Interactome of signaling networks in wheat: the protein–protein interaction between TaRAR1 and TaSGT1

RAR1 and SGT1 are required for development and disease resistance in plants. In many cases, RAR1 and SGT1 regulate the resistance (R)-gene-mediated defense signaling pathways. Lr21 is the first identified NBS-LRR-type R protein in wheat and is required for resistance to the leaf rust pathogen. The Lr21-mediated signaling pathways require the wheat homologs of RAR1, SGT1, and HSP90. However, the molecular mechanisms of the Lr21-mediated signaling networks remain unknown. Here I present the DNA and protein sequences of TaRAR1 and TaSGT1, and demonstrate for the first time a direct protein-protein interaction between them.     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Analysis of the aplyronine A-induced protein–protein interaction between actin and tubulin by surface plasmon resonance

The antitumor macrolide aplyronine A induces protein–protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin–aplyronine A complex, and the dissociation constant K_D was 1.84 μM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.     

Identification of a disruptor of the MDM2-p53 protein–protein interaction facilitated by high-throughput in silico docking

NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein–protein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it well suited for lead optimization studies that can result in more potent analogs with improved drug-like properties. Its synthesis was achieved using an acid catalyzed condensation reaction from commercially available benzothiazole hydrazine and pyridyl phenyl ketone in refluxing methanol. Stereochemical implications for this compound are described.     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

On the functional and structural characterization of hubs in protein–protein interaction networks

A number of interesting issues have been addressed on biological networks about their global and local properties. The connection between the topological properties of proteins in Protein–Protein Interaction (PPI) networks and their biological relevance has been investigated focusing on hubs, i.e. proteins with a large number of interacting partners. We will survey the literature trying to answer the following questions: Do hub proteins have special biological properties? Do they tend to be more essential than non-hub proteins? Are they more evolutionarily conserved? Do they play a central role in modular organization of the protein interaction network? Are there structural properties that characterize hub proteins?     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

Identification of proliferative diabetic retinopathy-associated genes on the protein–protein interaction network by using heat diffusion algorithm

Diabetic retinopathy is a common complication of diabetes mellitus that causes pathogenic damage to the retina. Particularly, the proliferative diabetic retinopathy (PDR) state can cause abnormal angiogenesis in the retina tissues and trigger the retina destruction in advanced stage. In the clinic, the symptoms during the initiation and progression of PDR are relatively unrecognizable. Therefore, various studies have focused on the pathogenesis of PDR. According to published literature, genetic contributions play an irreplaceable role in the initiation and progression of PDR. Although many computational methods, such as shortest path- and random walk with restart-based methods, have been applied in screening the potential pathogenic factors of PDR, advanced computational methods, which may provide essential supplements for previous ones, are still widely needed. In this study, a novel computational method was presented to infer novel PDR-associated genes. Different from previous methods, the method used in this work employed a different network algorithm, that is, the Laplacian heat diffusion algorithm. This algorithm was applied on the protein–protein interaction network reported in the STRING database. Three screening tests were performed to filter the most likely inferred genes. A total of 26 genes were accessed using the proposed method. Compared with the two previous predictions, most of the identified genes were novel, and only one gene was shared. Several inferred genes, such as CSF3, COL18A1, CXCR2, CCR1, FGF23, CXCL11, and IL13, were related to the pathogenesis of PDR.     

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein–protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.     

The protein–protein interaction network of the human Sirtuin family

Protein–protein interaction networks are useful for studying human diseases and to look for possible health care through a holistic approach. Networks are playing an increasing and important role in the understanding of physiological processes such as homeostasis, signaling, spatial and temporal organizations, and pathological conditions. In this article we show the complex system of interactions determined by human Sirtuins (Sirt) largely involved in many metabolic processes as well as in different diseases. The Sirtuin family consists of seven homologous Sirt-s having structurally similar cores but different terminal segments, being rather variable in length and/or intrinsically disordered. Many studies have determined their cellular location as well as biological functions although molecular mechanisms through which they act are actually little known therefore, the aim of this work was to define, explore and understand the Sirtuin-related human interactome. As a first step, we have integrated the experimentally determined protein–protein interactions of the Sirtuin-family as well as their first and second neighbors to a Sirtuin-related sub-interactome. Our data showed that the second-neighbor network of Sirtuins encompasses 25% of the entire human interactome, and exhibits a scale-free degree distribution and interconnectedness among top degree nodes. Moreover, the Sirtuin sub interactome showed a modular structure around the core comprising mixed functions. Finally, we extracted from the Sirtuin sub-interactome subnets related to cancer, aging and post-translational modifications for information on key nodes and topological space of the subnets in the Sirt family network.     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.     

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvβ3

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvβ3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA–integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA–integrin binding was specific to sPLA2-IIA and not to the integrin.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Identification of PrP sequences essential for the interaction between the PrP polymers and Aβ peptide in a yeast-based assay

Alzheimer disease is associated with the accumulation of oligomeric amyloid β peptide (Aβ), accompanied by synaptic dysfunction and neuronal death. Polymeric form of prion protein (PrP), PrP^Sc, is implicated in transmissible spongiform encephalopathies (TSEs). Recently, it was shown that the monomeric cellular form of PrP (PrP^C), located on the neuron surface, binds Aβ oligomers (and possibly other β-rich conformers) via the PrP_23–27 and PrP_90–110 segments, acting as Aβ receptor. On the other hand, PrP^Sc polymers efficiently bind to Aβ monomers and accelerate their oligomerization. To identify specific PrP sequences that are essential for the interaction between PrP polymers and Aβ peptide, we have co-expressed Aβ and PrP (or its shortened derivatives), fused to different fluorophores, in the yeast cell. Our data show that the 90–110 and 28–89 regions of PrP control the binding of proteinase-resistant PrP polymers to the Aβ peptide, whereas the 23–27 segment of PrP is dispensable for this interaction. This indicates that the set of PrP fragments involved in the interaction with Aβ depends on PrP conformational state.     

Identification of mitogen-activated protein kinase kinase gene family and MKK–MAPK interaction network in maize

Plant mitogen-activated protein kinases (MAPK) are involved in important processes, including stress signaling and development. MAPK kinases (MAPKK, MKK) have been investigated in several plant species including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Brachypodium distachyon. In the present study, nine putative maize MKK genes have been identified. Analysis of the conserved protein motifs, exon–intron junctions and intron phase has revealed high levels of conservation within the phylogenetic groups. Next, we defined four new ZmMKK–ZmMPK interactions using yeast two-hybrid. Finally, we examined the biological functions of the ZmMKK4 gene. Overexpression of ZmMKK4 in Arabidopsis conferred tolerance to oxidative stress by increased germination rate and early seedling growth compared with WT plants. Taken together, we provide a comprehensive bioinformatics analysis of the MKK gene family in maize genome and our data provide an important foundation for further functional study of MAPK and MKK families in maize.