A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction

We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.     

Identification and characterization of novel ERC‐55 interacting proteins: Evidence for the existence of several ERC‐55 splicing variants; including the cytosolic ERC‐55‐C

ERC‐55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC‐55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC‐55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC‐55 splicing variants including ERC‐55‐C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub‐cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin‐6, kininogen and lysozyme with ERC‐55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca²⁺] of ～10^?7 M or greater, while calcyclin interaction requires [Ca²⁺] of >10^?5 M. Interaction with peroxiredoxin‐6 is independent of Ca²⁺. Co‐localization of lactoferrin, S100P and calcyclin with ERC‐55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC‐55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ‐coupled 2‐D LC‐MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV‐induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2‐D LC‐MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell‐based HBV replication was established in both RPHs and HepG2 cells. HBV replication‐induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl‐tRNA synthetase, were identified by 2‐D LC‐MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis‐associated proteins identified in our study may eventually lead to novel anti‐angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid,C8‐β‐D‐lactosylceramide

Plasma membrane (PM) microdomains, including caveolae and other cholesterol‐enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D‐erythro‐octanoyl‐lactosylceramide (C8‐D ‐e‐LacCer), stimulates endocytosis via caveolae and induces the appearance of micron‐size microdomains on the PM. To further understand the effects of C8‐D ‐e‐LacCer treatment on PM microdomains, we used a detergent‐free method to isolate microdomain‐enriched membranes from fibroblasts treated ±C8‐D ‐e‐LacCer, and performed 2‐DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain‐enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho‐associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D ‐e‐LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8‐D ‐e‐LacCer‐treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8‐L ‐threo‐lactosylceramide, a glycosphingolipid with non‐natural stereochemistry. This study identifies new microdomain‐associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.     

Tissue proteomics of the low‐molecular weight proteome using an integrated cLC‐ESI‐QTOFMS approach

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500–5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC‐MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.     

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.     

Identification of additional proteins in differential proteomics using protein interaction networks

In this study, we developed a novel computational approach based on protein–protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein–protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest‐ranked proteins, beta‐arrestin 1, and beta‐arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost‐effective means to identify additional proteins that remain elusive for current 2D gel‐based proteomic profiling techniques.     

Coupling proteomics and transcriptomics in the quest of subtype‐specific proteins in breast cancer

Breast‐cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype‐specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, MS‐based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5200 non‐redundant proteins were identified with 23, four, and four proteins identified uniquely in basal, HER2‐neu‐amplified, and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype‐specific signature) showed significant correlation with estrogen receptor (ER) expression. A MS‐based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of ER‐positive and ER‐negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with ER status at both mRNA and protein levels: ABAT, PDZK1, and PTX3, with the former showing significant prognostic potential.     

The Interactorium: Visualising proteins,complexes and interaction networks in a virtual 3‐D cell

Here, we describe the Interactorium, a tool in which a Virtual Cell is used as the context for the seamless visualisation of the yeast protein interaction network, protein complexes and protein 3‐D structures. The tool has been designed to display very complex networks of up to 40 000 proteins or 6000 multiprotein complexes and has a series of toolboxes and menus to allow real‐time data manipulation and control the manner in which data are displayed. It incorporates new algorithms that reduce the complexity of the visualisation by the generation of putative new complexes from existing data and by the reduction of edges through the use of protein “twins” when they occur in multiple locations. Since the Interactorium permits multi‐level viewing of the molecular biology of the cell, it is a considerable advance over existing approaches. We illustrate its use for Saccharomyces cerevisiae but note that it will also be useful for the analysis of data from simpler prokaryotes and higher eukaryotes, including humans. The Interactorium is available for download at http://www.interactorium.net .     

Identification of 14‐3‐3zeta associated protein networks in oral cancer

Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra‐ and intercellular communication and tumor–host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14‐3‐3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14‐3‐3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14‐3‐3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14‐3‐3ζ associated protein networks in oral cancer cell lines using IP–MS/MS and bioinformatics. A total of 287 binding partners of 14‐3‐3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14‐3‐3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14‐3‐3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14‐3‐3ζ in oral cancer progression and metastasis.     

Identification of prolyl hydroxylation modifications in mammalian cell proteins

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen‐dependent regulation of hypoxia inducible factor‐α (HIF‐α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF‐α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506‐binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C‐1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.     

Proteomic identification of the candidate target proteins of 15‐deoxy‐delta12,14‐prostaglandin J2

15‐Deoxy‐delta12, 14‐prostaglandin J₂ (15d‐PGJ₂) is an endogenous anti‐inflammatory lipid derived from PGD₂. One potential mechanism for its activity is the covalent modification of cellular proteins, via a reactive α,β‐unsaturated carbonyl group in its cyclopentenone ring, which in turn alters protein function. In order to identify the candidate target proteins covalently modified by 15d‐PGJ₂ in human aortic endothelial cell (EC), EC was treated with biotinylated‐15d‐PGJ₂, the modified proteins extracted by Neutravidin affinity‐purification and the proteins identified by LTQ Orbitrap mass spectrometer. Classification of the 358 identified proteins was performed using PANTHER classification system ( www.pantherdb.org ), showing that the proteins mapped to metabolic process, cellular process, and transport activity. This protein data set highlights the potential for 15d‐PGJ₂ to covalently modify cellular proteins and provides a source of data that will aid further studies on the mechanism of action of this endogenous regulator of inflammation.     

酵母双杂交筛选与GsCBRLK相互作用的蛋白质

GsCBRLK(calcium/calmodulin-binding receptor-like kinase from Glycine soja)在ABA及盐胁迫诱导的钙离子信号通路中起到关键的调节作用。为深入研究GsCBRLK蛋白的作用机制, 文章采用膜酵母双杂交系统, 以GsCBRLK为诱饵蛋白, 筛选与其相互作用的蛋白质。通过构建野生大豆盐胁迫条件下的cDNA文库、膜酵母双杂交系统筛选、复筛、回转验证、生物信息学分析以及酵母体内互作验证等手段, 最终获得2个(SNARE 和 14-3-3 蛋白)与GsCBRLK诱饵蛋白相互作用的蛋白质。