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.     

丙型肝炎病毒蛋白作用于细胞信号转导途径的研究进展

细胞信号转导异常往往与人类疾病的发生、发展密切相关。一些病毒致病和感染机制即为病毒抗原蛋白作用宿主细胞信号转导途径,导致宿主细胞内信号转导发生紊乱。丙型肝炎病毒（HCV）是引发慢性丙型肝炎,导致肝硬化和肝细胞癌发生的主要病原体,但目前HCV的致病机制与宿主内持续感染机制尚不清楚。HCV致病机制可能与HCV表达的蛋白质干扰宿主细胞信号转导途径而导致异常的细胞信号转导有关。研究HCV蛋白对宿主细胞信号转导途径的影响不仅有助于阐明其致病机制,还能为新药设计和寻找新的治疗方法提供新思路和新靶点。本文主要综述了近年来国内外有关HCV蛋白作用细胞信号转导途径的研究进展。     

Proteomics and leukocytes: an approach to understanding potential molecular mechanisms of inflammatory responses

Leukocytes play an important role in the progression of disease and leukocyte-derived proteins are associated with the pathogenesis of the disease. Leukocyte activation causes production of inflammatory mediators, over-expression of cell surface adhesion molecules, and an increase in migration and infiltration, phagocytosis, and degranulation, as well as receptor phosphorylation and signal transduction. An increasing number of studies on the application of leukocyte proteomics have appeared in mapping protein profiles of inflammatory cells, contributing to the understanding of potential mechanisms involved in leukocyte function. Together with improvements in proteomic technology in leukocyte research, leukocyte proteomic analysis becomes more simple, rapid, flexible, sensitive, and specific. This enables proteomic investigation of activated or non-activated leukocytes to be highly focused on defined suborgans or specific signaling pathways. Research in leukocyte proteomics is progressing from fingerprinting to functioning, human cell lines to primary leukocytes, non-activated cells to inflammatory mediator-stimulated cells, in vitro culture to in vivo challenge, and animal models to human disease. A number of newly identified proteins from leukocyte proteomics may offer new mechanism-orientated targets for drug discovery and development.     

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).     

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.     

In silico reconstruction of nutrient-sensing signal transduction pathways in Aspergillus nidulans

We report here probable nutrient-sensing signal transduction pathways in Aspergillus nidulans, a model filamentous fungus, based on sequence homology studies with known Saccharomyces cerevisiae and Schizosaccharomyces pombe proteins. Specifically, we identified A. nidulans homologs for yeast proteins involved in (1) filamentation-invasion, (2) cAMP-PKA, (3) pheromone response, (4) cell integrity and (5) TOR signaling pathways. We have also studied autophagy, one of the most important cellular responses regulated by TOR signaling. The Basic Local Alignment Search Tool program "blastp" was used to assess the homology of proteins. We note that by using a highly conservative approach, 70% of the S. cerevisiae signal transduction proteins (107 proteins out of 153 proteins studied) have significant homologs in A. nidulans. Using a slightly less conservative approach, we are able to identify homologs for as high as 91% of the S. cerevisiae signal transduction proteins (139 proteins out of 153 proteins studied). The filamentation-invasion, cell integrity and TOR signaling pathways showed greatest similarity with S. cerevisiae, while the cAMP-PKA and pheromone response pathways showed greater similarity with S. pombe. Based on these results, probable pathways in A. nidulans were constructed using well-established S. cerevisiae and S. pombe models.     

Understanding signaling in yeast: Insights from network analysis

Reconstruction of protein interaction networks that represent groups of proteins contributing to the same cellular function is a key step towards quantitative studies of signal transduction pathways. Here we present a novel approach to reconstruct a highly correlated protein interaction network and to identify previously unknown components of a signaling pathway through integration of protein-protein interaction data, gene expression data, and Gene Ontology annotations. A novel algorithm is designed to reconstruct a highly correlated protein interaction network which is composed of the candidate proteins for signal transduction mechanisms in yeast Saccharomyces cerevisiae. The high efficiency of the reconstruction process is proved by a Receiver Operating Characteristic curve analysis. Identification and scoring of the possible linear pathways enables reconstruction of specific sub-networks for glucose-induction signaling and high osmolarity MAPK signaling in S. cerevisiae. All of the known components of these pathways are identified together with several new "candidate" proteins, indicating the successful reconstructions of two model pathways involved in S. cerevisiae. The integrated approach is hence shown useful for (i) prediction of new signaling pathways, (ii) identification of unknown members of documented pathways, and (iii) identification of network modules consisting of a group of related components that often incorporate the same functional mechanism.     

Src 在胃癌中的作用

Src是非受体型酪氨酸激酶家族成员之一,通过广泛的信号转导通路参与HP感染、胃癌细胞粘附、受体调节、增殖和血管形成等。近年来研究表明,Src在胃癌组织中异常表达或活性增高,与胃癌的发生发展密切相关。因此,研究Src和胃癌的关系有着重要意义。目前,针对Src的靶向抑制剂已经进入临床试验。大量研究发现,Src抑制剂包括Dasatinib、AZD0530、Sunitinib等对胃癌有抑制作用,这将为胃癌的治疗开辟新的治疗途径。     

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus–host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.     

Lipopolysaccharide-responsive phosphoproteins in Nicotiana tabacum cells.

Mounting evidence is merging to affirm the effectiveness of bacterial lipopolysaccharides (LPS) as biological control agents, inducers of innate immunity, and to stimulate/potentiate the development of defense responses in plants through protein phosphorylation-mediated signal perception/transduction responses. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins. Substantial differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. In this study, qualitative and quantitative changes in phosphoproteins of Nicotiana tabacum suspension cells during elicitation by LPS from the Gram-negative bacteria, Burkholderia cepacia, were analyzed using two-dimensional electrophoresis in combination with a phosphoprotein-specific gel stain. Trypsin digested phosphoproteins were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and nano-electrospray-ionization liquid chromatography tandem mass spectrometry (nano-ESI-LC/MS/MS). A total of 27 phosphoproteins were identified from 23 excised gel spots. The identified phosphoproteins indicate that LPS(B.cep)-induced signal perception/transduction involves G-protein coupled receptor signaling, Ca(2+)/calmodulin-dependent signaling pathways, H(+)-ATPase regulation of intracellular pH, thioredoxin-mediated signaling and phosphorylation of 14-3-3 regulatory proteins. Other targets of LPS(B.cep)-responsive phosphorylation included NTP pool maintenance, heat shock proteins, protein biosynthesis and chaperones as well as cytoskeletal tubulin. The results add novel insights into the biochemical process of LPS perception and resulting signal transduction.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling     

应用磷酸化蛋白质组学方法初步研究喉癌相关基因 LCRG1 的功能

mRNA 差异显示技术克隆的喉癌相关基因 LCRG1 ,对不表达该基因的喉癌细胞系 (Hep-2) 的生长具有明显抑制作用 . 软件分析推测, LCRG1 可能在细胞信号传导中发挥作用 . 为进一步地研究 LCRG1 的功能,应用 RT-PCR 和平板克隆形成实验证实,经多次传代的 Hep-2/LCRG1 细胞,仍表达 LCRG1 ,且 LCRG1 具有显著的抑制细胞增殖的能力 . 抽提 Hep-2/LCRG1 和 Hep-2/pcDNA3.1(+) 细胞系总蛋白质,应用固相 pH 梯度 (IPG) 双向凝胶电泳 (2DGE) ,结合抗酪氨酸磷酸化抗体的免疫印迹和基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF-MS) ,鉴定酪氨酸磷酸化的蛋白质 . 得到了分辨率较高、重复性较好的 Hep-2/LCRG1 和 Hep-2/pcDNA3.1(+) 细胞系的总蛋白质双向凝胶电泳图谱;结合免疫印迹反应、软件分析和质谱技术识别并鉴定了 13 个差异反应的酪氨酸磷酸化的蛋白质 . 这些蛋白质参与了细胞信号传导和细胞代谢等过程 . 推测 LCRG1 可能是通过调节这些蛋白质的酪氨酸磷酸化、去磷酸化状态,参与细胞增殖、代谢和凋亡等过程的调控,而发挥抑瘤作用 . 这为全面、真实地揭示 LCRG1 抑瘤作用的分子机理提供了新思路 .     

The kinase domain of CK1δ can be phosphorylated by Chk1

ABSTRACT

Members of the casein kinase 1 (CK1) family are key regulators in numerous cellular signal transduction pathways and in order to prevent the development of certain diseases, CK1 kinase activity needs to be tightly regulated. Modulation of kinase activity by site-specific phosphorylation within the C-terminal regulatory domain of CK1δ has already been shown for several cellular kinases. By using biochemical methods, we now identified residues T161, T174, T176, and S181 within the kinase domain of CK1δ as target sites for checkpoint kinase 1 (Chk1). At least residues T176 and S181 show full conservation among CK1δ orthologues from different eukaryotic species. Enzyme kinetic analysis furthermore led to the hypothesis that site-specific phosphorylation within the kinase domain finally contributes to fine-tuning of CK1δ kinase activity. These data provide a basis for the extension of our knowledge about the role of site-specific phosphorylation for regulation of CK1δ and associated signal transduction pathways.     

Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic analysis of SET-binding proteins

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2-DE and identified by MS using a MALDI-TOF. This experimental approach allowed the recognition of a number of SET-binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2alpha, glycogen phosphorylase (GP), and TCP1-beta) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein-protein interactions.