Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein–protein interactions and complexes

Miyamoto‐Sato, Etsuko; Ishizaka, Masamichi; Horisawa, Kenichi; Tateyama, Seiji; Takashima, Hideaki; Fuse, Shinichiro; Sue, Kaori; Hirai, Naoya; Masuoka, Kazuyo; Yanagawa, Hiroshi

doi:10.1101/gr.3510505

Cited by 44 publications

(90 citation statements)

References 35 publications

(62 reference statements)

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“…IVV screening is a powerful tool for identifying biologic macromolecules that participate in protein-protein interactions (15)(16)(17). By using this approach, we identified PKM2 as a mediator of the promotion of the glycolytic phenotype of cancer cells by CD44.…”

Section: Discussionmentioning

confidence: 99%

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Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

et al. 2012

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An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance. Cancer Res; 72(6); 1438-48. Ó2012 AACR.

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Section: Discussionmentioning

confidence: 99%

“…IVV screening and GST pull-down assay IVV selection and glutathione S-transferase (GST) pulldown assay were conducted as described previously (15)(16)(17). The cDNA library for screening was obtained from U251MG cells.…”

Section: Cell Proliferation Assaymentioning

confidence: 99%

Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

et al. 2012

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“…This method has added advantage of post-translation modification of the bait protein, such as tyrosine phosphorylation of SHPS-1, prior to selection as compared with other methods such as in vitro virus (24). We determined that SHPS-1 interacts with several hitherto uncharacterized binding partners, which represent several functional categories including stress response proteins, protein kinases and phosphatases, GTP-binding proteins, cytoskeletal proteins, and transcription regulatory proteins.…”

Section: Shps-1/cd Is Required For Igf-i-dependent Protein Synthesis-mentioning

confidence: 99%

“…The role of STAT-1 and SUPT6H is less clear. The differences in the interactions of STAT1 and SUPT6H with SHPS-1 in vitro and in vivo could be explained by the intracellular concentrations of these proteins or by the presence of other co-factors that are present in vivo that regulate their recruitment (19,21,24). However, The Novel Role of SHPS-1 in Mediating IGF-I Signaling further study will be required to determine the significance of their association with or dissociation from SHPS-1 for downstream signaling.…”

Section: Shps-1/cd Is Required For Igf-i-dependent Protein Synthesis-mentioning

confidence: 99%

“…Two approaches recently used to identify protein binding partners are mRNA display techniques, such as mRNA-protein fusions (17)(18)(19)(20)(21) and in vitro virus, based on cell-free co-translation and affinity selection (22)(23)(24) and tandem affinity purification (TAP)-mass spectrometry methods. These methods are well suited for identifying the components of complexes that form in near physiological conditions.…”

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Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Shen

Radhakrishnan

et al. 2009

Molecular & Cellular Proteomics

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Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2⅐p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF

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Integration of cell‐free protein coexpression with an enzyme‐linked immunosorbent assay enables rapid analysis of protein–protein interactions directly from DNA

Layton

Hellinga

2011

Protein Science

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Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody-affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed.

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Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein–protein interactions and complexes

Cited by 44 publications

References 35 publications

Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Integration of cell‐free protein coexpression with an enzyme‐linked immunosorbent assay enables rapid analysis of protein–protein interactions directly from DNA

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