Showing papers on "Protein–protein interaction published in 2018"

Pi-Pi contacts are an overlooked protein feature relevant to phase separation.

Abstract: Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore Multivalent interactions of modular binding domains and their target motifs can drive phase separation However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing

Extreme disorder in an ultrahigh-affinity protein complex

Abstract: Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

The m6A reader protein YTHDC2 interacts with the small ribosomal subunit and the 5'-3' exoribonuclease XRN1.

Abstract: N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m6A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5'-3' exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a "fast-track" expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation.

A proximity-tagging system to identify membrane protein–protein interactions

Abstract: The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes.

Abstract: Understanding the dynamics of endogenous protein–protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples.

N-glycan microheterogeneity regulates interactions of plasma proteins.

Abstract: Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein–protein, or protein–small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin–hemoglobin (Hp–Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp–Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein–drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

Abstract: Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved β4-β8 cleft of its alpha-crystallin domain. The β4-β8 region is also the site of Hsp27-Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the β4-β8 groove acts as a tunable sensor for clients.

Surface Charge Modulates Protein-Protein Interactions in Physiologically Relevant Environments.

Abstract: Protein–protein interactions are fundamental to biology yet are rarely studied under physiologically relevant conditions where the concentration of macromolecules can exceed 300 g/L. These high concentrations cause cosolute–complex contacts that are absent in dilute buffer. Understanding such interactions is important because they organize the cellular interior. We used 19F nuclear magnetic resonance, the dimer-forming A34F variant of the model protein GB1, and the cosolutes bovine serum albumin (BSA) and lysozyme to assess the effects of repulsive and attractive charge–charge dimer–cosolute interactions on dimer stability. The interactions were also manipulated via charge-change variants and by changing the pH. Charge–charge repulsions between BSA and GB1 stabilize the dimer, and the effects of lysozyme indicate a role for attractive interactions. The data show that chemical interactions can regulate the strength of protein–protein interactions under physiologically relevant crowded conditions and sugges...

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice.

Abstract: The voltage‐gated sodium channel Na V 1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na V 1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na V 1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo . The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na V 1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na V 1.7 channel.

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

Abstract: Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

Abstract: Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

Abstract: Summary During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein–protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.

Cytosolic proteins can exploit membrane localization to trigger functional assembly.

Abstract: Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10-1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding.

Capturing Post-Translational Modification-Triggered Protein–Protein Interactions Using Dual Noncanonical Amino Acid Mutagenesis

Abstract: Reversible post-translational modification (PTM) is a powerful and ubiquitous mechanism to regulate protein function. The mechanistic basis of the associated functional regulation by PTMs often involves the recruitment of interaction partners that selectively bind the modified protein. Identifying such functionally important protein–protein interactions that are uniquely triggered by PTMs remains difficult due to several technical challenges. To address this, here we develop technology to site-specifically incorporate two distinct noncanonical amino acids into recombinant proteins: one modeling a PTM of interest and the second harboring a photoaffinity probe. Using lysine-23 acetylation of histone 3 as a model system, we show that such dual-labeled “protein probes” can covalently capture its “reader” protein.

Analysis of lncRNA-Protein Interactions by RNA-Protein Pull-Down Assays and RNA Immunoprecipitation (RIP)

Abstract: Long noncoding RNAs (lncRNAs) have important roles in shaping chromatin by targeting chromatin-modifying enzymes to distinct genomic sites. This section covers two methods to analyze lncRNA-protein interactions. The RNA-protein pull-down assays use either bead-bound proteins to capture in vitro transcripts, or immobilized synthetic RNAs to bind proteins from cell lysates. In the RNA immunoprecipitation (RIP) assay, endogenous RNAs are co-immunoprecipitated with a protein of interest. Both the methods can be applied to material from proliferating and quiescent cells, thus providing insights into how lncRNA-protein interactions are altered between these two cellular states.

High-Affinity Peptidomimetic Inhibitors of the DCN1-UBC12 Protein–Protein Interaction

Abstract: The Cullin-RING ligases (CRLs) regulate the turnover of approximately 20% of the proteins in mammalian cells and are emerging therapeutic targets in human diseases The activation of CRLs requires the neddylation of their cullin subunit, which is controlled by an activation complex consisting of Cullin-RBX1-UBC12-NEDD8-DCN1 Herein, we describe the design, synthesis, and evaluation of peptidomimetics targeting the DCN1-UBC12 protein–protein interaction Starting from a 12-residue UBC12 peptide, we have successfully obtained a series of peptidomimetic compounds that bind to DCN1 protein with KD values of <10 nM Determination of a cocrystal structure of a potent peptidomimetic inhibitor complexed with DCN1 provides the structural basis for their high-affinity interaction Cellular investigation of one potent DCN1 inhibitor, compound 36 (DI-404), reveals that it effectively and selectively inhibits the neddylation of cullin 3 over other cullin members Further optimization of DI-404 may yield a new class of

Protein-Protein Interactions in DNA Base Excision Repair

Abstract: The system of base excision repair (BER) ensures correction of the most abundant DNA damages in mammalian cells and plays an important role in maintaining genome stability. Enzymes and protein factors participate in the multistage BER in a coordinated fashion, which ensures repair efficiency. The suggested coordination mechanisms are based on formation of protein complexes stabilized via either direct or indirect DNA-mediated interactions. The results of investigation of direct interactions of the proteins participating in BER with each other and with other proteins are outlined in this review. The known protein partners and sites responsible for their interaction are presented for the main participants as well as quantitative characteristics of their affinity. Information on the mechanisms of regulation of protein–protein interactions mediated by DNA intermediates and posttranslational modification is presented. It can be suggested based on all available data that the multiprotein complexes are formed on chromatin independent of the DNA damage with the help of key regulators of the BER process – scaffold protein XRCC1 and poly(ADP-ribose) polymerase 1. The composition of multiprotein complexes changes dynamically depending on the DNA damage and the stage of BER process.

Multiple components of the nuclear pore complex interact with the amino-terminus of MX2 to facilitate HIV-1 restriction.

Abstract: Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4+ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1.

Real-Time In-Cell Nuclear Magnetic Resonance: Ribosome-Targeted Antibiotics Modulate Quinary Protein Interactions.

Abstract: How ribosome antibiotics affect a wide range of biochemical pathways is not well understood; changes in RNA-mediated protein quinary interactions and consequent activity inside the crowded cytosol may provide one possible mechanism. We developed real-time (RT) in-cell nuclear magnetic resonance (NMR) spectroscopy to monitor temporal changes in protein quinary structure, for ≥24 h, in response to external and internal stimuli. RT in-cell NMR consists of a bioreactor containing gel-encapsulated cells inside a 5 mm NMR tube, a gravity siphon for continuous exchange of medium, and a horizontal drip irrigation system to supply nutrients to the cells during the experiment. We showed that adding antibiotics that bind to the small ribosomal subunit results in more extensive quinary interactions between thioredoxin and mRNA. The results substantiate the idea that RNA-mediated modulation of quinary protein interactions may provide the physical basis for ribosome inhibition and other regulatory pathways.

Systematic characterization of pan-cancer mutation clusters

Abstract: Cancer genome sequencing has shown that driver genes can often be distinguished not only by the elevated mutation frequency but also by specific nucleotide positions that accumulate changes at a high rate. However, properties associated with a residue9s potential to drive tumorigenesis when mutated have not yet been systematically investigated. Here, using a novel methodological approach, we identify and characterize a compendium of 180 hotspot residues within 160 human proteins which occur with a significant frequency and are likely to have functionally relevant impact. We find that such mutations (i) are more prominent in proteins that can exist in the on and off state, (ii) reflect the identity of a tumor of origin, and (iii) often localize within interfaces which mediate interactions with other proteins or ligands. Following, we further examine structural data for human protein complexes and identify a number of additional protein interfaces that accumulate cancer mutations at a high rate. Jointly, these analyses suggest that disruption and dysregulation of protein interactions can be instrumental in switching functions of cancer proteins and activating downstream changes.