Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Emmott, Edward; Goodfellow, Ian

doi:10.3791/51656

Cited by 22 publications

(20 citation statements)

References 16 publications

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“…The cutoff for differentially regulated proteins was set as described previously (22). Briefly, the Gaussian distribution of protein ratios was analyzed, and proteins with ratios deviating from the mean of the normally distributed data by 1.96 standard deviations (SDs) were considered differentially regulated ( Fig.…”

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confidence: 99%

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection

Xin

Deng

Chen

et al. 2017

J Virol

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Zika virus (ZIKV) is an emerging arbovirus belonging to the genus of the family During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes and are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

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confidence: 99%

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection

Xin

Deng

Chen

et al. 2017

J Virol

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“…Based on the Gaussian distribution of the quantitative ratio (log 2 value) list, the mean and the standard deviations value of the Gaussian distribution were calculated as previously described method (29).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses*

Zhou

Qin

et al. 2015

Molecular & Cellular Proteomics

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Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cyclerelated proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons. Molecular & Cellular

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“…HEK293T cells were cultured in Arg/Lys-free DMEM, supplemented with light (R0K0), medium (R6K4) or heavy (R10K8) amino acids, as described (32). 1x10 7 cells were transfected with 10 μg plasmid DNA using Lipofectamine 2000 (ThermoFisher).…”

Section: Stable Isotope Labelling With Amino Acids In Cell Culture Anmentioning

confidence: 99%

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Fleith

Mears

Emmott

et al. 2018

Preprint

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word count: 197Text word count: 9541 author/funder. All rights reserved. No reuse allowed without permission. AbstractInterferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and subsequent 40S recruitment. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 interacts rapidly and strongly with IFIT3 forming a stable heterotetramer. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that associates with IFIT1, forming an IFIT1:IFIT2:IFIT3 trimer. Site-directed mutagenesis revealed a C-terminal 'YxxxL' motif in IFIT1 that mediates its association with IFIT3. Using various reporter mRNAs, we demonstrate for the first time that IFIT3 stabilises IFIT1 binding to cap0-mRNA and enhances its translation inhibition activity. Disrupting the binding interface between IFIT1 and IFIT3 abrogated this enhancement. This work reveals molecular aspects of IFIT assembly and provides an important 'missing link' between IFIT interaction and function.author/funder. All rights reserved. No reuse allowed without permission. doi: bioRxiv preprint PCR amplified to contain 5' NdeI and 3' XhoI restriction sites for cloning into pET28b (Novagen) producing a full-length protein with thrombin cleavable N-terminal 6-His tag. For reporter RNA transcription, the firefly luciferase reporter gene (Fluc) was PCR amplified using primers containing the 5' UTR and 3' UTR sequences of human -globin (NM_000518.4), including a 5' T7 promotor and EcoRI and PstI sites to facilitate cloning into pUC57. pUC57-ZIKV-Fluc was previously described (30). Protein expression and purificationRecombinant IFITs were expressed in Rosetta 2 (DE3) pLysS (Novagen). Cells were grown to an OD600 of approximately 1 in 2x TY media at 37 C. Expression was induced by adding 1 mM isopropyl β-D-1thiogalactopyranoside. The induced culture was incubated at 22 C for 16 hours. Cells were harvested and lysed in a buffer containing 20 mM Tris-Cl pH 7.5, 400 mM KCl, 5 % glycerol, 1 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mg/ml lysozyme (from hen egg). IFITs were isolated by affinity chromatography on Ni-NTA Agarose beads (Qiagen). IFIT1 and IFIT1 mutants were additionally purified by FPLC on MonoQ (Q buffer: 20 mM Tris-Cl pH 7.5, 5 % glycerol, 1 mM DTT and 100-500 mM KCl), followed by MonoS 5/50 GL (S buffer: 30 mM HEPES pH 7.5, 5 % glycerol, 1 mM DTT and 100-500 mM KCl). IFIT2 and IFIT3 were treated with thrombin (from bovine plasma). author/funder. All rights reserved. No reuse allowed with...

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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Cited by 22 publications

References 16 publications

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection

Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses*

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

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