scholarly journals A Comprehensive Proteomics Analysis of the JC Virus (JCV) Large and Small Tumor Antigen Interacting Proteins: Large T Primarily Targets the Host Protein Complexes with V-ATPase and Ubiquitin Ligase Activities While Small t Mostly Associates with Those Having Phosphatase and Chromatin-Remodeling Functions

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1192
Author(s):  
Sami Saribas ◽  
Mahmut Safak
Keyword(s):  
Tumor Antigens ◽  
Jc Virus ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Host Protein ◽  
Proteomics Data ◽  
Host Proteins ◽  
Oncogenic Potential ◽  
Oncogenic Pathways ◽  
Biochemical Assays

The oncogenic potential of both the polyomavirus large (LT-Ag) and small (Sm t-Ag) tumor antigens has been previously demonstrated in both tissue culture and animal models. Even the contribution of the MCPyV tumor antigens to the development of an aggressive human skin cancer, Merkel cell carcinoma, has been recently established. To date, the known primary targets of these tumor antigens include several tumor suppressors such as pRb, p53, and PP2A. However, a comprehensive list of the host proteins targeted by these proteins remains largely unknown. Here, we report the first interactome of JCV LT-Ag and Sm t-Ag by employing two independent “affinity purification/mass spectroscopy” (AP/MS) assays. The proteomics data identified novel targets for both tumor antigens while confirming some of the previously reported interactions. LT-Ag was found to primarily target the protein complexes with ATPase (v-ATPase and Smc5/6 complex), phosphatase (PP4 and PP1), and ligase (E3-ubiquitin) activities. In contrast, the major targets of Sm t-Ag were identified as Smarca1/6, AIFM1, SdhA/B, PP2A, and p53. The interactions between “LT-Ag and SdhB”, “Sm t-Ag and Smarca5”, and “Sm t-Ag and SDH” were further validated by biochemical assays. Interestingly, perturbations in some of the LT-Ag and Sm t-Ag targets identified in this study were previously shown to be associated with oncogenesis, suggesting new roles for both tumor antigens in novel oncogenic pathways. This comprehensive data establishes new foundations to further unravel the new roles for JCV tumor antigens in oncogenesis and the viral life cycle.

scholarly journals Insights Into the Polerovirus–Plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry

2015 ◽  
Vol 28 (4) ◽  
pp. 467-481 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Richard Johnson ◽  
Jaclyn Mahoney ◽  
Alexander Karasev ◽  
Stewart M. Gray ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Fixation ◽  
Dynamic Range ◽  
High Resolution Mass Spectrometry ◽  
Protein Complexes ◽  
Interaction Network ◽  
Host Protein ◽  
Host Proteins ◽  
Leafroll Virus ◽  
Readthrough Protein

Identification of host proteins interacting with the aphidborne Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step toward understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to coimmunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells, overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and consisted of host proteins interacting directly or indirectly with virions, as well as the nonincorporated readthrough protein (RTP) and three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGO and STRING showed that plant proteins in the PLRV protein interaction network regulate key biochemical processes, including carbon fixation, amino acid biosynthesis, ion transport, protein folding, and trafficking.

scholarly journals A SARS-CoV-2 – host proximity interactome

2020 ◽  
Author(s):  
Payman Samavarchi-Tehrani ◽  
Hala Abdouni ◽  
James D.R. Knight ◽  
Audrey Astori ◽  
Reuben Samson ◽  
...  
Keyword(s):  
Host Cell ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Drug Repurposing ◽  
Viral Proteins ◽  
Host Protein ◽  
Virus Protein ◽  
Host Proteins ◽  
Biochemical Purification ◽  
Cell Functions

AbstractViral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

scholarly journals The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiao-Tao Zeng ◽  
Xiao-Ti Yu ◽  
Wei Cheng
Keyword(s):  
Subcellular Localization ◽  
Hela Cells ◽  
Virus Entry ◽  
Affinity Purification ◽  
Cell Types ◽  
Host Protein ◽  
Cysteine Residues ◽  
Host Proteins ◽  
S Protein ◽  
Affinity Purification Mass Spectrometry

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein’s subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear. Methods The palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase. Results In this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein’s subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2. Conclusions ZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

Affinity Tags for Protein Purification

2020 ◽  
Vol 21 (8) ◽  
pp. 821-830
Author(s):  
Vibhor Mishra
Keyword(s):  
Protein Purification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Domain ◽  
Affinity Tag ◽  
Small Peptides ◽  
Affinity Tags ◽  
C Terminus ◽  
Solubility Enhancers ◽  
As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

scholarly journals Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

2015 ◽  
Vol 118 ◽  
pp. 81-94 ◽  
Author(s):  
Jean-Philippe Lambert ◽  
Monika Tucholska ◽  
Christopher Go ◽  
James D.R. Knight ◽  
Anne-Claude Gingras
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Interactome Mapping

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

scholarly journals Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

10.3791/58532 ◽  
2018 ◽  
Author(s):  
Venkatasalam Shanmugabalaji ◽  
Véronique Douet ◽  
Birgit Agne ◽  
Felix Kessler
Keyword(s):  
Protein Complexes ◽  
Affinity Purification

scholarly journals Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

2015 ◽  
Vol 16 (9) ◽  
pp. 22456-22472 ◽  
Author(s):  
Yangchao Dong ◽  
Jing Yang ◽  
Wei Ye ◽  
Yuan Wang ◽  
Chuantao Ye ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Streptavidin Aptamer ◽  
Rna Protein Complexes

scholarly journals RGEN-seq For Highly Sensitive Amplification-free Screen Of Off-target Sites Of Gene Editors

2021 ◽  
Author(s):  
Alexander Kuzin ◽  
Brendan Redler ◽  
Jaya Onuska ◽  
Alexei Slesarev
Keyword(s):  
Target Detection ◽  
Dna Cleavage ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Detailed Comparison ◽  
Detection Methods ◽  
Guide Rnas ◽  
Coverage Bias ◽  
Biochemical Assays ◽  
Target Sites

Abstract Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain.. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

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