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Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated KC channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases. [ABSTRACT FROM AUTHOR]

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin-binding peptide (CBP) and two immunoglobulin G (IgG)-binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG-coated beads, TEV protease cleavage releases CBP-tagged bait protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: Heterochromatin protein 1 (HP1) was first described in Drosophila melanogaster as a heterochromatin associated protein required for epigenetic gene silencing. Most eukaryotes have at least three HP1 homologs that play differential roles in heterochromatin and euchromatin. However, despite the fact that the three HP1 proteins bind to different regions of the genome, several studies show that most of the interactions occur in a manner specific to HP1a. In addition, little is known about the overall interaction network of the three Drosophila HP1 homologs, HP1a, HP1b, and HP1c. Here, we performed the first comprehensive proteomic analysis of Drosophila HP1 homologs by coupling a double-affinity purification approach with MudPIT analysis to identify interacting proteins of Drosophila HP1. We discovered 160–310 proteins co-eluted with HP1, including a number of novel HP1-binding partners along with the previously identified HP1 binding proteins. Finally, we showed that slight and unique binding preferences might exist between the three HP1 proteins in Drosophila. These studies are the first to systematically analyze the interactome of HP1 paralogs and provide the basic clues as to the molecular mechanism by which HP1 might control cellular processes. Biological significance: Most eukaryotes have at least three HP1 homologs with similar domain structures but with differential roles in heterochromatin and euchromatin. However, little is known about the overall interactome of the three Drosophila HP1 homologs, HP1a, HP1b, and HP1c. The present study compared interacting proteins of three HP1 homologs in Drosophila. To better understand the underlying mechanisms for gene regulation of HP1, a double-affinity purification and MudPIT mass spectrometry were employed to identify differential proteins as well as common binding proteins of HP1. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the HP1 homolog-specific function. [Copyright &y& Elsevier]

Abstract: We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. [Copyright &y& Elsevier]

Abstract: Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby messenger ribonucleoprotein (mRNP) assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes, suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus. [Copyright &y& Elsevier]

Protein-protein interactions play a pivotal role in both inter- and intra-cellular signaling. Identification of signaling protein complexes can thus shed important new insights into cell communications. We developed a parallel affinity precipitation protocol to overcome the disadvantages of the tandem affinity purification procedure, such as the potential disruption of target protein conformation, subcellular localization or function by epitope tags, the potential need of large amounts of cell culture or generation of stable cell lines, and relatively long duration the two-step precipitation takes. This new simplified assay of protein interaction is quick, economic and specific. This paper describes the details in the design and method of the assay. [ABSTRACT FROM AUTHOR]

In fat and muscle cells, insulin-stimulated glucose uptake is mainly mediated by glucose transporter 4 (GLUT4), which translocates from intracellular compartments to the cell surface in response to insulin stimulation. AS160 is one of the substrates of Akt and plays important roles in insulin-regulated GLUT4 translocation. In this study, RuvB-like protein 2 (RUVBL2) is identified as a new AS160-binding protein using mammalian tandem affinity purification (TAP) combined with mass spectrometry. In 3T3-L1 adipocytes, RUVBL2 is highly expressed and is mainly distributed in the cytosol. Depletion of RUVBL2 in adipocytes inhibits insulin-stimulated GLUT4 translocation and glucose uptake through reducing insulin-stimulated AS160 phosphorylation. However, introduction of human RUVBL2 can reverse this inhibitory effect. These data suggest that RUVBL2 plays an important role in insulin-stimulated GLUT4 translocation through its interaction with AS160. [ABSTRACT FROM AUTHOR]

Abstract: TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1β and TNFα signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1β-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1β-dependent manner. Overexpression of FBXW5 inhibited IL-1β-induced activation of JNK/p38 MAPKs and NF-κB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1β stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1β signaling pathway. [Copyright &y& Elsevier]

Abstract: Association of serine/threonine phosphatases with regulatory proteins is a key component of their specificity, and the identification of these binding partners is critical to understanding phosphatases function and regulation. Affinity-purification/mass spectrometry (AP-MS) approaches have been and continue to be instrumental in identifying these interactors. Here, we review the general principles of AP-MS, and present two affinity-purification protocols compatible with subsequent mass spectrometry, namely FLAG purification, and the tandem affinity purification (TAP). We have successfully used these protocols for the identification of binding partners for PP2A, PP4 and PP6, and they should be amenable to the analysis of interactors for other phosphatases. [Copyright &y& Elsevier]

The identification and characterization of protein interactions is a key topic in current life science research; a huge variety of methodologies have been established in recent years to expedite research in this area. Generic methods have been established for monitoring protein interactions in vivo by protein fragment complementation and for screening protein interactions in vitro by highly parallel solid-phase techniques. Substantial progress has been made in identifying and characterizing interactions with and between membrane proteins. Studying protein interactions on the single-molecule level has become an important tool for understanding protein function in vivo and in vitro. [Copyright &y& Elsevier]

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved. [ABSTRACT FROM AUTHOR]

Recent technological advances in genomics, proteomics and bioinformatics have offered new insights into the molecular mechanisms that underlie lymphocyte signaling and function, and the development of new tools in these areas has opened up new avenues for biological investigation. By adding a quantitative dimension to lymphocyte proteome profiling, molecular machines and spatiotemporal regulatory processes can now be analyzed using such discovery-driven approaches. Biologists employing genomic and proteomic tools are gathering data at increasing speed and their struggle to extract maximal biological information is helped by new software tools that enable the detailed comparison of multiple datasets. [Copyright &y& Elsevier]

Recent large-scale studies of protein complexes in yeast have demonstrated that the wide majority of proteins exist in the cell as parts of multicomponent assemblies, mostly novel and of unknown function. The structural and functional analysis of these complexes should be a priority for structural biologists in coming years. In silico methods such as docking simulations, which may contribute to this analysis, are being tested in the CAPRI community-wide experiment, which assesses blind predictions of the structure of protein–protein complexes. [Copyright &y& Elsevier]

Computational methods play an important role at all stages of the process of determining protein–protein interactions. They are used to predict potential interactions, to validate the results of high-throughput interaction screens and to analyze the protein networks inferred from interaction databases. [Copyright &y& Elsevier]

The availability of complete, annotated genome sequences for a variety of eukaryotic organisms has paved the way for a paradigm shift in biomedical research from the ‘one gene–one hypothesis’ approach to more global, systematic strategies that analyse genes or proteins on a genome- and proteome-wide scale. One daunting task in the post-genome era is to determine how the complement of expressed cellular proteins — the proteome — is organised into functional, higher-order networks, by mapping all constitutive and dynamic protein–protein interactions. Traditionally, reductionist approaches have typically focused on a few, selected gene products and their interactions in a particular physiological context. In contrast, more holistic strategies aim at understanding complex biological systems, for example global protein–protein interaction networks on a cellular or organismal level. Several large-scale proteomics technologies have been developed to generate comprehensive, cellular protein–protein interaction maps. [Copyright &y& Elsevier]

The availability of complete genome sequences for a variety of organisms, coupled with novel approaches that allow evaluation of the functions of thousands of genes in parallel, have the potential to greatly impact on cell biology research. Functional genomic approaches in Saccharomyces cerevisiae are beginning to make significant contributions to the understanding of complex biological systems. [Copyright &y& Elsevier]

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.