 1 subunit, and ribosomal protein S12) via two-dimensional electrophoresis analysis. These results imply that differentially expressed proteins under the LG condition may provide further information on the aging and differentiation of stem cells.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Proteomic analysis of bovine sperm YWHA binding partners identify proteins involved in signaling and metabolism

Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.