Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.

Identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using tandem affinity purification and mass spectrometry.

BACKGROUND: According to the World Health Organization Report, depressive disorders affect about 10% of the population. The molecular mechanism of the pathogenesis of depression is still not well understood. The new findings point to phosphatases as potential targets for effective depression therapy. The aim of the present work was the development of a method that would enable the identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using a proteomic approach. METHODS: The research was carried out using the PC12 cell line, often used as a model for neurobiological research. The use of the procedure for efficient purification of protein complexes-tandem affinity purification (TAP) will facilitate the identification of proteins interacting with MKP-1, a potential goal of effective antidepressant therapy. RESULTS: Identified proteins belong to various groups: cytoskeletal, ribosomal, nucleic acid binding, chaperones, and enzymes and may potentially be involved in the molecular mechanism of depression. CONCLUSIONS: The presented protocol for the purification of protein complexes is universal and can be successfully used in different mammalian cell lines. Proteins identified in the present work have been reported in the literature concerning studies on depressive disorders, which speaks in favour of their role in depression.

Isolation of Protein Complexes from Tobacco Leaves by a Two-Step Tandem Affinity Purification.

Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non-model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein-packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two-step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single-particle cryo-electron microscopy-we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1-XopQ complex.

A protein interaction landscape of breast cancer.

Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purification­mass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.

A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species.

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.

Tandem Affinity Purification and Mass Spectrometry (TAP-MS) for the Analysis of Protein Complexes.

Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

C9orf140, a novel Axin1-interacting protein, mediates the negative feedback loop of Wnt/ß-catenin signaling.

Wnt/ß-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants. However, the molecular components and the regulatory mechanisms involved in its fine-tuning remain to be determined. Here, we identified C9orf140, a tumor-specific protein, as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry. We further showed that C9orf140 is a negative regulator of Wnt/ß-catenin signaling in cultured cells as well as in zebrafish embryos. It functions upstream of ß-catenin, outcompetes PP2A for binding to Axin1, influences the balance between phosphorylation and de-phosphorylation of ß-catenin, and ultimately compromises Wnt3A-induced ß-catenin accumulation. Interestingly, Wnt-induced C9orf140 expression via ß-catenin. We propose that C9orf140 mediates a negative feedback loop of Wnt/ß-catenin signaling by interacting with Axin1. Our results advance the current understanding of the exquisite control of Wnt/ß-catenin signaling cascade, and provide evidence of the new role of C9orf140.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Systematic Analysis of Human Protein Phosphatase Interactions and Dynamics.

Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in contrast to kinases, much less is known about protein phosphatase functions and their interactions and complexes. Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors. We identified 838 high-confidence interactions, of which 631, to our knowledge, have not been reported before. We show that inhibiting the activity of phosphatases PP1 and PP2A by okadaic acid disrupts their specific interactions, supporting the potential of therapeutics that target these proteins. Additional analyses revealed candidate physical and functional interaction links to phosphatase-based regulation of several signaling pathways and to human cancer. Our study provides an initial glimpse of the protein interaction landscape of phosphatases and their functions in cellular regulation.