Co-evolutionary analysis reveals insights into protein-protein interactions

Protein-protein interactions play crucial roles in biological processes. Experimental methods have been developed to survey the proteome for interacting partners and some computational approaches have been developed to extend the impact of these experimental methods. Computational methods are routinely applied to newly discovered genes to infer protein function and plausible protein-protein interactions. Here, we develop and extend a quantitative method that identifies interacting proteins based upon the correlated behavior of the evolutionary histories of protein ligands and their receptors. We have studied six families of ligand-receptor pairs including: the syntaxin/Unc-18 family, the GPCR/G-alpha's, the TGF-beta/TGF-beta receptor system, the immunity/colicin domain collection from bacteria, the chemokine/chemokine receptors, and the VEGF/VEGF receptor family. For correlation scores above a defined threshold, we were able to find an average of 79% of all known binding partners. We then applied this method to find plausible binding partners for proteins with uncharacterized binding specificities in the syntaxin/Unc-18 protein and TGF-beta/TGF-beta receptor families. Analysis of the results shows that co-evolutionary analysis of interacting protein families can reduce the search space for identifying binding partners by not only finding binding partners for uncharacterized proteins but also recognizing potentially new binding partners for previously characterized proteins. We believe that correlated evolutionary histories provide a route to exploit the wealth of whole genome sequences and recent systematic proteomic results to extend the impact of these studies and focus experimental efforts to categorize physiologically or pathologically relevant protein-protein interactions.     

Computational prediction for the protein interactions of tyrosinase: Protein experimental interactome MAP

Tyrosinase (EC 1.14.18.1) is a diversely distributed enzyme in various organisms with physiological roles related to pigment production. Tyrosinase has gained the attention of researchers due to its biological functions and potential applications. In this regard, studies on the partner proteins of tyrosinase are important. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein–protein interactions between tyrosinase and binding partners by using the PEIMAP algorithm. As a result, we found that tyrosinase is predicted to bind with G protein-related proteins, potassium voltage-gated channel-related proteins, and vesicle/sorting-related proteins. In particular, GIPC1, GIPC2, GIPC3, TYRP1, and DCT were predicted to primarily bind with tyrosinase. Interacting proteins (103) were secondarily bound to these 5 interacting proteins in the PEIMAP network of tyrosinase. An involvement in melanogenesis was also newly predicted by associating the predicted binding proteins. We simulated the protein–protein bindings by probing the residues of each complex facing toward the predicted site of interaction with tyrosinase. Among the interacting residues, some were predicted to dock to the active site of tyrosinase, which could affect its activity directly. Our computational predictions will be useful for elucidating the protein–protein interactions of tyrosinase and for studying its binding mechanisms and the melanin biosynthesis pathway.     

Exploiting the co-evolution of interacting proteins to discover interaction specificity

Protein interactions are fundamental to the functioning of cells, and high throughput experimental and computational strategies are sought to map interactions. Predicting interaction specificity, such as matching members of a ligand family to specific members of a receptor family, is largely an unsolved problem. Here we show that by using evolutionary relationships within such families, it is possible to predict their physical interaction specificities. We introduce the computational method of matrix alignment for finding the optimal alignment between protein family similarity matrices. A second method, 3D embedding, allows visualization of interacting partners via spatial representation of the protein families. These methods essentially align phylogenetic trees of interacting protein families to define specific interaction partners. Prediction accuracy depends strongly on phylogenetic tree complexity, as measured with information theoretic methods. These results, along with simulations of protein evolution, suggest a model for the evolution of interacting protein families in which interaction partners are duplicated in coupled processes. Using these methods, it is possible to successfully find protein interaction specificities, as demonstrated for >18 protein families.     

Poxvirus Proteomics and Virus-Host Protein Interactions

Summary: Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.     

14-3-3ε couples protein kinase A to semaphorin signaling and silences plexin RasGAP-mediated axonal repulsion

The biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are among the largest families of axon guidance cues and utilize Plexin (Plex) receptors to exert repulsive effects on axon extension. However, Semaphorin repulsion can be silenced by other distinct cues and signaling cascades, raising questions of the logic underlying these events. We now uncover a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras/Rap family GTPase activating proteins (GAPs) and we find that the PlexA GAP domain is phosphorylated by the cAMP-dependent protein kinase (PKA). This PlexA phosphorylation generates a specific binding site for 14-3-3ε,?a phospho-binding protein that we find to be necessary for axon guidance. These PKA-mediated Plexin-14-3-3ε interactions prevent PlexA from interacting with its Ras family GTPase substrate and antagonize Semaphorin repulsion. Our results indicate that these?interactions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules.     

Plexin B1 inhibits integrin-dependent pp125FAK and Rho activity in melanoma

Semaphorins are secreted and membrane bound proteins that regulate axon guidance through receptors Plexins and neuropilins. Plexin B1, the Semaphorin 4D receptor, is a recently described tumor suppressor protein for melanoma. We recently showed that Plexin B1 abrogates activation of the oncogenic receptor, c-Met, by its ligand, hepatocyte growth factor (HGF), in melanoma. We have now investigated the effect of Plexin B1 on integrin-dependent pp125(FAK) activation, and the small GTP-binding protein Rho, in melanoma. Integrin receptors and Rho play critical roles in melanoma progression, through regulation of migration, proliferation and apoptosis. We engineered two human melanoma cell lines expressing Plexin B1 and analyzed integrin-dependent migration, integrin-dependent pp125(FAK) activation, and Rho activity. Results show that Plexin B1 abrogates integrin-dependent migration and activation of pp125(FAK). We also show that Rho activity is significantly reduced in cells expressing Plexin B1, and that Plexin B1 suppresses HGF-dependent Rho activation.     

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.     

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.     

PICK1：一个功能多样的药靶蛋白

蛋白质是生命功能的执行者.生命体中某些关键蛋白的功能异常往往是导致疾病发生的根本原因.这些疾病相关蛋白极有可能成为药物靶点,为新药研发和疾病治疗提供重要线索. PICK1蛋白（protein interacting with Cα kinase 1）结合能力广泛、功能多样以及在多种重要疾病（如：癌症、精神分裂症、疼痛、帕金森综合症等）的发生发展过程中发挥潜在的作用,使其成为一个可能的药靶蛋白. PICK1与绝大多数配体蛋白的相互作用是通过其PDZ结构域与配体C末端区域的结合介导的,使PICK1的PDZ结构域成为一个潜在的药物靶点.因此,可以利用生物小分子物质特异性地结合PICK1的PDZ结构域,干扰或阻断PICK1与配体蛋白的天然相互作用,最终达到治疗相关疾病的目的.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.     

Beauty Is in the Eye of the Beholder: Proteins Can Recognize Binding Sites of Homologous Proteins in More than One Way

Understanding the mechanisms of protein–protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein–protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A) and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein–protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.     

Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing

Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.     

Molecular Characterization of the Multiple Interactions of SpsD,a Surface Protein from Staphylococcus pseudintermedius,with Host Extracellular Matrix Proteins

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.     

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.