Characterization Of Protein Complexes Research Articles

The conditional mitochondrial protein complexome in the Arabidopsis thaliana root and shoot

Protein complexes are important for almost all biological processes. Hence, to fully understand how cells work, it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues. Moreover, the dynamics of protein interaction play crucial roles in regulating the (dis)association of protein complexes and, in turn, regulating biological processes such as metabolism. Here, mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic (dis)associations. Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment. These included changes in enzymatic protein complexes involving γ-amino butyric acid transaminase (GABA-T), Δ-ornithine aminotransferase (Δ-OAT), or proline dehydrogenase 1 (POX1) that are expected to affect proline metabolism. Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid (TCA) cycle and the abundance of complexes of the oxidative phosphorylation pathway. In addition, we compared the mitochondrial complexes of roots and shoots. Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus, the formation of super-complexes in the oxidative phosphorylation pathway, and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.

Multiple pathways for Fig4 contributions to cellular homeostasis in yeast

Mutations in the lipid phosphatase Fig4 are associated with multiple neurodegenerative diseases including a rare and severe form of Charcot‐Marie‐Tooth disease known as type 4J (CMT4J). CMT4J has been attributed to dysregulation of the production and turnover of Fig4’s lipid substrate, the phosphoinositide signaling lipid, PI3,5P2 through disruption of its well characterized protein complex, the Fab1‐Vac14‐Fig4 complex. Fig4 is known to play roles in the cell independent of its catalytic function. Our work in yeast suggests that the presence of Fig4 in cells can confer a growth advantage under stress and that this growth advantage is conferred in a dominant manner, independent of Fig4 catalytic activity or its participation in the Fab1‐Vac14‐Fig4 complex. Importantly, we have found that Fig4 variants associated with multiple demyelinating diseases, including those involved in CMT4J, leukodystrophy and amyotrophic lateral sclerosis (ALS), display similar behavior. These data suggest that Fig4 influences cellular homeostasis, and perhaps human disease, through multiple pathways.

An Effective Link-Based Clustering Algorithm for Detecting Overlapping Protein Complexes in Protein-Protein Interaction Networks

Protein complexes are one most important kind of functional modules for biological processes in cells. In this regard, their detection is vital for understanding the principle of cell organization and function. A variety of clustering algorithms have been developed to detect protein complexes from protein-protein interaction (PPI) networks. However, most of them are based on a certain clustering criterion. Given the fact that proteins should interact with each other rather than act independently, we reason that clustering upon interactions can better characterize protein complexes than upon proteins, thus improving the detection accuracy. To this end, a link-based clustering algorithm has been proposed in this paper to effectively detect overlapping protein complexes. It first measures the similarity between pairwise interactions from the perspectives of network topology and Gene Ontology. The problem of protein complex detection is then formulated as an optimization problem of link-based clustering, which is resolved by the proposed algorithm. This proposed algorithm explores the intrinsic correlation between protein complexes and interactions for detecting functionally significant protein complexes. Experimental results on five independent PPI datasets collected from the species of yeast and human demonstrate that compared with state-of-the-art algorithms, the proposed algorithm has significantly improved the detection accuracy for protein complexes.

Protein Complexes Form a Basis for Complex Hybrid Incompatibility.

Proteins are the workhorses of the cell and execute many of their functions by interacting with other proteins forming protein complexes. Multi-protein complexes are an admixture of subunits, change their interaction partners, and modulate their functions and cellular physiology in response to environmental changes. When two species mate, the hybrid offspring are usually inviable or sterile because of large-scale differences in the genetic makeup between the two parents causing incompatible genetic interactions. Such reciprocal-sign epistasis between inter-specific alleles is not limited to incompatible interactions between just one gene pair; and, usually involves multiple genes. Many of these multi-locus incompatibilities show visible defects, only in the presence of all the interactions, making it hard to characterize. Understanding the dynamics of protein-protein interactions (PPIs) leading to multi-protein complexes is better suited to characterize multi-locus incompatibilities, compared to studying them with traditional approaches of genetics and molecular biology. The advances in omics technologies, which includes genomics, transcriptomics, and proteomics can help achieve this end. This is especially relevant when studying non-model organisms. Here, we discuss the recent progress in the understanding of hybrid genetic incompatibility; omics technologies, and how together they have helped in characterizing protein complexes and in turn multi-locus incompatibilities. We also review advances in bioinformatic techniques suitable for this purpose and propose directions for leveraging the knowledge gained from model-organisms to identify genetic incompatibilities in non-model organisms.

Mapping a Conformational Epitope of Hemagglutinin A Using Native Mass Spectrometry and Ultraviolet Photodissociation.

As the importance of effective vaccines and the role of protein therapeutics in the drug industry continue to expand, alternative strategies to characterize protein complexes are needed. Mass spectrometry (MS) in conjunction with enzymatic digestion or chemical probes has been widely used for mapping binding epitopes at the molecular level. However, advances in instrumentation and application of activation methods capable of accessing higher energy dissociation pathways have recently allowed direct analysis of protein complexes. Here we demonstrate a workflow utilizing native MS and ultraviolet photodissociation (UVPD) to map the antigenic determinants of a model antibody-antigen complex involving hemagglutinin (HA), the primary immunogenic antigen of the influenza virus, and the D1 H1-17/H3-14 antibody which has been shown to confer potent protection to lethal infection in mice despite lacking neutralization activity. Comparison of sequence coverages upon UV photoactivation of HA and of the HA·antibody complex indicates the elimination of some sequence ions that originate from backbone cleavages exclusively along the putative epitope regions of HA in the presence of the antibody. Mapping the number of sequence ions covering the HA antigen versus the HA·antibody complex highlights regions with suppressed backbone cleavage and allows elucidation of unknown epitopes. Moreover, examining the observed fragment ion types generated by UVPD demonstrates a loss in diversity exclusively along the antigenic determinants upon MS/MS of the antibody-antigen complex. UVPD-MS shows promise as a method to rapidly map epitope regions along antibody-antigen complexes as novel antibodies are discovered or developed.

Comparing the Conformational Stability of Pyruvate Kinase in the Gas Phase and in Solution

Collision induced unfolding (CIU) is increasingly used to characterize protein complexes in the gas phase and is often employed to detect ligand binding-induced conformational stabilization. However, the extent to which gas-phase conformational stabilities measured by CIU reflect analogous parameters in solution is not yet clear, particularly for systems where conformational and protein complex stability are modulated by point mutation. Here, we compare CIU-derived relative stabilities of four point mutants of the homotetramer pyruvate kinase to solution stabilities measured by differential scanning fluorimetry (DSF) and solution conformational dynamics measured by time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX). Our results demonstrate that both destabilization of the tetrameric state and generally reduced conformational stability of the monomer in solution are well correlated to lower onset energies for specific unfolding transitions observed in CIU. However, this correlation not fully retained when comparing CIU to HDX data, where the latter measurement is strongly impacted by conformational dynamics within the tetramer.

Identifying Heteroprotein Complexes in the Nuclear Envelope

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

Unveiling the Impact of the Core Circadian Clock Protein FREQUENCY on Output in Neurospora crassa

Circadian rhythms are cellular oscillations that tune an organism's biology to the 24‐hour light/dark cycle on Earth. This cellular time‐keeping mechanism aids in the anticipation of environmental changes and increases organismal survival. These rhythms are maintained by a transcriptional/translational negative feedback loop, called the core circadian clock, that is evolutionarily conserved throughout most branches of life. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins. The negative arms proteins act back on the positive arm, in a time specific manner, to halt the negative arm's activation before degradation and re‐initiation, thus setting the period of the clock. Beyond this core clock, positive arm proteins also act to transcriptionally activate a variety of other genes that do not participate in the core clock mechanism in a time dependent manor, referred to as clock‐controlled genes (ccgs.) Most research into the mechanisms of circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the regulation of circadian output is also accomplished via post‐translational mechanisms, which we hypothesize are imparted by the negative arm proteins.In the model organism Neurospora crassa, the negative arm comprises FREQUENCY (FRQ), Frequency‐Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK‐1). Importantly, FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a single, predetermined three‐dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart post‐translational circadian regulation. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manner, to determine the impact these interactions could impart post‐translationally on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co‐immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in post‐translational regulation on metabolism, the cell cycle, and a variety of other functions.Support or Funding InformationThis work was funded through the support of the NIGMS/NIH R35GM128687 grant and the Rensselaer Polytechnic Institue Start Up Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electron microscopy snapshots of single particles from single cells

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

Unveiling the Impact of the Negative Arm of the Circadian Clock on Output in Neurospora crassa

Circadian rhythms are cellular oscillations that tune biology to Earth's 24-hour light/dark cycle, aiding in the anticipation of environmental changes. These rhythms are maintained by an evolutionarily conserved transcriptional/translational negative feedback loop, referred to as the core circadian clock. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins that act back on the positive arm to halt the negative arm's activation before degradation and re-initiation, thus setting the period of the clock. Most research into circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the post-translational regulation of circadian output exists, which we hypothesize is imparted by the negative arm proteins. In the model organism Neurospora crassa, the negative arm includes FREQUENCY (FRQ). FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a predetermined three-dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart circadian regulation post-translationally. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manor to determine the impact these interactions could impart on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co-immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in regulation on metabolism, the cell cycle, and a variety of other functions through novel protein interactions.

CORUM: the comprehensive resource of mammalian protein complexes-2019.

CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (67%), mouse (15%) and rat (10%). Given the vital functions of these macromolecular machines, their identification and functional characterization is foundational to our understanding of normal and disease biology. The new CORUM 3.0 release encompasses 4274 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 4473 different genes, representing 22% of the protein coding genes in humans. Protein complexes are described by a protein complex name, subunit composition, cellular functions as well as the literature references. Information about stoichiometry of subunits depends on availability of experimental data. Recent developments include a graphical tool displaying known interactions between subunits. This allows the prediction of structural interconnections within protein complexes of unknown structure. In addition, we present a set of 58 protein complexes with alternatively spliced subunits. Those were found to affect cellular functions such as regulation of apoptotic activity, protein complex assembly or define cellular localization. CORUM is freely accessible at http://mips.helmholtz-muenchen.de/corum/.

Targeted deletion of the AAA-ATPase Ruvbl1 in mice disrupts ciliary integrity and causes renal disease and hydrocephalus

Ciliopathies comprise a large number of hereditary human diseases and syndromes caused by mutations resulting in dysfunction of either primary or motile cilia. Both types of cilia share a similar architecture. While primary cilia are present on most cell types, expression of motile cilia is limited to specialized tissues utilizing ciliary motility. We characterized protein complexes of ciliopathy proteins and identified the conserved AAA-ATPase Ruvbl1 as a common novel component. Here, we demonstrate that Ruvbl1 is crucial for the development and maintenance of renal tubular epithelium in mice: both constitutive and inducible deletion in tubular epithelial cells result in renal failure with tubular dilatations and fewer ciliated cells. Moreover, inducible deletion of Ruvbl1 in cells carrying motile cilia results in hydrocephalus, suggesting functional relevance in both primary and motile cilia. Cilia of Ruvbl1-negative cells lack crucial proteins, consistent with the concept of Ruvbl1-dependent cytoplasmic pre-assembly of ciliary protein complexes.

Shieldin the ends for 53BP1.

Shieldin is a newly characterized protein complex that functions downstream of 53BP1 in promoting NHEJ

Interaction proteomics by using in-cell NMR spectroscopy

A synopsis of in-cell NMR spectroscopic approaches to study interaction proteomics in prokaryotic and eukaryotic cells is presented. We describe the use of in-cell NMR spectroscopy to resolve high resolution protein structures, discuss methodologies for determining and analyzing high and low affinity protein-target structural interactions, including intrinsically disordered proteins, and detail important functional interactions that result from these interactions. SignificanceThe ultimate goal of structural and biochemical research is to understand how macromolecular interactions give rise to and regulate biological activity in living cells. The challenge is formidable due to the complexity that arises not only from the number of proteins (genes) expressed by the organism, but also from the combinatorial interactions between them. Despite ongoing efforts to decipher the complex nature of protein interactions, new methods for structurally characterizing protein complexes are needed to fully understand molecular networks. With the onset of in-cell NMR spectroscopy, molecular structures and interactions can be studied under physiological conditions shedding light on the structural underpinning of biological activity.

Protein Complexes and Virus-Like Particle Technology.

Virus-like particle (VLP) technologies are based on virus-inspired artificial structures and the intrinsic ability of viral proteins to self-assemble at controlled conditions. Therefore, the basic knowledge about the mechanisms of viral particle formation is highly important for designing of industrial applications. As an alternative to genetic and chemical processes, different physical methods are frequently used for VLP construction, including well characterized protein complexes for introduction of foreign molecules in VLP structures.This chapter shortly discusses the mechanisms how the viruses form their perfectly ordered structures as well as the principles and most interesting application examples, how to exploit the structural and assembly/disassembly properties of viral structures for creation of new nanomaterials.

Catching global interactions in vivo

Histone proteins and transcription factors (TFs) play critical roles in gene transcription and development of multicellular organisms. Although antibody mediated protein isolation couple with mass spectrometry approach has been a standard method to identify TF interacting partners and characterize their functional molecular complexes, it becomes urge to develop a robust method to functional characterize how these transcription factors act during biological process in the post-human genome project era. Here, Dr. Zhao and his colleagues in the National Heart, Lung, and Blood Institute of NIH develop a sensitive and robust strategy to globally identify and characterize in vivo protein–protein interactions termed bait protein–protein interaction-sequencing (bPPI-seq) (Zhang et al. in Cell Res doi:10.1038/cr.2017.112, 2017). As a proof-of-principle, they demonstrated that genome-wide interacting partners of histone variant H2A.Z are mainly involved in transcriptional regulation which is distinct from the interacting proteins of canonical histone H2A. Thus, their results suggest that bPPI-seq can be widely used to globally characterize protein complexes especially transcription factor interacting partners and molecular networks formed.

Simple and Effective Affinity Purification Procedures for Mass Spectrometry-Based Identification of Protein-Protein Interactions in Cell Signaling Pathways.

Identification of protein-protein interactions can be a critical step in understanding the function and regulation of a particular protein and for exploring intracellular signaling cascades. Affinity purification coupled to mass spectrometry (APMS) is a powerful method for isolating and characterizing protein complexes. This approach involves the tagging and subsequent enrichment of a protein of interest along with any stably associated proteins that bind to it, followed by the identification of the interacting proteins using mass spectrometry. The protocol described here offers a quick and simple method for routine sample preparation for APMS analysis of suitably tagged human cell lines.

An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

The dynamic duo: Combining NMR and small angle scattering in structural biology

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X-ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well-suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR-derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state-of-the-art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.

Application of shotgun proteomics for discovery-driven protein-protein interaction.

Affinity purification of protein complexes and identification of co-purified proteins by mass spectrometry is a powerful method to discover novel protein-protein interactions. Application of this method to the study of biological systems often requires the ability to process a large number of samples. Hence, there is great need to generate proteomic workflows compatible with large-scale studies. The major goal of this protocol is to present a fast, reliable, and scalable method to characterize protein complexes by mass spectrometry to overcome the limitations of conventional geLC-MS/MS or MudPIT protocols. This method was successfully employed for the discovery and characterization of novel protein complexes in cultured yeast, mammalian cells, and mice.