Dataset Information

Evaluation of a bead-free immunoprecipitation technique coupled with tandem mass spectrometry for identification of plant-virus protein interactions (DeBlasio_JBT_2017)

ABSTRACT: Potato leafroll virus (PLRV) is an aphid-borne, RNA virus in the Luteoviridae that causes significant loss to potato production worldwide. Precision methods to block virus infection in plants or the transmission of a virus by the insect represent new approaches to virus disease management and require an in depth knowledge of the protein interactions used by viruses in their plant hosts and aphid vectors. In this study, we evaluated the feasibility of using an inexpensive, bead-free assay to identify plant proteins that interact with wild-type (WT) and PLRV mutant form. Comparison of host and viral proteins identified as enriched ³2-fold in both WT and mutant experiments using the bead-free method revealed a ~65% overlap with proteins identified as forming high-confident interactions with PLRV using antibody coated-magnetic beads with 19 of these proteins also detected as significant interactions in the bead-free assay. An additional 18 virus-host interactions were only identified in the bead-free assay. Two prey proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2, were identified as having a weakened or lost association with the mutant form of the virus in both immunoprecipitation assays, showing that the method is sensitive enough to detect quantitative differences between different, yet closely related viral bait proteins. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to reproducibly identify plant and viral proteins interacting with virions.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Nicotiana Benthamiana

TISSUE(S): Plant

SUBMITTER: Stacy DeBlasio

LAB HEAD: Michelle Cilia

PROVIDER: PXD006338 | Pride | 2018-10-24

REPOSITORIES: Pride

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Publications

Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

DeBlasio Stacy L SL Bereman Michael S MS Mahoney Jaclyn J Thannhauser Theodore W TW Gray Stewart M SM MacCoss Michael J MJ Cilia Heck Michelle M

Journal of biomolecular techniques : JBT 20170724 3

Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way ...[more]

PMID: 28785175

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Project description:Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated the kinetic transcriptional profile of gene expression in macrophages infected with Brucella melitensis strain 16M. A key aspect of Brucella virulence is their ability to proliferate within professional and nonprofessional phagocytic host cells, thereby successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells. Defining the interaction between a host cell and Brucella is crucial to understanding the infectious process. Most researchers have studied the pathogens, but the host plays a very important role during infections. To date, relatively few host factors have been shown important in Brucella infections. However, little is known about the host networks that mediate infection. The objective of the study is to analyze the genes and cellular components related to the innate immunity response to determine the mechanisms through which Brucella avoids the host innate immunity. A total of 3576 and 3962 genes that are differentially expressed between 0 and 4 h and between 0 and 24 h were identified. The identified genes are related to immune processes, signal transduction, inflammation, apoptosis, cell membrane, transcriptional regulation, and intracellular trafficking. Our data have added to the current understanding of different host gene expressions during different infection phases by Brucella spp. The RAW264.7 cells were seeded into 24-well plates at 5-105 cells/well, incubated in 5% CO2 at 37 M-BM-0C for 24 h, and then infected with Brucella at a multiplicity of infection of 200. To synchronize the infection, the infected plates were centrifuged at 200 ug for 5 min at room temperature and then incubated at 37 M-BM-0C for 20 minutes. The infected cell monolayers were washed three times with PBS, overlaid with 0.5 ml of DMEM containing 100 mg/ml of ampicillin and 50 mg/ml of kanamycin at 37 M-BM-0C for 0, 4, and 24 h. At the end of the incubation period, the culture medium was removed and centrifuged at 1500 rpm for 15 min. The cell pellet and adherent cells were resuspended in TRIzol. Total RNA were extracted from the cells at 0, 4 and 24 h post infection.

Project description:Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level are known to take part in the control of bone formation and bone resorption. Recently, targeted secretion of miRNAs from cells originating from various tissues has been described, which allows for their minimal-invasive detection in serum/plasma and use as biomarkers for presence and progression of pathological conditions. One pilot study has reported circulating miRNAs in serum and tissue of fracture patients. However, further studies are required to explore whether a dysbalance in bone homeostasis of fracture patients can reliably be reflected by specific circulating miRNAs, and whether these miRNAs might serve as drugable targets. Here, we report results from a comprehensive multiplex study of 175 miRNAs in serum samples obtained from 7 patients with osteoporotic fractures at the femoral neck, and 7 age-matched controls. Following elaborate quality control statistical analysis of this exploratory dataset identified 9 microRNAs with altered serum levels in response to fracture (adjusted p-value < 0.1). Of these, hsa-miR-10a/b gave excellent discrimination of both groups (AUC = 1.0), and clustering of samples based on the top10 miRNAs confirmed the high discriminatory power of circulating microRNAs for osteoporotic fractures. In the next step 3 miRNAs with unknown roles in osteogenic differentiation and 4 miRNA from a previous study were tested for their effects on osteogenic differentiation. Of these, 3 miRNAs showed robust effects on osteogenic differentiation. Overall, these data provide important insights into changes in serum miRNA in post-traumatic patients. Future studies will show, whether this knowledge can be used to improve current diagnostic methodologies to predict fracture risk and design novel treatment strategies for osteoporosis patients. Two groups with n=7 per group; one groups represents cases with osteoporotic fractures, the control group is age-matched without fractures

Dataset Information

Evaluation of a bead-free immunoprecipitation technique coupled with tandem mass spectrometry for identification of plant-virus protein interactions (DeBlasio_JBT_2017)

Publications

Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

Similar Datasets

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