Proteomic approaches to the analysis of multiprotein signaling complexes

Signal transduction is one of the most active fields in modern biomedical research. Increasing evidence has shown that signaling proteins associate with each other in characteristic ways to form large signaling complexes. These diverse structures operate to boost signaling efficiency, ensure specificity and increase sensitivity of the biochemical circuitry. Traditional methods of protein analysis are inadequate to fully characterize and understand these structures, which are intricate, contain many components and are highly dynamic. Instead, proteomics technologies are currently being applied to investigate the nature and composition of multimeric signaling complexes. This review presents commonly used and potential proteomic methods of analyzing diverse protein complexes along with a discussion and a brief evaluation of alternative approaches. Challenges associated with proteomic analysis of signaling complexes are also discussed.     

The integration of signaling by multiprotein complexes containing Raf kinases

In vivo, eukaryotic cells are subjected simultaneously to a broad array of signals ranging from mitogens and inflammatory inputs to environmental stresses and developmental cues. The combinatorial nature of cellular signaling necessitates that a cell integrate its signal transduction pathways so as to implement rapidly and efficiently an appropriate suite of responses. Emerging evidence indicates that, over the course of evolution, cells have developed multiprotein signaling complexes, or "signalosomes" that mediate the coordinate regulation of different signaling pathways. Such molecular signal integration contrasts with the classical notion of signaling complexes assembled by scaffold proteins-entities that function to segregate specific pathways from one another. This review will focus on two signal integrating multiprotein complexes that involve Raf family kinases: the MLK3-B-Raf-Raf-1 complex and the Raf-1-Mst-2 complex.     

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.     

Baculovirus expression system for heterologous multiprotein complexes

The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.     

MultiBac: expanding the research toolbox for multiprotein complexes

Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.     

Disassembly of intact multiprotein complexes in the gas phase

The observation of multiprotein complexes by mass spectrometry formerly relied upon chemical cross-linking to maintain interactions. Recent technological developments have enabled the observation of intact macromolecular complexes without modification. These assemblies, with masses far in excess of those measured previously, can be examined through controlled dissociation in the mass spectrometer, revealing information about their subunit interactions and topology.     

Mapping multiprotein complexes by affinity purification and mass spectrometry

The combination of affinity purification and tandem mass spectrometry (MS) has emerged as a powerful approach to delineate biological processes. In particular, the use of epitope tags has allowed this approach to become scaleable and has bypassed difficulties associated with generation of antibodies. Single epitope tags and tandem affinity purification (TAP) tags have been used to systematically map protein complexes generating protein interaction data at a near proteome-wide scale. Recent developments in the design of tags, optimisation of purification conditions, experimental design and data analysis have greatly improved the sensitivity and specificity of this approach. Concomitant developments in MS, including high accuracy and high-throughput instrumentation together with quantitative MS methods, have facilitated large-scale and comprehensive analysis of multiprotein complexes.     

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.     

Proteomics of multiprotein complexes: answering fundamental questions in neuroscience

Proteomics tools offer new ways to analyse networks of proteins that control important neurobiological phenomena such as learning and memory. In this review, we discuss how a combined proteomic, pharmacological and genetic approach reveals that multiprotein complexes process neural information and encode memories. Simultaneous analysis of multiple proteins enables the development of new concepts and approaches for neuroscience research.     

Polycystins as components of large multiprotein complexes of polycystin interactors

Naturally occurring mutations in two separate genes, PKD1 and PKD2, are responsible for the vast majority of all cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases affecting 1 in 1000 Americans. The hallmark of ADPKD is the development of epithelial cysts in the kidney, liver, and pancreas. PKD1 encodes a large plasma membrane protein (PKD1, PC1, or Polycystin-1) with a long extracellular domain and has been speculated to function as an atypical G protein coupled receptor. PKD2 encodes an ion channel of the Transient Receptor Potential superfamily (TRPP2, PKD2, PC2, or Polycystin-2). Despite the identification of these genes more than 20 years ago, the molecular function of their encoded proteins and the mechanism(s) by which mutations in PKD1 and PKD2 cause ADPKD remain elusive. Genetic, biochemical, and functional evidence suggests they form a multiprotein complex present in multiple locations in the cell, including the plasma membrane, endoplasmic reticulum, and the primary cilium. Over the years, numerous interacting proteins have been identified using directed and unbiased approaches, and shown to modulate function, cellular localization, and protein stability and turnover of Polycystins. Delineation of the molecular composition of the Polycystin complex can have a significant impact on understanding their cellular function in health and disease states and on the identification of more specific and effective therapeutic targets.     

Proteomics of multiprotein complexes: answering fundamental questions in neuroscience

Proteomics tools offer new ways to analyse networks of proteins that control important neurobiological phenomena such as learning and memory. In this review, we discuss how a combined proteomic, pharmacological and genetic approach reveals that multiprotein complexes process neural information and encode memories. Simultaneous analysis of multiple proteins enables the development of new concepts and approaches for neuroscience research.     

Molecular basis for chromatin assembly and modification by multiprotein complexes

Abstract: Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post‐translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.     

Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding

Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A–ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.     

Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles

Among bacterial cell envelopes, the Borrelia burgdorferi outer membrane (OM) is structurally unique in that the identities of many protein complexes remain unknown; however, their characterization is the first step toward our understanding of membrane protein interactions and potential functions. Here, we used two-dimensional blue native/SDS-PAGE/mass spectrometric analysis for a global characterization of protein-protein interactions as well as to identify protein complexes in OM vesicles isolated from multiple infectious sensu stricto isolates of B. burgdorferi. Although we uncovered the existence of at least 10 distinct OM complexes harboring several unique subunits, the complexome is dominated by the frequent occurrence of a limited diversity of membrane proteins, most notably P13, outer surface protein (Osp) A, -B, -C, and -D and Lp6.6. The occurrence of these complexes and specificity of subunit interaction were further supported by independent two-dimensional immunoblotting and coimmunoprecipitation assays as well as by mutagenesis studies, where targeted depletion of a subunit member (P66) selectively abolished a specific complex. Although a comparable profile of the OM complexome was detected in two major infectious isolates, such as B31 and 297, certain complexes are likely to occur in an isolate-specific manner. Further assessment of protein complexes in multiple Osp-deficient isolates showed loss of several protein complexes but revealed the existence of additional complex/subunits that are undetectable in wild-type cells. Together, these observations uncovered borrelial antigens involved in membrane protein interactions. The study also suggests that the assembly process of OM complexes is specific and that the core or stabilizing subunits vary between complexes. Further characterization of these protein complexes including elucidation of their biological significance may shed new light on the mechanism of pathogen persistence and the development of preventative measures against the infection.