A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins

Brown, Hakeenah F.; Wang, Ling; Khadka, Sudip; Fields, Stanley; LaCount, Douglas

doi:10.1016/j.molbiopara.2011.04.005

Cited by 6 publications

(8 citation statements)

References 20 publications

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“…Equivalent amounts of the in vitro translated proteins were added to each binding reaction, incubated for 4 hours at 4° C with rotation, washed three times, eluted by boiling in Laemmli SDS-PAGE sample loading buffer, and subjected to western blot analysis. Glutathione S-transferase (GST) pull-down assays were performed with GST or GST-tagged PF3D7_0402000 (GST-D90c) that was in vitro translated using wheat germ extracts [40]. GST or GST-D90c were mixed with 3XFLAG-C-FLuc tagged ANK1 and 4.1R, incubated with GST beads (Thermo Scientific) overnight at 4° C in presence of protease inhibitor (PI) cocktail (Roche), washed three times with PBS+0.1% TritonX-100, eluted by boiling in Laemmli SDS-PAGE sample loading buffer, and subjected to western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

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The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

Shakya

Penn

Nakayasu

et al. 2017

Molecular and Biochemical Parasitology

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Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.

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Section: Methodsmentioning

confidence: 99%

“…“Vector only” indicates negative control diploid yeast expressing empty AD + DBD-D90c. Diploids containing PFE1350c and PFC0255c were included as a positive (+) control [34, 40]. …”

Section: Figmentioning

confidence: 99%

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

Shakya

Penn

Nakayasu

et al. 2017

Molecular and Biochemical Parasitology

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“…Split‐luciferase assays were performed essentially as described in Ref. , except that the binding reactions were incubated for 1 h. For an interaction to be considered significant, the luminescent signal must be higher than both control reactions as determined by a one‐tailed t ‐test ( P < 0.05).…”

Section: Methodsmentioning

confidence: 99%

Intrinsic disorder mediates hepatitis C virus core–host cell protein interactions

et al. 2014

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Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein-protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N-terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two-hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site-directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.

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“…Split-luciferase Assays-The split-luciferase assay was performed as described (50). Full length NS3 and NS5 were cloned into plasmid p424-BYDV-NFLUC by in vivo homologous recombination.…”

Section: Methodsmentioning

confidence: 99%

“…For these experiments we used the N-and C-terminal firefly luciferase fragments optimized for their ability to confer high signal to noise ratios and robust enzymatic activity (74). We previously adapted this system for use with in vitro translated proteins produced in wheat germ extracts (50). DENV proteins fused to the N-terminal luciferase fragment (NFLUC) and human proteins fused to the C-terminal luciferase fragment (CFLUC) were expressed in wheat germ extracts, mixed, and tested for luciferase activity after overnight incubation.…”

Section: Mapping the Denv-human Protein Interactionmentioning

confidence: 99%

A Physical Interaction Network of Dengue Virus and Human Proteins

Khadka

Vangeloff

Zhang

et al. 2011

Molecular & Cellular Proteomics

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148

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Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in

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A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins

Cited by 6 publications

References 20 publications

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

Intrinsic disorder mediates hepatitis C virus core–host cell protein interactions

A Physical Interaction Network of Dengue Virus and Human Proteins

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