Composition Of Protein Complexes Research Articles

Cell Type-Specific Tandem Affinity Purification of the Mouse Hippocampal CB1 Receptor-Associated Proteome.

G protein coupled receptors (GPCRs) exert their effects through multiprotein signaling complexes. The cannabinoid receptor type 1 (CB1) is among the most abundant GPCRs in the mammalian brain and involved in a plethora of physiological functions. We used a combination of viral-mediated cell type-specific expression of a tagged CB1 fusion protein (CB1-SF), tandem affinity purification (TAP) and proteomics on hippocampal mouse tissue to analyze the composition and differences of CB1 protein complexes in glutamatergic neurons and in GABAergic interneurons. Purified proteins underwent tryptic digestion and were identified using deep-coverage data-independent acquisition with ion mobility separation-enhanced mass spectroscopy, leading to the identification of 951 proteins specifically enriched in glutamatergic and GABAergic CB1-SF TAP samples as compared to controls. Gene Ontology and protein network analyses showed an enrichment of single proteins and functional clusters of proteins involved in already well described domains of CB1 functions. Supported by this consistent data set we could confirm already known CB1 interactors, reveal new potentially interacting proteins and differences in cell type-specific signaling properties of CB1, thereby providing the foundation for further functional studies on differential CB1 signaling.

Identification of Bovine Sperm Surface Proteins Involved in Carbohydrate-mediated Fertilization Interactions

Glycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-toward-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (1) formation of the sperm reservoir in the oviductal epithelium, and (2) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of sperm. Based on these results and previous data, we suggest that sperm surface proteins play significant roles in fertilization events such as membrane remodeling, transport, protection and function, thus supporting the hypothesis that rather than a simple lock-and-key model, mammalian fertilization relies on a complex interactome involving multiple ligands/receptors and recognition/binding events.

Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress

To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress.

Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy

Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research.

Photosystem II Repair and Plant Immunity: Lessons Learned from Arabidopsis Mutant Lacking the THYLAKOID LUMEN PROTEIN 18.3.

Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415–425). Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII) repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Protein composition of 6K2-induced membrane structures formed during Potato virus A infection.

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.

SnapShot: SMC Protein Complexes Part II

This second of two SnapShots on SMC proteins depicts their roles at different stages of the eukaryotic cell cycle. The composition and architecture of SMC protein complexes and their regulators appear in SMC Protein Complexes Part I (available at http://www.cell.com/cell/pdf/S0092-8674%2815%2901690-6.pdf). To view this SnapShot, open or download the PDF.

Complex formation and turnover of mitochondrial transporters and ion channels

Mitochondria are responsible for many vital cellular functions in eukaryotic cells, such as ATP production, steroid synthesis and prosthetic group biogenesis. The vital functions of mitochondria are possible due to the compartmental nature of this organelle. Mitochondria form a dynamic network that can exist as a network throughout a cell or as distinct individual structures. Mitochondria are also composed of two membranes, an inner and outer membrane. The inner mitochondrial membrane (IMM) is significantly larger than the outer membrane and must fold upon itself to be contained within the outer mitochondrial membrane (OMM). These folds are known as cristae. Altogether these different membrane compartments specialize in different functions of the mitochondria. The OMM is responsible for passage of small metabolites into and out of the mitochondria while excluding macromolecules. The IMM is a highly selective barrier between the solutes of the cytosol and those within the mitochondrial matrix. Cristae specialize in oxidative phosphorylation. The functions of these membranes are afforded by membrane proteins that are able to transport specific solutes. The appropriate localization, assembly into multi-subunit protein complexes, and wild-type function of these membrane proteins therefore is vital for mitochondria to maintain appropriate function and support cellular survival. This review will address the composition and functions of mitochondrial membrane localized multi-subunit protein complexes along with how these proteins undergo degradation to maintain homeostatic functions of mitochondria in the context of mitochondria specific transporters and ion channels. Due to the large number of known mitochondrial membrane transporters and ion channels this review will focus on the topics presented at the Mitochondrial Ion Channels and Transporters Symposium hosted by the New York University College of Dentistry in September 2015 in honor of Casey Kinnally.

Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability.

Adhesion GPCR-Related Protein Networks.

Adhesion G protein-coupled receptors (aGPCRs/ADGRs) are unique receptors that combine cell adhesion and signaling functions. Protein networks related to ADGRs exert diverse functions, e.g., in tissue polarity, cell migration, nerve cell function, or immune response, and are regulated via different mechanisms. The large extracellular domain of ADGRs is capable of mediating cell-cell or cell-matrix protein interactions. Their intracellular surface and domains are coupled to downstream signaling pathways and often bind to scaffold proteins, organizing membrane-associated protein complexes. The cohesive interplay between ADGR-related network components is essential to prevent severe disease-causing damage in numerous cell types. Consequently, in recent years, attention has focused on the decipherment of the precise molecular composition of ADGR protein complexes and interactomes in various cellular modules. In this chapter, we discuss the affiliation of ADGR networks to cellular modules and how they can be regulated, pinpointing common features in the networks related to the diverse ADGRs. Detailed decipherment of the composition of protein networks should provide novel targets for the development of novel therapies with the aim to cure human diseases related to ADGRs.

Identification of protein complexes of microsomes in rat adipocytes by native gel coupled with LC-ESI-QTOF.

The study of the composition of microsome proteins/complexes/interactions in adipocytes provides useful information for researchers related to energy metabolism disorders. The native gel coupled with LC-ESI-QTOF approach was employed here for separating protein complexes. We found a series of proteins functionally clustered in biological processes of protein metabolism, cellular carbohydrate catabolism, response to stimulus and wounding, macromolecular complex subunit organization, positive regulation of molecular function, regulation of programmed cell death and biomolecule transport. According to clustering of proteins' electrophoresis profiles across native gel fractions and bioinformatics data retrieval, protein complexes/interactions involved in protein metabolism, cellular carbohydrate catabolism, macromolecular complex subunit organization and biomolecule transport were identified. Besides, the results also revealed some functional linkages, which may provide useful information for discovering previously unknown interactions. The interaction between SSAO and ALDH2 was verified by co-immunoprecipitation. The native gel combining mass spectrometry approach appeared to be a useful tool for investigating microsome proteins and complexes to complement the traditional electrophoresis approaches. The native gel strategy together with our findings should facilitate future studies of the composition of rat adipocyte microsome protein complexes under different conditions.

Protein-Protein Interaction Detection Via Mass Spectrometry-Based Proteomics.

Analysis of protein-protein interactions is one of the mainstays of mass spectrometry-based proteomics and recent developments, which have simplified the methodology, have permitted non-specialised laboratories to adopt the approach. We introduce and review three complimentary methods which allow for the targeted, global and site-specific analysis of protein complexes. Co-precipitation of endogenous or ectopically expressed proteins and their complexes followed by proteomic analysis allows for the discovery and accurate quantification of specific protein interactions. Whereas complimentary methods, such as co-purification of entire complexes based on physico-chemical attributes, can give a snap-shot of the composition and dynamics of protein complexes on a global scale. Cross-linking on the other hand can pinpoint the amino acids involved in protein-protein interactions to such a resolution that the likely complex can be reconstructed computationally.

SnapShot: SMC Protein Complexes Part I

This first of two SnapShots on SMC proteins depicts the composition and architecture of SMC protein complexes and their regulators. Their roles at different stages of the cell cycle will appear in Part II. To view this SnapShot, open or download the PDF.

In Vivo Radiolabeling of Arabidopsis Chloroplast Proteins and Separation of Thylakoid Membrane Complexes by Blue Native PAGE.

The investigation of membrane protein complex assembly and degradation is essential to understand cellular protein dynamics. Blue native PAGE provides a powerful tool to analyze the composition and formation of protein complexes. Combined with in vivo radiolabeling, the synthesis and decay of protein complexes can be monitored on a timescale ranging from minutes to several hours. Here, we describe a protocol to analyze thylakoid membrane complexes starting either with (35)S-methionine labeling of intact Arabidopsis leaves to investigate protein complex dynamics or with unlabeled leaf material to monitor steady-state complex composition.

Methionine restriction fundamentally supports health by tightening epithelial barriers

Dietary methionine restriction (MR) has been found to affect one of the most primary tissue-level functions of an organism: the efficiency with which the epithelial linings of major organs separate the fluid compartments that they border. This process, epithelial barrier function, is basic for proper function of all organs, including the lung, liver, gastrointestinal tract, reproductive tract, blood-brain barrier, and kidney. Specifically, MR has been found to modify the protein composition of tight junctional complexes surrounding individual epithelial cells in a manner that renders the complexes less leaky. This has been observed in both a renal epithelial cell culture model and in gastrointestinal tissue. In both cases, MR increased the transepithelial electrical resistance across the epithelium, while decreasing passive leak of small nonelectrolytes. However, the specific target protein modifications involved were unique to each case. Overall, this provides an example of the primary level on which MR functions to modify, and improve, an organism.

SYSTEMS BIOLOGY: Ancient protein complexes revealed.

A systematic analysis of protein-complex composition across a billion years of evolution reveals a spectrum of conservation.

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling.

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

Extending native mass spectrometry approaches to integral membrane proteins.

Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation.