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Sample records for block protein interactions

  1. The nature of protein interactions governing globular protein-polymer block copolymer self-assembly.

    PubMed

    Lam, Christopher N; Kim, Minkyu; Thomas, Carla S; Chang, Dongsook; Sanoja, Gabriel E; Okwara, Chimdimma U; Olsen, Bradley D

    2014-04-14

    The effects of protein surface potential on the self-assembly of protein-polymer block copolymers are investigated in globular proteins with controlled shape through two approaches: comparison of self-assembly of mCherry-poly(N-isopropylacrylamide) (PNIPAM) bioconjugates with structurally homologous enhanced green fluorescent protein (EGFP)-PNIPAM bioconjugates, and mutants of mCherry with altered electrostatic patchiness. Despite large changes in amino acid sequence, the temperature-concentration phase diagrams of EGFP-PNIPAM and mCherry-PNIPAM conjugates have similar phase transition concentrations. Both materials form identical phases at two different coil fractions below the PNIPAM thermal transition temperature and in the bulk. However, at temperatures above the thermoresponsive transition, mCherry conjugates form hexagonal phases at high concentrations while EGFP conjugates form a disordered micellar phase. At lower concentration, mCherry shows a two-phase region while EGFP forms homogeneous disordered micellar structures, reflecting the effect of changes in micellar stability. Conjugates of four mCherry variants with changes to their electrostatic surface patchiness also showed minimal change in phase behavior, suggesting that surface patchiness has only a small effect on the self-assembly process. Measurements of protein/polymer miscibility, second virial coefficients, and zeta potential show that these coarse-grained interactions are similar between mCherry and EGFP, indicating that coarse-grained interactions largely capture the relevant physics for soluble, monomeric globular protein-polymer conjugate self-assembly. PMID:24654888

  2. An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

    SciTech Connect

    Garcia, C.C.; Topisirovic, I.; Djavani, M.; Borden, K.L.B.; Damonte, E.B.; Salvato, M.S.

    2010-03-19

    The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

  3. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  4. A high throughput screening strategy to identify protein-protein interaction inhibitors that block the Fanconi anemia DNA repair pathway

    PubMed Central

    Voter, Andrew F.; Manthei, Kelly A.

    2016-01-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for re-sensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol. PMID:26962873

  5. Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

    PubMed Central

    Vucic, Domagoj; Kaiser, William J.; Miller, Lois K.

    1998-01-01

    Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins. PMID:9584170

  6. RVMAB: Using the Relevance Vector Machine Model Combined with Average Blocks to Predict the Interactions of Proteins from Protein Sequences.

    PubMed

    An, Ji-Yong; You, Zhu-Hong; Meng, Fan-Rong; Xu, Shu-Juan; Wang, Yin

    2016-01-01

    Protein-Protein Interactions (PPIs) play essential roles in most cellular processes. Knowledge of PPIs is becoming increasingly more important, which has prompted the development of technologies that are capable of discovering large-scale PPIs. Although many high-throughput biological technologies have been proposed to detect PPIs, there are unavoidable shortcomings, including cost, time intensity, and inherently high false positive and false negative rates. For the sake of these reasons, in silico methods are attracting much attention due to their good performances in predicting PPIs. In this paper, we propose a novel computational method known as RVM-AB that combines the Relevance Vector Machine (RVM) model and Average Blocks (AB) to predict PPIs from protein sequences. The main improvements are the results of representing protein sequences using the AB feature representation on a Position Specific Scoring Matrix (PSSM), reducing the influence of noise using a Principal Component Analysis (PCA), and using a Relevance Vector Machine (RVM) based classifier. We performed five-fold cross-validation experiments on yeast and Helicobacter pylori datasets, and achieved very high accuracies of 92.98% and 95.58% respectively, which is significantly better than previous works. In addition, we also obtained good prediction accuracies of 88.31%, 89.46%, 91.08%, 91.55%, and 94.81% on other five independent datasets C. elegans, M. musculus, H. sapiens, H. pylori, and E. coli for cross-species prediction. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-AB method is obviously better than the SVM-based method. The promising experimental results show the efficiency and simplicity of the proposed method, which can be an automatic decision support tool. To facilitate extensive studies for future proteomics research, we developed a freely

  7. RVMAB: Using the Relevance Vector Machine Model Combined with Average Blocks to Predict the Interactions of Proteins from Protein Sequences.

    PubMed

    An, Ji-Yong; You, Zhu-Hong; Meng, Fan-Rong; Xu, Shu-Juan; Wang, Yin

    2016-01-01

    Protein-Protein Interactions (PPIs) play essential roles in most cellular processes. Knowledge of PPIs is becoming increasingly more important, which has prompted the development of technologies that are capable of discovering large-scale PPIs. Although many high-throughput biological technologies have been proposed to detect PPIs, there are unavoidable shortcomings, including cost, time intensity, and inherently high false positive and false negative rates. For the sake of these reasons, in silico methods are attracting much attention due to their good performances in predicting PPIs. In this paper, we propose a novel computational method known as RVM-AB that combines the Relevance Vector Machine (RVM) model and Average Blocks (AB) to predict PPIs from protein sequences. The main improvements are the results of representing protein sequences using the AB feature representation on a Position Specific Scoring Matrix (PSSM), reducing the influence of noise using a Principal Component Analysis (PCA), and using a Relevance Vector Machine (RVM) based classifier. We performed five-fold cross-validation experiments on yeast and Helicobacter pylori datasets, and achieved very high accuracies of 92.98% and 95.58% respectively, which is significantly better than previous works. In addition, we also obtained good prediction accuracies of 88.31%, 89.46%, 91.08%, 91.55%, and 94.81% on other five independent datasets C. elegans, M. musculus, H. sapiens, H. pylori, and E. coli for cross-species prediction. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-AB method is obviously better than the SVM-based method. The promising experimental results show the efficiency and simplicity of the proposed method, which can be an automatic decision support tool. To facilitate extensive studies for future proteomics research, we developed a freely

  8. Blocking the Interactions between Calcium-Bound S100A12 Protein and the V Domain of RAGE Using Tranilast.

    PubMed

    Chiou, Jian Wei; Fu, Brian; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The receptor for advanced glycation end products (RAGE), a transmembrane receptor in the immunoglobulin superfamily, is involved in several inflammatory processes. RAGE induces cellular signaling pathways upon binding with various ligands, such as advanced glycation end products (AGEs), β-amyloids, and S100 proteins. The solution structure of S100A12 and the V ligand-binding region of RAGE have been reported previously. Using heteronuclear NMR spectroscopy to conduct 1H-15N heteronuclear single quantum coherence (HSQC) titration experiments, we identified and mapped the binding interface between S100A12 and the V domain of RAGE. The NMR chemical shift data were used as the constraints for the High Ambiguity Driven biomolecular DOCKing (HADDOCK) calculation to generate a structural model of the S100A12-V domain complex. In addition, tranilast (an anti-allergic drug) showed strong interaction with S100A12 in the 1H-15N HSQC titration, fluorescence experiments, and WST-1 assay. The results also indicated that tranilast was located at the binding site between S100A12 and the V domain, blocking interaction between these two proteins. Our results provide the mechanistic details for a structural model and reveal a potential precursor for an inhibitor for pro-inflammatory diseases, which could be useful for the development of new drugs. PMID:27598566

  9. Blocking the Interactions between Calcium-Bound S100A12 Protein and the V Domain of RAGE Using Tranilast

    PubMed Central

    Chiou, Jian Wei; Fu, Brian

    2016-01-01

    The receptor for advanced glycation end products (RAGE), a transmembrane receptor in the immunoglobulin superfamily, is involved in several inflammatory processes. RAGE induces cellular signaling pathways upon binding with various ligands, such as advanced glycation end products (AGEs), β-amyloids, and S100 proteins. The solution structure of S100A12 and the V ligand-binding region of RAGE have been reported previously. Using heteronuclear NMR spectroscopy to conduct 1H–15N heteronuclear single quantum coherence (HSQC) titration experiments, we identified and mapped the binding interface between S100A12 and the V domain of RAGE. The NMR chemical shift data were used as the constraints for the High Ambiguity Driven biomolecular DOCKing (HADDOCK) calculation to generate a structural model of the S100A12–V domain complex. In addition, tranilast (an anti-allergic drug) showed strong interaction with S100A12 in the 1H–15N HSQC titration, fluorescence experiments, and WST-1 assay. The results also indicated that tranilast was located at the binding site between S100A12 and the V domain, blocking interaction between these two proteins. Our results provide the mechanistic details for a structural model and reveal a potential precursor for an inhibitor for pro-inflammatory diseases, which could be useful for the development of new drugs. PMID:27598566

  10. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  11. Targeting Multiple Conformations Leads to Small Molecule Inhibitors of the uPAR·uPA Protein-Protein Interaction that Block Cancer Cell Invasion

    PubMed Central

    Khanna, May; Wang, Fang; Jo, Inha; Knabe, W. Eric; Wilson, Sarah M.; Li, Liwei; Bum-Erdene, Khuchtumur; Li, Jing; Sledge, George; Khanna, Rajesh; Meroueh, Samy O.

    2011-01-01

    Interaction of the urokinase receptor (uPAR) with its binding partners including the urokinase-type plasminogen activator (uPA) at the cell surface triggers a series of proteolytic and signaling events that promote invasion and metastasis. Here, we report the discovery of a small molecule (IPR-456) and its derivatives that inhibit the tight uPAR·uPA protein-protein interaction. IPR-456 was discovered by virtual screening against multiple conformations of uPAR sampled from explicit-solvent molecular dynamics simulations. Biochemical characterization reveal that the compound binds to uPAR with sub-micromolar affinity (Kd = 310 nM) and inhibits the tight protein-protein interaction with an IC50 of 10 μM. Free energy calculations based on explicit-solvent molecular dynamics simulations suggested the importance of a carboxylate moiety on IPR-456, which was confirmed by the activity of several derivatives including IPR-803. Immunofluorescence imaging showed that IPR-456 inhibited uPA binding to uPAR of breast MDA-MB-231 tumor cells with an IC50 of 8 μM. The compounds blocked MDA-MB-231 cell invasion, but IPR-456 showed little effect on MDA-MB-231 migration, and no effect on adhesion, suggesting that uPAR mediates these processes through its other binding partners. PMID:21875078

  12. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

    PubMed

    Voter, Andrew F; Manthei, Kelly A; Keck, James L

    2016-07-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

  13. Nonionic block copolymers assemble on the surface of protein bionanoparticle.

    PubMed

    Liu, Zhi; Gu, Jingxia; Wu, Man; Jiang, Shidong; Wu, Dayong; Wang, Qian; Niu, Zhongwei; Huang, Yong

    2012-08-21

    Efficient delivery of therapeutic proteins to a target site remains a challenge due to rapid clearance from the body. Here, we selected tobacco mosaic virus (TMV) as a model protein system to investigate the interactions between the protein and a nonionic block copolymer as a possible protecting agent for the protein. By varying the temperature, we were able to obtain core-shell structures based on hydrophobic interactions among PO blocks and noncovalent interactions between TMV and EO blocks. The protein-polymer interactions were characterized by dynamic light scattering and isothermal titration calorimetry. This study establishes principles for the possible design of clinically useful protein delivery systems.

  14. Block ground interaction of rockfalls

    NASA Astrophysics Data System (ADS)

    Volkwein, Axel; Gerber, Werner; Kummer, Peter

    2016-04-01

    During a rockfall the interaction of the falling block with the ground is one of the most important factors that define the evolution of a rockfall trajectory. It steers the rebound, the rotational movement, possibly brake effects, friction losses and damping effects. Therefore, if most reliable rockfall /trajectory simulation software is sought a good understanding of the block ground interaction is necessary. Today's rockfall codes enable the simulation of a fully 3D modelled block within a full 3D surface . However, the details during the contact, i.e. the contact duration, the penetration depth or the dimension of the marks in the ground are usually not part of the simulation. Recent field tests with rocks between 20 and 80 kg have been conducted on a grassy slope in 2014 [1]. A special rockfall sensor [2] within the blocks measured the rotational velocity and the acting accelerations during the tests. External video records and a so-called LocalPositioningSystem deliver information on the travel velocity. With these data not only the flight phases of the trajectories but also the contacts with the ground can be analysed. During the single jumps of a block the flight time, jump length, the velocity, and the rotation are known. During the single impacts their duration and the acting accelerations are visible. Further, the changes of rotational and translational velocity influence the next jump of the block. The change of the rotational velocity over the whole trajectory nicely visualizes the different phases of a rockfall regarding general acceleration and deceleration in respect to the inclination and the topography of the field. References: [1] Volkwein A, Krummenacher B, Gerber W, Lardon J, Gees F, Brügger L, Ott T (2015) Repeated controlled rockfall trajectory testing. [Abstract] Geophys. Res. Abstr. 17: EGU2015-9779. [2] Volkwein A, Klette J (2014) Semi-Automatic Determination of Rockfall Trajectories. Sensors 14: 18187-18210.

  15. DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation

    PubMed Central

    Koya, Junji; Kataoka, Keisuke; Sato, Tomohiko; Bando, Masashige; Kato, Yuki; Tsuruta-Kishino, Takako; Kobayashi, Hiroshi; Narukawa, Kensuke; Miyoshi, Hiroyuki; Shirahige, Katsuhiko; Kurokawa, Mineo

    2016-01-01

    Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias. PMID:27010239

  16. Azurin-like protein blocks invasion of Toxoplasma gondii through potential interactions with parasite surface antigen SAG1.

    PubMed

    Naguleswaran, Arunasalam; Fialho, Arsenio M; Chaudhari, Anita; Hong, Chang Soo; Chakrabarty, Ananda M; Sullivan, William J

    2008-02-01

    Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents. PMID:18070964

  17. Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

    PubMed

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-10-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

  18. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1

    PubMed Central

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants. PMID:26020533

  19. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

    PubMed

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants.

  20. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

    PubMed

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants. PMID:26020533

  1. MIBSA: Multi Interacting Blocks for Slope Analysis

    NASA Astrophysics Data System (ADS)

    Dattola, Giuseppe; Crosta, Giovanni; Castellanza, Riccardo; di Prisco, Claudio

    2016-04-01

    As it is well known, the slope instabilities have very important consequences in terms of human lives and activities. So predicting the evolution in time and space of slope mass movements becomes fundamental. This is even more relevant when we consider that the triggering mechanisms are a rising ground water level and the occurrence of earthquakes. Therefore, seasonal rainfall has a direct influence on the triggering of large rock and earthslide with a composite failure surface and causing differential behaviors within the sliding mass. In this contribution, a model describing the slope mass by means of an array of blocks that move on a prefixed failure surface, is defined. A shear band located at the base of each block, whose behavior is modelled via a viscous plastic model based on the Perzyna's approach, controls the slip velocity of the block. The motion of the blocks is obtained by solving the second balance equation in which the normal and tangential interaction forces are obtained by a specific interaction model. The model has been implemented in an original code and it is used to perform a parametric analysis that describes the effects of block interactions under a transient ground water oscillation. The numerical results confirm that the normal and tangential interactions between blocks can inhibit or induce the slope movements. The model is tested against some real case studies. This model is under development to add the dynamic effects generated by earthquake shaking.

  2. Responsive block copolymer photonics triggered by protein-polyelectrolyte coacervation.

    PubMed

    Fan, Yin; Tang, Shengchang; Thomas, Edwin L; Olsen, Bradley D

    2014-11-25

    Ionic interactions between proteins and polyelectrolytes are demonstrated as a method to trigger responsive transitions in block copolymer (BCP) photonic gels containing one neutral hydrophobic block and one cationic hydrophilic block. Poly(2-vinylpyridine) (P2VP) blocks in lamellar poly(styrene-b-2-vinylpyridine) block copolymer thin films are quaternized with primary bromides to yield swollen gels that show strong reflectivity peaks in the visible range; exposure to aqueous solutions of various proteins alters the swelling ratios of the quaternized P2VP (QP2VP) gel layers in the PS-QP2VP materials due to the ionic interactions between proteins and the polyelectrolyte. Parameters such as charge density, hydrophobicity, and cross-link density of the QP2VP gel layers as well as the charge and size of the proteins play significant roles on the photonic responses of the BCP gels. Differences in the size and pH-dependent charge of proteins provide a basis for fingerprinting proteins based on their temporal and equilibrium photonic response. The results demonstrate that the BCP gels and their photonic effect provide a robust and visually interpretable method to differentiate different proteins.

  3. Nanopatterning of recombinant proteins and viruses using block copolymer templates

    NASA Astrophysics Data System (ADS)

    Cresce, Arthur Von Wald

    The study of interfaces is important in understanding biological interactions, including cellular signaling and virus infection. This thesis is an original effort to examine the interaction between a block copolymer and both a protein and a virus. Block copolymers intrinsically form nanometer-scale structures over large areas without expensive processing, making them ideal for the synthesis of the nanopatterned surfaces used in this study. The geometry of these nanostructures can be easily tuned for different applications by altering the block ratio and composition of the block copolymer. Block copolymers can be used for controlled uptake of metal ions, where one block selectively binds metal ions while the other does not. 5-norbornene-2,3-dicarboxylic acid is synthesized through ring-opening metathesis polymerization. It formed spherical domains with spheres approximately 30 nm in diameter, and these spheres were then subsequently loaded with nickel ion. This norbornene block copolymer was tested for its ability to bind histidine-tagged green fluorescent protein (hisGFP), and it was found that the nickel-loaded copolymer was able to retain hisGFP through chelation between the histidine tag and the metal-containing portions of the copolymer surface. Poly(styrene-b-4-vinylpyridine) (PS/P4VP) was also loaded with nickel, forming a cylindrical microstructure. The binding of Tobacco mosaic virus and Tobacco necrosis virus was tested through Tween 20 detergent washes. Electron microscopy allowed for observation of both block copolymer nanostructures and virus particles. Results showed that Tween washes could not remove bound Tobacco mosaic virus from the surface of PS/P4VP. It was also seen that the size and tunability of block copolymers and the lack of processing needed to attain different structures makes them attractive for many applications, including microfluidic devices, surfaces to influence cellular signaling and growth, and as a nanopatterning surface for

  4. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  5. The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus

    SciTech Connect

    Ren Tao; Qu Feng; Morris, T. Jack . E-mail: jmorris@unlnotes.unl.edu

    2005-01-20

    We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

  6. Increased coverage of protein families with the blocks database servers.

    PubMed

    Henikoff, J G; Greene, E A; Pietrokovski, S; Henikoff, S

    2000-01-01

    The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBI's IMPALA program and 3D display of blocks on PDB structures.

  7. Visible Geology - Interactive online geologic block modelling

    NASA Astrophysics Data System (ADS)

    Cockett, R.

    2012-12-01

    Geology is a highly visual science, and many disciplines require spatial awareness and manipulation. For example, interpreting cross-sections, geologic maps, or plotting data on a stereonet all require various levels of spatial abilities. These skills are often not focused on in undergraduate geoscience curricula and many students struggle with spatial relations, manipulations, and penetrative abilities (e.g. Titus & Horsman, 2009). A newly developed program, Visible Geology, allows for students to be introduced to many geologic concepts and spatial skills in a virtual environment. Visible Geology is a web-based, three-dimensional environment where students can create and interrogate their own geologic block models. The program begins with a blank model, users then add geologic beds (with custom thickness and color) and can add geologic deformation events like tilting, folding, and faulting. Additionally, simple intrusive dikes can be modelled, as well as unconformities. Students can also explore the interaction of geology with topography by drawing elevation contours to produce their own topographic models. Students can not only spatially manipulate their model, but can create cross-sections and boreholes to practice their visual penetrative abilities. Visible Geology is easy to access and use, with no downloads required, so it can be incorporated into current, paper-based, lab activities. Sample learning activities are being developed that target introductory and structural geology curricula with learning objectives such as relative geologic history, fault characterization, apparent dip and thickness, interference folding, and stereonet interpretation. Visible Geology provides a richly interactive, and immersive environment for students to explore geologic concepts and practice their spatial skills.; Screenshot of Visible Geology showing folding and faulting interactions on a ridge topography.

  8. A Nonlinear Multi-Scale Interaction Model for Atmospheric Blocking: The Eddy-Blocking Matching Mechanism

    NASA Astrophysics Data System (ADS)

    Luo, Dehai; Cha, Jing; Zhong, Linhao; Dai, Aiguo

    2014-05-01

    In this paper, a nonlinear multi-scale interaction (NMI) model is used to propose an eddy-blocking matching (EBM) mechanism to account for how synoptic eddies reinforce or suppress a blocking flow. It is shown that the spatial structure of the eddy vorticity forcing (EVF) arising from upstream synoptic eddies determines whether an incipient block can grow into a meandering blocking flow through its interaction with the transient synoptic eddies from the west. Under certain conditions, the EVF exhibits a low-frequency oscillation on timescales of 2-3 weeks. During the EVF phase with a negative-over- positive dipole structure, a blocking event can be resonantly excited through the transport of eddy energy into the incipient block by the EVF. As the EVF changes into an opposite phase, the blocking decays. The NMI model produces life cycles of blocking events that resemble observations. Moreover, it is shown that the eddy north-south straining is a response of the eddies to a dipole- or Ω-type block. In our model, as in observations, two synoptic anticyclones (cyclones) can attract and merge with one another as the blocking intensifies, but only when the feedback of the blocking on the eddies is included. Thus, we attribute the eddy straining and associated vortex interaction to the feedback of the intensified blocking on synoptic eddies. The results illustrate the concomitant nature of the eddy deformation, whose role as a PV source for the blocking flow becomes important only during the mature stage of a block. Our EBM mechanism suggests that an incipient block flow is amplified (or suppressed) under certain conditions by the EVF coming from the upstream of the blocking region.

  9. Cordycepin Suppresses Thymic Stromal Lymphopoietin Expression via Blocking Caspase-1 and Receptor-Interacting Protein 2 Signaling Pathways in Mast Cells.

    PubMed

    Yoou, Myoung-schook; Jin, Mu Hyun; Lee, So Young; Lee, Sang Hwa; Kim, Byunghyun; Roh, Seok Seon; Choi, In Hwa; Lee, Myeong Soo; Kim, Hyung-Min; Jeong, Hyun-Ja

    2016-01-01

    Cordycepin (3'-deoxyadenosine) is one of the active components isolated from Cordyceps militaris, and has been shown to have anti-inflammatory, anti-oxidant, anti-aging, and anti-cancer effects. Mast cell-derived thymic stromal lymphopoietin (TSLP) plays an important role in the pathogenesis of allergic inflammatory reactions. Here, we investigated the regulatory effect and mechanisms of cordycepin on the expression of TSLP in the human mast cell line, HMC-1 cells, and in the human keratinocyte cell line, HaCaT cells. Cordycepin significantly decreased the production and mRNA expression of TSLP through the inhibition of caspase-1 and nuclear factor-κB activation. Cordycepin also significantly reduced the phosphorylation of receptor-interacting protein 2 and inhibitory kappa B (IκB) kinase β. Cordycepin significantly decreased the production and mRNA expression of interleukin (IL)-8, IL-1β, IL-6, and tumor necrosis factor-α in activated HMC-1 cells. Moreover, cordycepin significantly decreased the levels of TSLP in activated HaCaT cells. Our studies suggest that cordycepin can be applied to the treatment of allergic inflammatory diseases exacerbated by TSLP. PMID:26725432

  10. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  11. Cardiolipin Interactions with Proteins.

    PubMed

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E; Klein-Seetharaman, Judith

    2015-09-15

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.

  12. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  13. Cardiolipin Interactions with Proteins

    PubMed Central

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E.; Klein-Seetharaman, Judith

    2015-01-01

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions. PMID:26300339

  14. Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions

    PubMed Central

    Zervos, Thomas M.; Hernandez, Jean N.; Sutton, Patrick L.; Branch, Oralee H.

    2013-01-01

    SUMMARY The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections. PMID:22339946

  15. Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions.

    PubMed

    Zervos, Thomas M; Hernandez, Jean N; Sutton, Patrick L; Branch, Oralee H

    2012-05-01

    The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections.

  16. Ionic Interactions for Aqueous Templating of Biofunctional Molecules in Block Copolymer Nanostructures

    NASA Astrophysics Data System (ADS)

    Olsen, Bradley; Kim, Bokyung; Lam, Christopher; Stewart-Sloan, Charlotte; Gkikas, Emmanouil

    2013-03-01

    The use of ionic interactions to direct both biomolecular templating and block copolymer self-assembly into nanopatterned films with only aqueous processing conditions is demonstrated using block copolymers containing both thermally responsive and pH responsive blocks. Reversible addition-fragmentation chain transfer (RAFT) polymerization is employed to synthesize diblock copolymers with one neutral thermoresponsive and one polycationic block and the pH-dependnent complexation between model proteins or biomimetic J-aggregating chromophores and the polycationic block is demonstrated. Spin casting is used to prepare nanostructured films from the protein-block copolymer and chromophore-block copolymer coacervates. After film formation, the lower critical solution temperature (LCST) of the thermoresponsive block allows the nanomaterial to be effectively immobilized in aqueous environments at physiological temperatures, enabling use of the materials for biomolecule immobilization and controlled release. In the case of protein nanotemplating, the ionic environment in which the protein is confined enables the majority of the protein (80%) to retain its activity, even after having been dehydrated in vacuum and confined in the thin film.

  17. Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the Major Capsid Protein

    PubMed Central

    Matsushita, Misato; Fukui, Yoshiko; Yamada, Souichi; Tsuda, Mihoko; Higashi, Chizuka; Kaneko, Keiko; Hasegawa, Hideki; Yamaguchi, Toyofumi

    2012-01-01

    A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and -sensitive strains. In a plaque reduction assay under conditions in which the 50% effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25 μM, the 50% effective concentration of 35B2 was 0.75 μM. The selective index of the compound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open reading frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corresponding to the “floor” domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electron microscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are required to clarify the precise antiviral mechanism. PMID:22933294

  18. Interaction of Surfactants with Block Polyelectrolyte Gels

    NASA Astrophysics Data System (ADS)

    Crichton, Mark; Bhatia, Surita

    2002-03-01

    We present SANS and rheology for poly(styrene)-poly(acrylic acid) polymers in aqueous solutions. These polymers self-assemble to form spherical micelles in aqueous solutions, and the micelles associate to create elastic, transparent gels at moderate polymer concentrations. The addition of cationic and anionic surfactants (DTAB and SDS) can be used to modify the associative interactions and solution rheology. Addition of an anionic surfactant acts to screen attractive interactions and causes a monotonic decrease in the elastic modulus. However, the addition of a cationic surfactant appears to initially induce a stronger intermicellar attraction, leading to gels with a higher elastic modulus. At higher surfactant concentrations, the cationic surfactant begins to screen intermicellar association, leading to a decrease in the strength of the gel.

  19. Protein building blocks preserved by recombination.

    PubMed

    Voigt, Christopher A; Martinez, Carlos; Wang, Zhen-Gang; Mayo, Stephen L; Arnold, Frances H

    2002-07-01

    Borrowing concepts from the schema theory of genetic algorithms, we have developed a computational algorithm to identify the fragments of proteins, or schemas, that can be recombined without disturbing the integrity of the three-dimensional structure. When recombination leaves these schemas undisturbed, the hybrid proteins are more likely to be folded and functional. Crossovers found by screening libraries of several randomly shuffled proteins for functional hybrids strongly correlate with those predicted by this approach. Experimental results from the construction of hybrids of two beta-lactamases that share 40% amino acid identity demonstrate a threshold in the amount of schema disruption that the hybrid protein can tolerate. To the extent that introns function to promote recombination within proteins, natural selection would serve to bias their locations to schema boundaries. PMID:12042875

  20. Nanobiotechnology: protein-nanomaterial interactions.

    PubMed

    Kane, Ravi S; Stroock, Abraham D

    2007-01-01

    We review recent research that involves the interaction of nanomaterials such as nanoparticles, nanowires, and carbon nanotubes with proteins. We begin with a focus on the fundamentals of the structure and function of proteins on nanomaterials. We then review work in three areas that exploit these interactions: (1) sensing, (2) assembly of nanomaterials by proteins and proteins by nanomaterials, and (3) interactions with cells. We conclude with the identification of challenges and opportunities for the future. PMID:17335286

  1. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  2. Highly specific protein-protein interactions, evolution and negative design.

    PubMed

    Sear, Richard P

    2004-12-01

    We consider highly specific protein-protein interactions in proteomes of simple model proteins. We are inspired by the work of Zarrinpar et al (2003 Nature 426 676). They took a binding domain in a signalling pathway in yeast and replaced it with domains of the same class but from different organisms. They found that the probability of a protein binding to a protein from the proteome of a different organism is rather high, around one half. We calculate the probability of a model protein from one proteome binding to the protein of a different proteome. These proteomes are obtained by sampling the space of functional proteomes uniformly. In agreement with Zarrinpar et al we find that the probability of a protein binding a protein from another proteome is rather high, of order one tenth. Our results, together with those of Zarrinpar et al, suggest that designing, say, a peptide to block or reconstitute a single signalling pathway, without affecting any other pathways, requires knowledge of all the partners of the class of binding domains the peptide is designed to mimic. This knowledge is required to use negative design to explicitly design out interactions of the peptide with proteins other than its target. We also found that patches that are required to bind with high specificity evolve more slowly than those that are required only to not bind to any other patch. This is consistent with some analysis of sequence data for proteins engaged in highly specific interactions.

  3. Discovering interacting domains and motifs in protein-protein interactions.

    PubMed

    Hugo, Willy; Sung, Wing-Kin; Ng, See-Kiong

    2013-01-01

    Many important biological processes, such as the signaling pathways, require protein-protein interactions (PPIs) that are designed for fast response to stimuli. These interactions are usually transient, easily formed, and disrupted, yet specific. Many of these transient interactions involve the binding of a protein domain to a short stretch (3-10) of amino acid residues, which can be characterized by a sequence pattern, i.e., a short linear motif (SLiM). We call these interacting domains and motifs domain-SLiM interactions. Existing methods have focused on discovering SLiMs in the interacting proteins' sequence data. With the recent increase in protein structures, we have a new opportunity to detect SLiMs directly from the proteins' 3D structures instead of their linear sequences. In this chapter, we describe a computational method called SLiMDIet to directly detect SLiMs on domain interfaces extracted from 3D structures of PPIs. SLiMDIet comprises two steps: (1) interaction interfaces belonging to the same domain are extracted and grouped together using structural clustering and (2) the extracted interaction interfaces in each cluster are structurally aligned to extract the corresponding SLiM. Using SLiMDIet, de novo SLiMs interacting with protein domains can be computationally detected from structurally clustered domain-SLiM interactions for PFAM domains which have available 3D structures in the PDB database.

  4. Interactive-predictive detection of handwritten text blocks

    NASA Astrophysics Data System (ADS)

    Ramos Terrades, O.; Serrano, N.; Gordó, A.; Valveny, E.; Juan, A.

    2010-01-01

    A method for text block detection is introduced for old handwritten documents. The proposed method takes advantage of sequential book structure, taking into account layout information from pages previously transcribed. This glance at the past is used to predict the position of text blocks in the current page with the help of conventional layout analysis methods. The method is integrated into the GIDOC prototype: a first attempt to provide integrated support for interactive-predictive page layout analysis, text line detection and handwritten text transcription. Results are given in a transcription task on a 764-page Spanish manuscript from 1891.

  5. Designed, Helical Protein Nanotubes with Variable Diameters from a Single Building Block.

    PubMed

    Brodin, Jeffrey D; Smith, Sarah J; Carr, Jessica R; Tezcan, F Akif

    2015-08-26

    Due to their structural and mechanical properties, 1D helical protein assemblies represent highly attractive design targets for biomolecular engineering and protein design. Here we present a designed, tetrameric protein building block, Zn8R4, which assembles via Zn coordination interactions into a series of crystalline, helical nanotubes whose widths can be controlled by solution conditions. X-ray crystallography and transmission electron microscopy (TEM) measurements indicate that all classes of protein nanotubes are constructed through the same 2D arrangement of Zn8R4 tetramers held together by Zn coordination. The mechanical properties of these nanotubes are correlated with their widths. All Zn8R4 nanotubes are found to be highly flexible despite possessing crystalline order, owing to their minimal interbuilding-block interactions mediated solely by metal coordination.

  6. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  7. Aeolotopic interactions of globular proteins

    PubMed Central

    Lomakin, Aleksey; Asherie, Neer; Benedek, George B.

    1999-01-01

    Protein crystallization, aggregation, liquid–liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek aiolos (“variable”) and topos (“place”)] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization. PMID:10449715

  8. Bacteriophage Protein–Protein Interactions

    PubMed Central

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage–host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  9. Disentangling protein-silica interactions.

    PubMed

    Giussani, Lara; Tabacchi, Gloria; Gianotti, Enrica; Coluccia, Salvatore; Fois, Ettore

    2012-03-28

    We present the results of modelling studies aimed at the understanding of the interaction of a 7 nm sized water droplet containing a negatively charged globular protein with flat silica surfaces. We show how the droplet interaction with the surface depends on the electrostatic surface charge, and that adhesion of the droplet occurs when the surface is negatively charged as well. The key role of water and of the charge-balancing counter ions in mediating the surface-protein adhesion is highlighted. The relevance of the present results with respect to the production of bioinorganic hybrids via encapsulation of proteins inside mesoporous silica materials is discussed.

  10. How to Study Protein-protein Interactions.

    PubMed

    Podobnik, Marjetka; Kraševec, Nada; Bedina Zavec, Apolonija; Naneh, Omar; Flašker, Ajda; Caserman, Simon; Hodnik, Vesna; Anderluh, Gregor

    2016-01-01

    Physical and functional interactions between molecules in living systems are central to all biological processes. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Several powerful methodologies and techniques have been developed to study molecular interactions and thus help elucidate their nature and role in biology as well as potential ways how to interfere with them. All different techniques used in these studies have their strengths and weaknesses and since they are mostly employed in in vitro conditions, a single approach can hardly accurately reproduce interactions that happen under physiological conditions. However, complementary usage of as many as possible available techniques can lead to relatively realistic picture of the biological process. Here we describe several proteomic, biophysical and structural tools that help us understand the nature and mechanism of these interactions. PMID:27640371

  11. Interaction Trap/Two-Hybrid System to Identify Interacting Proteins

    PubMed Central

    Golemis, Erica A.; Serebriiskii, Ilya; Finley, Russell L.; Kolonin, Mikhail G.; Gyuris, Jeno; Brent, Roger

    2014-01-01

    The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins. PMID:18228339

  12. Nanopatterning of Viruses and Proteins Using Microphase Separated Block Copolymers

    NASA Astrophysics Data System (ADS)

    Cresce, Arthur; Lewandowski, Angela; Bentley, William; Kofinas, Peter

    2006-03-01

    Diblock copolymers containing nickel ions have been prepared that are capable of selectively adsorbing histidine-tagged green fluorescent protein (hisGFP), and also binding tobacco mosaic virus (TMV). A block copolymer of norbornene and norbornene dicarboxylic acid was synthesized using ring-opening metathesis polymerization. A 400/50 block ratio achieved a spherical microphase-separated morphology with roughly 20 nm diameter dicarboxylic acid spheres. The spherical phase was exposed to nickel ions in solution, templating the formation of nickel nanoparticles. This process gave a nickel-loaded diblock copolymer film whose surface was used to chelate hisGFP. Fluorescence spectroscopy and TEM confirmed the presence of the protein on the polymer surface. A sulfonated triblock copolymer was loaded with nickel ions using a similar solution-doping procedure. The morphology of this copolymer was lamellar, and its sulfonated block was loaded with nickel ions. TEM studies revealed the presence of the virus on the surface of the copolymer and showed that the bond between the TMV and the polymer surface can withstand severe detergent washes.

  13. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  14. The nucleocapsid protein of measles virus blocks host interferon response

    SciTech Connect

    Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira; Omi-Furutani, Mio; Sugai, Akihiro; Kanki, Keita; Yoneda, Misako; Kai, Chieko

    2012-03-01

    Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-{alpha}/{beta} and {gamma}-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

  15. Interactions of Pathological Hallmark Proteins

    PubMed Central

    Oláh, Judit; Vincze, Orsolya; Virók, Dezső; Simon, Dóra; Bozsó, Zsolt; Tőkési, Natália; Horváth, István; Hlavanda, Emma; Kovács, János; Magyar, Anna; Szűcs, Mária; Orosz, Ferenc; Penke, Botond; Ovádi, Judit

    2011-01-01

    The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ42 tightly bound to TPPP/p25 (Kd = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ42, α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease. PMID:21832049

  16. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  17. Control of Protein Affinity of Bioactive Nanocellulose and Passivation Using Engineered Block and Random Copolymers.

    PubMed

    Vuoriluoto, Maija; Orelma, Hannes; Zhu, Baolei; Johansson, Leena-Sisko; Rojas, Orlando J

    2016-03-01

    We passivated TEMPO-oxidized cellulose nanofibrils (TOCNF) toward human immunoglobulin G (hIgG) by modification with block and random copolymers of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA). The block copolymers reversibly adsorbed on TOCNF and were highly effective in preventing nonspecific interactions with hIgG, especially if short PDMAEMA blocks were used. In such cases, total protein rejection was achieved. This is in contrast to typical blocking agents, which performed poorly. When an anti-human IgG biointerface was installed onto the passivated TOCNF, remarkably high affinity antibody-antigen interactions were observed (0.90 ± 0.09 mg/m(2)). This is in contrast to the nonpassivated biointerface, which resulted in a significant false response. In addition, regeneration of the biointerface was possible by low pH aqueous wash. Protein A from Staphylococcus aureus was also utilized to successfully increase the sensitivity for human IgG recognition (1.28 ± 0.11 mg/m(2)). Overall, the developed system based on TOCNF modified with multifunctional polymers can be easily deployed as bioactive material with minimum fouling and excellent selectivity. PMID:26844956

  18. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  19. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  20. Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.

    PubMed

    Main, Ewan R G; Phillips, Jonathan J; Millership, Charlotte

    2013-10-01

    There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.

  1. Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides.

    PubMed

    Lin, Wei-Ling; Guu, Shih-Yun; Tsai, Chan-Chuan; Prakash, Ekambaranellore; Viswaraman, Mohan; Chen, Hsing-Bao; Chang, Chuan-Fa

    2015-01-01

    Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids.

  2. Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides

    PubMed Central

    Lin, Wei-Ling; Guu, Shih-Yun; Tsai, Chan-Chuan; Prakash, Ekambaranellore; Viswaraman, Mohan; Chen, Hsing-Bao; Chang, Chuan-Fa

    2015-01-01

    Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids. PMID:26076445

  3. Protein interactions in genome maintenance as novel antibacterial targets.

    PubMed

    Marceau, Aimee H; Bernstein, Douglas A; Walsh, Brian W; Shapiro, Walker; Simmons, Lyle A; Keck, James L

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  4. Insulin/poly(ethylene glycol)-block-poly(L-lysine) Complexes: Physicochemical Properties and Protein Encapsulation.

    PubMed

    Pippa, Natassa; Kalinova, Radostina; Dimitrov, Ivaylo; Pispas, Stergios; Demetzos, Costas

    2015-06-01

    Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides. PMID:25974620

  5. The protein-protein interaction map of Helicobacter pylori.

    PubMed

    Rain, J C; Selig, L; De Reuse, H; Battaglia, V; Reverdy, C; Simon, S; Lenzen, G; Petel, F; Wojcik, J; Schächter, V; Chemama, Y; Labigne, A; Legrain, P

    2001-01-11

    With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.

  6. Probing protein-sugar interactions.

    PubMed Central

    Ebel, C; Eisenberg, H; Ghirlando, R

    2000-01-01

    We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented. PMID:10620302

  7. Protein-protein interaction databases: keeping up with growing interactomes

    PubMed Central

    2009-01-01

    Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction database [MINT], the Biomolecular Interaction Network Database [BIND], the Database of Interacting Proteins [DIP], the IntAct molecular interaction database [IntAct] and the Human Protein Reference Database [HPRD]) differ in scope and content; integration of all datasets is non-trivial owing to differences in data annotation. With respect to human protein-protein interaction data, HPRD seems to be the most comprehensive. To obtain a complete dataset, however, interactions from all six databases have to be combined. To overcome this limitation, meta-databases such as the Agile Protein Interaction Database (APID) offer access to integrated protein-protein interaction datasets, although these also currently have certain restrictions. PMID:19403463

  8. Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies.

    PubMed

    Wright, Katherine E; Hjerrild, Kathryn A; Bartlett, Jonathan; Douglas, Alexander D; Jin, Jing; Brown, Rebecca E; Illingworth, Joseph J; Ashfield, Rebecca; Clemmensen, Stine B; de Jongh, Willem A; Draper, Simon J; Higgins, Matthew K

    2014-11-20

    Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN). Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite. PMID:25132548

  9. Proteins detection by polymer optical fibers sensitised with overlayers of block and random copolymers

    NASA Astrophysics Data System (ADS)

    El Sachat, Alexandros; Meristoudi, Anastasia; Markos, Christos; Pispas, Stergios; Riziotis, Christos

    2014-03-01

    A low cost and low complexity optical detection method of proteins is presented by employing a detection scheme based on electrostatic interactions, and implemented by sensitization of a polymer optical fibers' (POF) surface by thin overlayers of properly designed sensitive copolymer materials with predesigned charges. This method enables the fast detection of proteins having opposite charge to the overlayer, and also the effective discrimination of differently charged proteins like lysozyme (LYS) and bovine serum albumin (BSA). As sensitive materials the block and the random copolymers of the same monomers were employed, namely the block copolymer poly(styrene-b-2vinylpyridine) (PS-b- P2VP) and the corresponding random copolymer poly(styrene-r-2vinylpyridine) (PS-r-P2VP), of similar composition and molecular weights. Results show systematically different response between the block and the random copolymers, although of the same order of magnitude, drawing thus important conclusions on their applications' techno-economic aspects given that they have significantly different associated manufacturing method and costs. The use of the POF platform, in combination with those adaptable copolymer sensing materials could lead to efficient low cost bio-detection schemes.

  10. Protein Adsorption on Chemically Modified Block Copolymer Nanodomains: Influence of Charge and Flow.

    PubMed

    Silverstein, Joshua S; Casey, Brendan J; Kofinas, Peter; Dair, Benita J

    2016-02-01

    Understanding the interactions of biomacromolecules with nanoengineered surfaces is vital for assessing material biocompatibility. This study focuses on the dynamics of protein adsorption on nanopatterned block copolymers (BCPs). Poly(styrene)-block-poly(1,2-butadiene) BCPs functionalized with an acid, amine, amide, or captopril moieties were processed to produce nanopatterned films. These films were characterized using water contact angle measurements and atomic force microscopy in air and liquid to determine how the modification process affected. wettability and swelling. Protein adsorption experiments were conducted under static and dynamic conditions via a quartz crystal microbalance with dissipation. Proteins of various size, charge, and stability were investigated to determine whether their physical characteristics affected adsorption. Significantly decreased contact angles were caused by selective swelling of modified BCP domains. The results indicate that nanopatterned chemistry and experimental conditions strongly impact adsorption dynamics. Depending on the structural stability of the protein, polyelectrolyte surfaces significantly increased adsorption over controls. Further analysis suggested that protein stability may correlate with dissipation versus frequency plots. PMID:27433605

  11. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  12. Website Review: Protein-Protein Interactions on the Web

    PubMed Central

    Wixon, Jo

    2001-01-01

    We present a brief guide to resources on the Internet relating to Protein-Protein Interactions. These include databases containing experimentally verified and computationally inferred physical and functional interactions. There are also tools for predicting interactions and for extracting information on interactions from the literature, and organism specific databases. PMID:18629244

  13. Protein-protein interactions as a target for drugs in proteomics.

    PubMed

    Archakov, Alexander I; Govorun, Vadim M; Dubanov, Alexander V; Ivanov, Yuri D; Veselovsky, Alexander V; Lewi, Paul; Janssen, Paul

    2003-04-01

    Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization. PMID:12687606

  14. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  15. Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells.

    PubMed

    Rochette, Samuel; Diss, Guillaume; Filteau, Marie; Leducq, Jean-Baptiste; Dubé, Alexandre K; Landry, Christian R

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

  16. The aromatic stacking interactions between proteins and their macromolecular ligands.

    PubMed

    Rahman, Mohammad Mizanur; Muhseen, Ziyad Tariq; Junaid, Muhammad; Zhang, Houjin

    2015-01-01

    Aromatic stacking interactions arise from the attractive force between the π-electron clouds in the neighboring aromatic groups. The aromatic stacking is common between proteins and small molecules. The stacking interactions at the interfaces of proteins and other macromolecules are relatively rare. However it contributes to a significant portion of the stabilizing forces. In the proteinprotein complexes, aromatic interactions are involved in the protein oligomerization, such as dimer, trimer and tetramer formation. Also, aromatic residues can bind to nanoparticles through stacking interactions which offer them stronger affinity than other residues. These interactions play crucial roles in proteinnanoparticle conjugation. In the protein-nucleotide complexes, the specific recognitions are realized through stacking interactions between aromatic residues and the bases in the nucleotides. Many nucleoproteins use aromatic stacking to recognize binding site on DNA or RNA. Stacking interactions are involved in the process of mismatch repair, strand separation, deadenylation, degradation and RNA cap binding. They are proved to be important for the stability of complexes. The aromatic stacking is also the underlying reasons of many fatal diseases such as Alzheimer, cancer and cardiovascular diseases. The chemicals that can block the stacking interactions could have potential pharmaceutical values. In this review, we summarize recent finding regarding the functions of aromatic stacking interactions in the protein-macromolecule complexes. Our aim is to understand the mechanisms underlying the stacking-mediated complex formation and facilitate the development of drugs and other bio-products.

  17. Small molecules that target phosphorylation dependent protein-protein interaction.

    PubMed

    Watanabe, Nobumoto; Osada, Hiroyuki

    2016-08-01

    Protein-protein interaction is one of the key events in the signal transduction pathway. The interaction changes the conformations, activities, localization and stabilities of the proteins, and transduces the signal to the next step. Frequently, this interaction occurs upon the protein phosphorylation. When upstream signals are stimulated, protein kinase(s) is/are activated and phosphorylate(s) their substrates, and induce the phosphorylation dependent protein-protein interaction. For this interaction, several domains in proteins are known to specifically recognize the phosphorylated residues of target proteins. These specific domains for interaction are important in the progression of the diseases caused by disordered signal transduction such as cancer. Thus small molecules that modulate this interaction are attractive lead compounds for the treatment of such diseases. In this review, we focused on three examples of phosphorylation dependent protein-protein interaction modules (14-3-3, polo box domain of Plk1 and F-box proteins in SCF ubiquitin ligases) and summarize small molecules that modulate their interaction. We also introduce our original screening system to identify such small molecules.

  18. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  19. Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

    PubMed

    Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

    2015-02-23

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

  20. Fluorescence Studies of Protein Crystallization Interactions

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori; Forsythe, Elizabeth

    1999-01-01

    We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

  1. A Working Model of Protein Synthesis Using Lego(TM) Building Blocks.

    ERIC Educational Resources Information Center

    Templin, Mark A.; Fetters, Marcia K.

    2002-01-01

    Uses Lego building blocks to improve the effectiveness of teaching about protein synthesis. Provides diagrams and pictures for a 2-3 day student activity. Discusses mRNA, transfer RNA, and a protein synthesis model. (MVL)

  2. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  3. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    NASA Astrophysics Data System (ADS)

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  4. The protein interaction map of bacteriophage lambda

    PubMed Central

    2011-01-01

    Background Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. PMID:21943085

  5. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  6. Molecular simulations of lipid-mediated protein-protein interactions.

    PubMed

    de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

    2008-08-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved. PMID:18487292

  7. How do oncoprotein mutations rewire protein-protein interaction networks?

    PubMed

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  8. Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction.

    PubMed

    Basu, Avik; Chakrabarti, Abhijit

    2015-10-14

    In this report we have analyzed interacting partners of hemoglobin inside erythrocyte and sought possible implications of hemoglobin-spectrin interaction. Our list of identified cytosolic hemoglobin interacting proteins includes redox regulators like peroxiredoxin-2, Cu-Zn superoxide dismutase, catalase, aldehyde dehydrogenase-1, flavin reductase and chaperones like HSP70, α-hemoglobin stabilizing protein. Others include metabolic enzymes like carbonic anhydrase-1, selenium binding protein-1, purine nucleoside phosphorylase and nucleoside diphosphate kinase. Additionally, various membrane proteins like α and β spectrin, ankyrin, band3, protein4.1, actin and glyceraldehyde 3 phosphate dehydrogenase have been shown to interact with hemoglobin. Our result indicates that major membrane skeleton protein spectrin, that also has a chaperone like activity, helps to fold the unstable alpha-globin chains in vitro. Taken together our results could provide insight into a protein network evolved around hemoglobin molecule inside erythrocyte that may add a new perspective in understanding the hemoglobin function and homeostasis.

  9. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

  10. Peroxiredoxin 1 interacts with and blocks the redox factor APE1 from activating interleukin-8 expression.

    PubMed

    Nassour, Hassan; Wang, Zhiqiang; Saad, Amine; Papaluca, Arturo; Brosseau, Nicolas; Affar, El Bachir; Alaoui-Jamali, Moulay A; Ramotar, Dindial

    2016-01-01

    APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis. PMID:27388124

  11. Peroxiredoxin 1 interacts with and blocks the redox factor APE1 from activating interleukin-8 expression

    PubMed Central

    Nassour, Hassan; Wang, Zhiqiang; Saad, Amine; Papaluca, Arturo; Brosseau, Nicolas; Affar, El Bachir; Alaoui-Jamali, Moulay A.; Ramotar, Dindial

    2016-01-01

    APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis. PMID:27388124

  12. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  13. IFT-Cargo Interactions and Protein Transport in Cilia.

    PubMed

    Lechtreck, Karl F

    2015-12-01

    The motile and sensory functions of cilia and flagella are indispensable for human health. Cilia assembly requires a dedicated protein shuttle, intraflagellar transport (IFT), a bidirectional motility of multi-megadalton protein arrays along ciliary microtubules. IFT functions as a protein carrier delivering hundreds of distinct proteins into growing cilia. IFT-based protein import and export continue in fully grown cilia and are required for ciliary maintenance and sensing. Large ciliary building blocks might depend on IFT to move through the transition zone, which functions as a ciliary gate. Smaller, freely diffusing proteins, such as tubulin, depend on IFT to be concentrated or removed from cilia. As I discuss here, recent work provides insights into how IFT interacts with its cargoes and how the transport is regulated. PMID:26498262

  14. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  15. Electrostatic interactions as governing the fouling in protein microfiltration

    NASA Astrophysics Data System (ADS)

    Ouammou, M.; Tijani, N.; Calvo, J. I.; Palacio, L.; Prádanos, P.; Hernández, A.

    2005-03-01

    The influence of pH and electrostatic interactions on the fouling mechanism during protein dead-end microfiltration (MF) has been investigated for two charged membranes. Polyethersulfone acidic membranes (ICE-450), being negatively charged, and basic ones (SB-6407), these positively charged, both from Pall Co., have been used in the investigations. BSA and Lysozyme solutions at different pH values (3.0, 5.0, 7.0, 8.5 and 10.0) were microfiltered through the membranes at a constant applied transmembrane pressure. Results have been analysed in terms of usual blocking filtration laws and a substantial change in the fouling behaviour has been observed when solution pH and/or membrane charge as the pressure was changed, this change being clearly related with the specific membrane-protein and protein-protein interactions.

  16. NOXclass: prediction of protein-protein interaction types

    PubMed Central

    Zhu, Hongbo; Domingues, Francisco S; Sommer, lngolf; Lengauer, Thomas

    2006-01-01

    Background Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Results Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. Conclusion NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at . PMID:16423290

  17. The role of synoptic/planetary-scale interactions during the development of a blocking anticyclone

    NASA Technical Reports Server (NTRS)

    Tsou, Chih-Hua; Smith, Phillip J.

    1990-01-01

    Consideration is given to the development of a blocking anticyclone over the North Atlantic preceded by explosive cyclogenesis about 500 km south of Nova Scotia during January 19-21, 1979. The general behavior of the block is studied using the extended height tendency equation. The relative importance of planetary-scale, synoptic-scale, and scale-interaction forcing of the block is examined.

  18. Protein interaction networks from literature mining

    NASA Astrophysics Data System (ADS)

    Ihara, Sigeo

    2005-03-01

    The ability to accurately predict and understand physiological changes in the biological network system in response to disease or drug therapeutics is of crucial importance in life science. The extensive amount of gene expression data generated from even a single microarray experiment often proves difficult to fully interpret and comprehend the biological significance. An increasing knowledge of protein interactions stored in the PubMed database, as well as the advancement of natural language processing, however, makes it possible to construct protein interaction networks from the gene expression information that are essential for understanding the biological meaning. From the in house literature mining system we have developed, the protein interaction network for humans was constructed. By analysis based on the graph-theoretical characterization of the total interaction network in literature, we found that the network is scale-free and semantic long-ranged interactions (i.e. inhibit, induce) between proteins dominate in the total interaction network, reducing the degree exponent. Interaction networks generated based on scientific text in which the interaction event is ambiguously described result in disconnected networks. In contrast interaction networks based on text in which the interaction events are clearly stated result in strongly connected networks. The results of protein-protein interaction networks obtained in real applications from microarray experiments are discussed: For example, comparisons of the gene expression data indicative of either a good or a poor prognosis for acute lymphoblastic leukemia with MLL rearrangements, using our system, showed newly discovered signaling cross-talk.

  19. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949

  20. Nonparametric Tests for Analyzing Interactions among Intra-Block Ranks in Multiple Group Repeated Measures Designs.

    ERIC Educational Resources Information Center

    Beasley, T. Mark

    2000-01-01

    Developed an extension of the Hollander and Sethuraman (M. Hollander and J. Sethuraman, 1978) statistic (B squared) for testing discordance among intra-block rankings of K elements for multiple groups of raters. Simulation results confirmed the usefulness of B squared as an omnibus test of interaction among intra-block ranks and demonstrated its…

  1. Replication forks blocked by protein-DNA complexes have limited stability in vitro.

    PubMed

    McGlynn, Peter; Guy, Colin P

    2008-08-29

    There are many barriers that replication forks must overcome in order to duplicate a genome in vivo. These barriers include damage to the template DNA and proteins bound to this template. If replication is halted by such a block, then the block must be either removed or bypassed for replication to continue. If continuation of replication employs the original fork, avoiding the need to reload the replication apparatus, then the blocked replisome must retain functionality. In vivo studies of Escherichia coli replication forks suggest that replication forks blocked by protein-DNA complexes retain the ability to resume replication upon removal of the block for several hours. Here we tested the functional stability of replication forks reconstituted in vitro and blocked by lac repressor-operator complexes. Once a fork comes to a halt at such a block, it cannot continue subsequently to translocate through the block until addition of IPTG induces repressor dissociation. However, the ability to resume replication is retained only for 4-6 min regardless of the topological state of the template DNA. Comparison of our in vitro data with previous in vivo data suggests that either accessory factors that stabilise blocked forks are present in vivo or the apparent stability of blocked forks in vivo is due to continual reloading of the replication apparatus at the site of the block.

  2. A method for interactive specification of multiple-block topologies

    NASA Technical Reports Server (NTRS)

    Sorenson, Reese L.; Mccann, Karen M.

    1991-01-01

    A method is presented for dealing with the vast amount of topological and other data which must be specified to generate a multiple-block computational grid. Specific uses of the graphical capabilities of a powerful scientific workstation are described which reduce the burden on the user of collecting and formatting such large amounts of data. A program to implement this method, 3DPREP, is described. A plotting transformation algorithm, some useful software tools, notes on programming, and a database organization are also presented. Example grids developed using the method are shown.

  3. A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

    PubMed

    Athuluri-Divakar, Sai Krishna; Vasquez-Del Carpio, Rodrigo; Dutta, Kaushik; Baker, Stacey J; Cosenza, Stephen C; Basu, Indranil; Gupta, Yogesh K; Reddy, M V Ramana; Ueno, Lynn; Hart, Jonathan R; Vogt, Peter K; Mulholland, David; Guha, Chandan; Aggarwal, Aneel K; Reddy, E Premkumar

    2016-04-21

    Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling. PMID:27104980

  4. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  5. An Interactive Introduction to Protein Structure

    ERIC Educational Resources Information Center

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  6. Analysis of Stable and Transient Protein-Protein Interactions

    PubMed Central

    Byrum, Stephanie; Smart, Sherri K.; Larson, Signe; Tackett, Alan J.

    2012-01-01

    The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challenging as many of the techniques used for isolation result in the purification of only the most stable components and the transient interactions are lost. A technology called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) has been developed to identify these transiently interacting proteins as well as the stable interactions. Described here are the detailed methodological approaches used for a transient I-DIRT analysis of a multi-subunit complex, NuA3, that acetylates histone H3 and functions to activate gene transcription. Transcription is known to involve a concert of protein assemblies performing different activities on the chromatin/gene template, thus understanding the less stable or transient protein interactions with NuA3 will shed light onto the protein complexes that function synergistically, or antagonistically, to regulate gene transcription and chromatin remodeling. PMID:22183593

  7. Noninvasive imaging of protein-protein interactions in living animals

    NASA Astrophysics Data System (ADS)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  8. Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.

    PubMed

    Ambroz, Kristi L H; Zhang, Yonghong; Schutz-Geschwender, Amy; Olive, D Michael

    2008-06-01

    Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments. PMID:18563731

  9. DIP: The Database of Interacting Proteins

    DOE Data Explorer

    The DIP Database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent set of protein-protein interactions. By interaction, the DIP Database creators mean that two amino acid chains were experimentally identified to bind to each other. The database lists such pairs to aid those studying a particular protein-protein interaction but also those investigating entire regulatory and signaling pathways as well as those studying the organisation and complexity of the protein interaction network at the cellular level. The data stored within the DIP database were curated, both, manually by expert curators and also automatically using computational approaches that utilize the knowledge about the protein-protein interaction networks extracted from the most reliable, core subset of the DIP data. It is a relational database that can be searched by protein, sequence, motif, article information, and pathBLAST. The website also serves as an access point to a number of projects related to DIP, such as LiveDIP, The Database of Ligand-Receptor Partners (DLRP) and JDIP. Users have free and open access to DIP after login. [Taken from the DIP Guide and the DIP website] (Specialized Interface) (Registration Required)

  10. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  11. Structure and Interactions of Isolated Biomolecular Building Blocks.

    NASA Astrophysics Data System (ADS)

    de Vries, Mattanjah

    2006-03-01

    We investigate biomolecular building blocks and their clusters with each other and with water on a single molecular level. The motivation is the need to distinguish between intrinsic molecular properties and those that result from the biological environment. This is achieved by a combination of laser desorption and jet cooling, applied to aromatic amino acids, small peptides containing those, nucleobases and nucleosides. This approach is coupled with a number of laser spectroscopic techniques, including resonant multi-photon ionization, spectral hole burning and infra-red ion-dip spectroscopy. We will discuss examples illustrating how information can be obtained on spatial structure of individual biomolecules, including peptide conformations and details of DNA base-pairing.

  12. QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition.

    PubMed

    Tung, Chi-Hua; Chen, Chi-Wei; Guo, Ren-Chao; Ng, Hui-Fuang; Chu, Yen-Wei

    2016-01-01

    Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition. Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions. Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins. PMID:27610389

  13. QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition.

    PubMed

    Tung, Chi-Hua; Chen, Chi-Wei; Guo, Ren-Chao; Ng, Hui-Fuang; Chu, Yen-Wei

    2016-01-01

    Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition. Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions. Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins.

  14. QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition

    PubMed Central

    Tung, Chi-Hua; Chen, Chi-Wei; Guo, Ren-Chao; Ng, Hui-Fuang

    2016-01-01

    Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition. Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions. Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins. PMID:27610389

  15. QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition

    PubMed Central

    Tung, Chi-Hua; Chen, Chi-Wei; Guo, Ren-Chao; Ng, Hui-Fuang

    2016-01-01

    Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition. Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions. Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins.

  16. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  17. Capturing the Interaction Potential of Amyloidogenic Proteins

    SciTech Connect

    Javid, Nadeem; Vogtt, Karsten; Winter, Roland; Krywka, Christina; Tolan, Metin

    2007-07-13

    Experimentally derived static structure factors obtained for the aggregation-prone protein insulin were analyzed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek potential. The data reveal that the protein self-assembles into equilibrium clusters already at low concentrations. Furthermore, striking differences regarding interaction forces between aggregation-prone proteins such as insulin in the preaggregated regime and natively stable globular proteins are found.

  18. Quantitative interaction proteomics of neurodegenerative disease proteins.

    PubMed

    Hosp, Fabian; Vossfeldt, Hannes; Heinig, Matthias; Vasiljevic, Djordje; Arumughan, Anup; Wyler, Emanuel; Landthaler, Markus; Hubner, Norbert; Wanker, Erich E; Lannfelt, Lars; Ingelsson, Martin; Lalowski, Maciej; Voigt, Aaron; Selbach, Matthias

    2015-05-19

    Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.

  19. How Hofmeister ion interactions affect protein stability.

    PubMed Central

    Baldwin, R L

    1996-01-01

    Model compound studies in the literature show how Hofmeister ion interactions affect protein stability. Although model compound results are typically obtained as salting-out constants, they can be used to find out how the interactions affect protein stability. The null point in the Hofmeister series, which divides protein denaturants from stabilizers, arises from opposite interactions with different classes of groups: Hofmeister ions salt out nonpolar groups and salt in the peptide group. Theories of how Hofmeister ion interactions work need to begin by explaining the mechanisms of these two classes of interactions. Salting-out nonpolar groups has been explained by the cavity model, but its use is controversial. When applied to model compound data, the cavity model 1) uses surface tension increments to predict the observed values of the salting-out constants, within a factor of 3, and 2) predicts that the salting-out constant should increase with the number of carbon atoms in the aliphatic side chain of an amino acid, as observed. The mechanism of interaction between Hofmeister ions and the peptide group is not well understood, and it is controversial whether this interaction is ion-specific, or whether it is nonspecific and the apparent specificity resides in interactions with nearby nonpolar groups. A nonspecific salting-in interaction is known to occur between simple ions and dipolar molecules; it depends on ionic strength, not on position in the Hofmeister series. A theory by Kirkwood predicts the strength of this interaction and indicates that it depends on the first power of the ionic strength. Ions interact with proteins in various ways besides the Hofmeister ion interactions discussed here, especially by charge interactions. Much of what is known about these interactions comes from studies by Serge Timasheff and his co-workers. A general model, suitable for analyzing diverse ion-protein interactions, is provided by the two-domain model of Record and co

  20. Signature Product Code for Predicting Protein-Protein Interactions

    SciTech Connect

    Martin, Shawn B.; Brown, William M.

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictions about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.

  1. Signature Product Code for Predicting Protein-Protein Interactions

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictionsmore » about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.« less

  2. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    PubMed Central

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  3. Reconstitution of the membrane protein OmpF into biomimetic block copolymer-phospholipid hybrid membranes.

    PubMed

    Bieligmeyer, Matthias; Artukovic, Franjo; Nussberger, Stephan; Hirth, Thomas; Schiestel, Thomas; Müller, Michaela

    2016-01-01

    Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  4. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    PubMed Central

    Torres, Jaume; Surya, Wahyu; Li, Yan; Liu, Ding Xiang

    2015-01-01

    Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i) the envelope protein in coronaviruses and (ii) the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity. PMID:26053927

  5. Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling.

    PubMed

    Neacsu, Maria Victoria; Matei, Iulia; Micutz, Marin; Staicu, Teodora; Precupas, Aurica; Popa, Vlad Tudor; Salifoglou, Athanasios; Ionita, Gabriela

    2016-05-12

    The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements, and differential scanning microcalorimetry (μDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational changes to albumin if it is organized in a micellar form, while albumin influences the self-assembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The μDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the

  6. Yeast protein-protein interaction assays and screens.

    PubMed

    de Folter, Stefan; Immink, Richard G H

    2011-01-01

    Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein's function and the molecular mechanism underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and identify the composition of protein complexes. This in vivo based system makes use of two functional protein domains of the GAL4 transcription factor, each fused to a protein of interest. Upon interaction between the two proteins under study, a transcriptional activator gets reconstituted and reporter genes get activated, allowing the yeast to grow on selective medium. In this chapter protocols are given for Y2H library screening, directed Y2H screening, Y2H matrix screening, and YnH screening involving more than two proteins. PMID:21720951

  7. Protein-phospholipid interactions in blood clotting.

    PubMed

    Morrissey, James H; Davis-Harrison, Rebecca L; Tavoosi, Narjes; Ke, Ke; Pureza, Vincent; Boettcher, John M; Clay, Mary C; Rienstra, Chad M; Ohkubo, Y Zenmei; Pogorelov, Taras V; Tajkhorshid, Emad

    2010-04-01

    Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions. PMID:20129649

  8. Protein-Phospholipid Interactions in Blood Clotting

    PubMed Central

    Morrissey, James H.; Davis-Harrison, Rebecca L.; Tavoosi, Narjes; Ke, Ke; Pureza, Vincent; Boettcher, John M.; Clay, Mary C.; Rienstra, Chad M.; Ohkubo, Y. Zenmei; Pogorelov, Taras V.; Tajkhorshid, Emad

    2010-01-01

    Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions. PMID:20129649

  9. Characterization of protein-protein interactions by isothermal titration calorimetry.

    PubMed

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2015-01-01

    The analysis of protein-protein interactions has attracted the attention of many researchers from both a fundamental point of view and a practical point of view. From a fundamental point of view, the development of an understanding of the signaling events triggered by the interaction of two or more proteins provides key information to elucidate the functioning of many cell processes. From a practical point of view, understanding protein-protein interactions at a quantitative level provides the foundation for the development of antagonists or agonists of those interactions. Isothermal Titration Calorimetry (ITC) is the only technique with the capability of measuring not only binding affinity but the enthalpic and entropic components that define affinity. Over the years, isothermal titration calorimeters have evolved in sensitivity and accuracy. Today, TA Instruments and MicroCal market instruments with the performance required to evaluate protein-protein interactions. In this methods paper, we describe general procedures to analyze heterodimeric (porcine pancreatic trypsin binding to soybean trypsin inhibitor) and homodimeric (bovine pancreatic α-chymotrypsin) protein associations by ITC.

  10. RNA Protein Interaction in Neurons

    PubMed Central

    Darnell, Robert B.

    2013-01-01

    Neurons have their own systems for regulating RNA. Several multigene families encode RNA binding proteins (RNABPs) that are uniquely expressed in neurons, including the well-known neuron-specific markers ELAV and NeuN, and the disease antigen NOVA. New technologies have emerged in recent years to assess the function of these proteins in vivo, and the answers are yielding insights into how and why neurons may regulate RNA in special ways—to increase cellular complexity, to spatially localize mRNA, and to regulate their expression in response to synaptic stimuli. The functions of such restricted neuronal proteins is likely to be complimented by more widely expressed RNABPs that may themselves have developed specialized functions in neurons, including Argonaute/miRNAs. Here we review what is known about such RNABPs, and explore the potential biologic and neurologic significance of neuronal RNA regulatory systems. PMID:23701460

  11. Characterizing carbohydrate-protein interactions by NMR

    PubMed Central

    Bewley, Carole A.; Shahzad-ul-Hussan, Syed

    2013-01-01

    Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792

  12. Embryonic stem cells: protein interaction networks*

    PubMed Central

    Ng, Patricia Miang-Lon; Lufkin, Thomas

    2012-01-01

    Embryonic stem cells have the ability to differentiate into nearly all cell types. However, the molecular mechanism of its pluripotency is still unclear. Oct3/4, Sox2 and Nanog are important factors of pluripotency. Oct3/4 (hereafter referred to as Oct4), in particular, has been an irreplaceable factor in the induction of pluripotency in adult cells. Proteins interacting with Oct4 and Nanog have been identified via affinity purification and mass spectrometry. These data, together with iterative purifications of interacting proteins allowed a protein interaction network to be constructed. The network currently includes 77 transcription factors, all of which are interconnected in one network. In-depth studies of some of these transcription factors show that they all recruit the NuRD complex. Hence, transcription factor clustering and chromosomal remodeling are key mechanism used by embryonic stem cells. Studies using RNA interference suggest that more pluripotency genes are yet to be discovered via protein-protein interactions. More work is required to complete and curate the embryonic stem cell protein interaction network. Analysis of a saturated protein interaction network by system biology tools can greatly aid in the understanding of the embryonic stem cell pluripotency network. PMID:22639699

  13. Predicting the fission yeast protein interaction network.

    PubMed

    Pancaldi, Vera; Saraç, Omer S; Rallis, Charalampos; McLean, Janel R; Převorovský, Martin; Gould, Kathleen; Beyer, Andreas; Bähler, Jürg

    2012-04-01

    A systems-level understanding of biological processes and information flow requires the mapping of cellular component interactions, among which protein-protein interactions are particularly important. Fission yeast (Schizosaccharomyces pombe) is a valuable model organism for which no systematic protein-interaction data are available. We exploited gene and protein properties, global genome regulation datasets, and conservation of interactions between budding and fission yeast to predict fission yeast protein interactions in silico. We have extensively tested our method in three ways: first, by predicting with 70-80% accuracy a selected high-confidence test set; second, by recapitulating interactions between members of the well-characterized SAGA co-activator complex; and third, by verifying predicted interactions of the Cbf11 transcription factor using mass spectrometry of TAP-purified protein complexes. Given the importance of the pathway in cell physiology and human disease, we explore the predicted sub-networks centered on the Tor1/2 kinases. Moreover, we predict the histidine kinases Mak1/2/3 to be vital hubs in the fission yeast stress response network, and we suggest interactors of argonaute 1, the principal component of the siRNA-mediated gene silencing pathway, lost in budding yeast but preserved in S. pombe. Of the new high-quality interactions that were discovered after we started this work, 73% were found in our predictions. Even though any predicted interactome is imperfect, the protein network presented here can provide a valuable basis to explore biological processes and to guide wet-lab experiments in fission yeast and beyond. Our predicted protein interactions are freely available through PInt, an online resource on our website (www.bahlerlab.info/PInt).

  14. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  15. A hierarchical protein folding scheme based on the building block folding model.

    PubMed

    Haspel, Nurit; Wainreb, Gilad; Inbar, Yuval; Tsai, Hui-Hsu; Tsai, Chung-Jung; Wolfson, Haim J; Nussinov, Ruth

    2007-01-01

    The building block protein folding model states that the native protein structure is the product of a combinatorial assembly of relatively structurally independent contiguous parts of the protein that possess a hydrophobic core, i.e., building blocks (BBs). According to this model, our group proposed a three-stage scheme for a feasible time-wise semi ab-intio protein structure prediction. Given a protein sequence, at the first stage of the prediction scheme, we propose cutting the sequence into structurally assigned BBs. Next, we perform a combinatorial assembly and attempt to predict the relative three-dimensional arrangement of the BBs. In the third stage, we refine and rank the assemblies. The scheme has proven to be very promising in reducing the complexity of the protein folding problem and gaining insight into the protein folding process. In this chapter, we describe the different stages of the scheme and discuss a possible application of the model to protein design. PMID:16957324

  16. Structure Activity Relationships of Monocyte Chemoattractant Proteins in Complex with a Blocking Antibody

    SciTech Connect

    Reid,C.; Rushe, M.; Jarpe, M.; Van Vlijmen, H.; Dolinski, B.; Qian, F.; Cachero, T.; Cuervo, H.; Yanachkova, M.; et al.

    2006-01-01

    Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 Angstroms structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody- chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.

  17. Interaction proteomics of synapse protein complexes

    PubMed Central

    Klemmer, Patricia; Smit, August B.

    2010-01-01

    The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome. PMID:20361179

  18. Multivalent Protein Assembly Using Monovalent Self-Assembling Building Blocks

    PubMed Central

    Petkau-Milroy, Katja; Sonntag, Michael H.; Colditz, Alexander; Brunsveld, Luc

    2013-01-01

    Discotic molecules, which self-assemble in water into columnar supramolecular polymers, emerged as an alternative platform for the organization of proteins. Here, a monovalent discotic decorated with one single biotin was synthesized to study the self-assembling multivalency of this system in regard to streptavidin. Next to tetravalent streptavidin, monovalent streptavidin was used to study the protein assembly along the supramolecular polymer in detail without the interference of cross-linking. Upon self-assembly of the monovalent biotinylated discotics, multivalent proteins can be assembled along the supramolecular polymer. The concentration of discotics, which influences the length of the final polymers at the same time dictates the amount of assembled proteins. PMID:24152447

  19. TSEMA: interactive prediction of protein pairings between interacting families.

    PubMed

    Izarzugaza, José M G; Juan, David; Pons, Carles; Ranea, Juan A G; Valencia, Alfonso; Pazos, Florencio

    2006-07-01

    An entire family of methodologies for predicting protein interactions is based on the observed fact that families of interacting proteins tend to have similar phylogenetic trees due to co-evolution. One application of this concept is the prediction of the mapping between the members of two interacting protein families (which protein within one family interacts with which protein within the other). The idea is that the real mapping would be the one maximizing the similarity between the trees. Since the exhaustive exploration of all possible mappings is not feasible for large families, current approaches use heuristic techniques which do not ensure the best solution to be found. This is why it is important to check the results proposed by heuristic techniques and to manually explore other solutions. Here we present TSEMA, the server for efficient mapping assessment. This system calculates an initial mapping between two families of proteins based on a Monte Carlo approach and allows the user to interactively modify it based on performance figures and/or specific biological knowledge. All the explored mappings are graphically shown over a representation of the phylogenetic trees. The system is freely available at http://pdg.cnb.uam.es/TSEMA. Standalone versions of the software behind the interface are available upon request from the authors.

  20. Website on Protein Interaction and Protein Structure Related Work

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  1. Novel computational methods to design protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Zhou, Alice Qinhua; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite the abundance of structural data, we still cannot accurately predict the structural and energetic changes resulting from mutations at protein interfaces. The inadequacy of current computational approaches to the analysis and design of protein-protein interactions has hampered the development of novel therapeutic and diagnostic agents. In this work, we apply a simple physical model that includes only a minimal set of geometrical constraints, excluded volume, and attractive van der Waals interactions to 1) rank the binding affinity of mutants of tetratricopeptide repeat proteins with their cognate peptides, 2) rank the energetics of binding of small designed proteins to the hydrophobic stem region of the influenza hemagglutinin protein, and 3) predict the stability of T4 lysozyme and staphylococcal nuclease mutants. This work will not only lead to a fundamental understanding of protein-protein interactions, but also to the development of efficient computational methods to rationally design protein interfaces with tunable specificity and affinity, and numerous applications in biomedicine. NSF DMR-1006537, PHY-1019147, Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, and Howard Hughes Medical Institute.

  2. Teaching Noncovalent Interactions Using Protein Molecular Evolution

    ERIC Educational Resources Information Center

    Fornasari, Maria Silvina; Parisi, Gustavo; Echave, Julian

    2008-01-01

    Noncovalent interactions and physicochemical properties of amino acids are important topics in biochemistry courses. Here, we present a computational laboratory where the capacity of each of the 20 amino acids to maintain different noncovalent interactions are used to investigate the stabilizing forces in a set of proteins coming from organisms…

  3. 21 CFR 520.2380a - Thiabendazole top dressing and mineral protein block.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Thiabendazole top dressing and mineral protein... § 520.2380a Thiabendazole top dressing and mineral protein block. (a) Chemical name. 2-(4-Thiazolyl..., Ostertagia and Cooperia). (iv) Limitations. Administer to cattle on pasture or range accustomed to...

  4. 21 CFR 520.2380a - Thiabendazole top dressing and mineral protein block.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Thiabendazole top dressing and mineral protein... § 520.2380a Thiabendazole top dressing and mineral protein block. (a) Chemical name. 2-(4-Thiazolyl..., Ostertagia and Cooperia). (iv) Limitations. Administer to cattle on pasture or range accustomed to...

  5. 21 CFR 520.2380a - Thiabendazole top dressing and mineral protein block.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Thiabendazole top dressing and mineral protein... § 520.2380a Thiabendazole top dressing and mineral protein block. (a) Chemical name. 2-(4-Thiazolyl..., Ostertagia and Cooperia). (iv) Limitations. Administer to cattle on pasture or range accustomed to...

  6. 21 CFR 520.2380a - Thiabendazole top dressing and mineral protein block.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Thiabendazole top dressing and mineral protein... § 520.2380a Thiabendazole top dressing and mineral protein block. (a) Chemical name. 2-(4-Thiazolyl..., Ostertagia and Cooperia). (iv) Limitations. Administer to cattle on pasture or range accustomed to...

  7. 21 CFR 520.2380a - Thiabendazole top dressing and mineral protein block.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Thiabendazole top dressing and mineral protein... § 520.2380a Thiabendazole top dressing and mineral protein block. (a) Chemical name. 2-(4-Thiazolyl..., Ostertagia and Cooperia). (iv) Limitations. Administer to cattle on pasture or range accustomed to...

  8. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    ERIC Educational Resources Information Center

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  9. Quantitative study of protein-protein interactions by quartz nanopipettes.

    PubMed

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-09-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

  10. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    SciTech Connect

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui; Xiao, Gengfu

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  11. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  12. Non-interacting surface solvation and dynamics in protein-protein interactions.

    PubMed

    Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

    2015-03-01

    Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. PMID:25524313

  13. Nascent peptides that block protein synthesis in bacteria

    PubMed Central

    Woolstenhulme, Christopher J.; Parajuli, Shankar; Healey, David W.; Valverde, Diana P.; Petersen, E. Nicholas; Starosta, Agata L.; Guydosh, Nicholas R.; Johnson, W. Evan; Wilson, Daniel N.; Buskirk, Allen R.

    2013-01-01

    Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene. Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins. PMID:23431150

  14. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

    PubMed

    Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

    2015-09-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

  15. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

    PubMed Central

    Checco, James W.; Lee, Erinna F.; Evangelista, Marco; Sleebs, Nerida J.; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J.; Eddinger, Geoffrey A.; Belair, David G.; Wilson, Julia L.; Eller, Chelcie H.; Raines, Ronald T.; Murphy, William L.; Smith, Brian J.; Gellman, Samuel H.; Fairlie, W. Douglas

    2015-01-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of L-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues (“α/β-peptides”) manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous “α-peptides”. This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a “stapled” Bim BH3 α-peptide, which contains a hydrocarbon crosslink to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain crosslinking to produce synergistic benefits. PMID:26317395

  16. Proteomic analysis of SETD6 interacting proteins

    PubMed Central

    Cohn, Ofir; Chen, Ayelet; Feldman, Michal; Levy, Dan

    2016-01-01

    SETD6 (SET-domain-containing protein 6) is a mono-methyltransferase that has been shown to methylate RelA and H2AZ. Using a proteomic approach we recently identified several new SETD6 substrates. To identify novel SETD6 interacting proteins, SETD6 was immunoprecipitated (IP) from Human erythromyeloblastoid leukemia K562 cells. SETD6 binding proteins were subjected to mass-spectrometry analysis resulting in 115 new SETD6 binding candidates. STRING database was used to map the SETD6 interactome network. Network enrichment analysis of biological processes with Gene Ontology (GO) database, identified three major groups; metabolic processes, muscle contraction and protein folding. PMID:26937450

  17. Targeting protein-protein interactions in hematologic malignancies: still a challenge or a great opportunity for future therapies?

    PubMed Central

    Cierpicki, Tomasz; Grembecka, Jolanta

    2015-01-01

    Summary Over the past several years, there has been an increasing research effort focused on inhibition of protein-protein interactions (PPIs) to develop novel therapeutic approaches for cancer, including hematologic malignancies. These efforts have led to development of small molecule inhibitors of PPIs, some of which already advanced to the stage of clinical trials while others are at different stages of pre-clinical optimization, emphasizing PPIs as an emerging and attractive class of drug targets. Here, we review several examples of recently developed inhibitors of protein-protein interactions highly relevant to hematologic cancers. We address the existing skepticism about feasibility of targeting PPIs and emphasize potential therapeutic benefit from blocking PPIs in hematologic malignancies. We then use these examples to discuss the approaches for successful identification of PPI inhibitors and provide analysis of the protein-protein interfaces, with the goal to address ‘druggability’ of new PPIs relevant to hematology. We discuss lessons learned to improve the success of targeting new protein-protein interactions and evaluate prospects and limits of the research in this field. We conclude that not all PPIs are equally tractable for blocking by small molecules, and detailed analysis of PPI interfaces is critical for selection of those with the highest chance of success. Together, our analysis uncovers patterns that should help to advance drug discovery in hematologic malignancies by successful targeting of new protein-protein interactions. PMID:25510283

  18. Water-protein interactions from high-resolution protein crystallography.

    PubMed Central

    Nakasako, Masayoshi

    2004-01-01

    To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level. PMID:15306376

  19. Scale interactions and diabatic processes during the development of an Atlantic blocking event

    NASA Technical Reports Server (NTRS)

    Morgan, Michael C.; Neilley, Peter P.; Dole, Randall M.

    1991-01-01

    Results of a case study of the development of a major blocking event over the North Atlantic Ocean are reported with emphasis on the potential roles of interactions between synoptic-scale systems and diabatic processes in the large-scale development. The approach used here involves the analysis of the evolution of the potential temperature on a constant potential vorticity (PV) surface, where the value of PV is chosen to be representative of that of the tropopause. The approach is shown to provide a simple way to qualitatively identify significant features in the development of the block, including scale interactions and diabatic processes.

  20. Pauli blocking and final-state interaction in electron-nucleus quasielastic scattering

    SciTech Connect

    Liu, Lon-chang

    2008-01-01

    The nucleon final-state interaction in electron-nucleus quasielastic scattering is studied. Based on the unitarity equation satisfied by the scattering-wave operators, a doorway model is developed to implement the Pauli-blocking of nucleon knockout. The model is complementary to the commonly used nuclear Fermi gas model which can not be applied with confidence to light- and medium-mass nuclei. Pauli blocking in these latter nuclei is illustrated with the case of Coulomb interaction. Significant effects are noted for beam energies below {approx} 350 MeV/c. Extension of the model to high-energy hadron-nucleus quasielastic scatterings is discussed.

  1. Predicting protein-peptide interactions from scratch

    NASA Astrophysics Data System (ADS)

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin; Zou lab Team

    Protein-peptide interactions play an important role in many cellular processes. The ability to predict protein-peptide complex structures is valuable for mechanistic investigation and therapeutic development. Due to the high flexibility of peptides and lack of templates for homologous modeling, predicting protein-peptide complex structures is extremely challenging. Recently, we have developed a novel docking framework for protein-peptide structure prediction. Specifically, given the sequence of a peptide and a 3D structure of the protein, initial conformations of the peptide are built through protein threading. Then, the peptide is globally and flexibly docked onto the protein using a novel iterative approach. Finally, the sampled modes are scored and ranked by a statistical potential-based energy scoring function that was derived for protein-peptide interactions from statistical mechanics principles. Our docking methodology has been tested on the Peptidb database and compared with other protein-peptide docking methods. Systematic analysis shows significantly improved results compared to the performances of the existing methods. Our method is computationally efficient and suitable for large-scale applications. Nsf CAREER Award 0953839 (XZ) NIH R01GM109980 (XZ).

  2. Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.

    PubMed Central

    Fearon, E R; Finkel, T; Gillison, M L; Kennedy, S P; Casella, J F; Tomaselli, G F; Morrow, J S; Van Dang, C

    1992-01-01

    We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions. Images PMID:1387709

  3. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  4. Predicting protein-protein interactions in the post synaptic density.

    PubMed

    Bar-shira, Ossnat; Chechik, Gal

    2013-09-01

    The post synaptic density (PSD) is a specialization of the cytoskeleton at the synaptic junction, composed of hundreds of different proteins. Characterizing the protein components of the PSD and their interactions can help elucidate the mechanism of long-term changes in synaptic plasticity, which underlie learning and memory. Unfortunately, our knowledge of the proteome and interactome of the PSD is still partial and noisy. In this study we describe a computational framework to improve the reconstruction of the PSD network. The approach is based on learning the characteristics of PSD protein interactions from a set of trusted interactions, expanding this set with data collected from large scale repositories, and then predicting novel interaction with proteins that are suspected to reside in the PSD. Using this method we obtained thirty predicted interactions, with more than half of which having supporting evidence in the literature. We discuss in details two of these new interactions, Lrrtm1 with PSD-95 and Src with Capg. The first may take part in a mechanism underlying glutamatergic dysfunction in schizophrenia. The second suggests an alternative mechanism to regulate dendritic spines maturation.

  5. DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions.

    PubMed

    Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

    2016-01-01

    Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction's mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein-protein interactions or protein-DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1,040,000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43,000 RNA-mediated interactions, and ∼12,000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

  6. Side-Chain Conformational Preferences Govern Protein-Protein Interactions.

    PubMed

    Watkins, Andrew M; Bonneau, Richard; Arora, Paramjit S

    2016-08-24

    Protein secondary structures serve as geometrically constrained scaffolds for the display of key interacting residues at protein interfaces. Given the critical role of secondary structures in protein folding and the dependence of folding propensities on backbone dihedrals, secondary structure is expected to influence the identity of residues that are important for complex formation. Counter to this expectation, we find that a narrow set of residues dominates the binding energy in protein-protein complexes independent of backbone conformation. This finding suggests that the binding epitope may instead be substantially influenced by the side-chain conformations adopted. We analyzed side-chain conformational preferences in residues that contribute significantly to binding. This analysis suggests that preferred rotamers contribute directly to specificity in protein complex formation and provides guidelines for peptidomimetic inhibitor design.

  7. A novel peptide can mimic extracellular fibrinogen-binding protein to block the activation of complement system.

    PubMed

    Gao, Ya-ping; Dong, Jie; Zhang, Xin; Liu, Yu; Lu, Qiang; Feng, Jian-nan; Tan, Xiao-rong; Yang, Guang

    2013-07-01

    Extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus (S. aureus) is a bi-functional protein, which can specifically bind fibrinogen with its N terminus and inhibit deposition of C3b on the surface of S. aureus with its C terminus. Here, we screened the epitopes of Efb using phage display. Four peptides with consensus motif were screened. This consensus motif was identical to C terminus (161-164) of Efb. In the further investigation, it was found the synthesized peptide EC1 (154-165aa of Efb) could specifically bind C3/C3b and subsequently to block the activation of complement. Meanwhile, EC1 could inhibit the interaction between Efb and C3/C3b. Moreover, the interaction between the mutant protein of EmC1 (Efb without EC1) and C3 was decreased. And, the effect on the complement system of the mutant protein was dramatically declined compared with Efb. Our finding suggested that the peptide EC1 could mimic Efb to block complement system activation via binding C3.

  8. Interactions between plasma and block copolymers used in directed self-assembly patterning

    NASA Astrophysics Data System (ADS)

    Sirard, Stephen; Azarnouche, Laurent; Gurer, Emir; Durand, William; Maher, Michael; Mori, Kazunori; Blachut, Gregory; Janes, Dustin; Asano, Yusuke; Someya, Yasunobu; Hymes, Diane; Graves, David; Ellison, Christopher J.; Willson, C. Grant

    2016-03-01

    The directed self-assembly (DSA) of block copolymers offers a promising route for scaling feature sizes below 20 nm. At these small dimensions, plasmas are often used to define the initial patterns. It is imperative to understand how plasmas interact with each block in order to design processes with sufficient etch contrast and pattern fidelity. Symmetric lamella forming block copolymers including, polystyrene-b-poly(methyl methacrylate) and several high-χ silicon-containing and tin-containing block copolymers were synthesized, along with homopolymers of each block, and exposed to various oxidizing, reducing, and fluorine-based plasma processes. Etch rate kinetics were measured, and plasma modifications of the materials were characterized using XPS, AES, and FTIR. Mechanisms for achieving etch contrast were elucidated and were highly dependent on the block copolymer architecture. For several of the polymers, plasma photoemissions were observed to play an important role in modifying the materials and forming etch-resistant protective layers. Furthermore, it was observed for the silicon- and tin-containing polymers that an initial transient state exists, where the polymers exhibit an enhanced etch rate, prior to the formation of the etch-resistant protective layer. Plasma developed patterns were demonstrated for the differing block copolymer materials with feature sizes ranging from 20 nm down to approximately 5 nm.

  9. Thermosensing via transmembrane protein-lipid interactions.

    PubMed

    Saita, Emilio A; de Mendoza, Diego

    2015-09-01

    Cell membranes are composed of a lipid bilayer containing proteins that cross and/or interact with lipids on either side of the two leaflets. The basic structure of cell membranes is this bilayer, composed of two opposing lipid monolayers with fascinating properties designed to perform all the functions the cell requires. To coordinate these functions, lipid composition of cellular membranes is tailored to suit their specialized tasks. In this review, we describe the general mechanisms of membrane-protein interactions and relate them to some of the molecular strategies organisms use to adjust the membrane lipid composition in response to a decrease in environmental temperature. While the activities of all biomolecules are altered as a function of temperature, the thermosensors we focus on here are molecules whose temperature sensitivity appears to be linked to changes in the biophysical properties of membrane lipids. This article is part of a Special Issue entitled: Lipid-protein interactions.

  10. Ac-susceptibility investigations of superspin blocking and freezing in interacting magnetic nanoparticle ensembles

    NASA Astrophysics Data System (ADS)

    Botez, Cristian E.; Morris, Joshua L.

    2016-03-01

    We have investigated the effect of dipolar interactions on the superspin blocking and freezing of 9 nm average size Fe3O4 magnetic nanoparticle ensembles. Our dynamic susceptibility data reveals a two-regime behavior of the blocking temperature, T B, upon diluting a Fe3O4/hexane magnetic fluid. As the nanoparticle volume ratio, Φ, is reduced from an as-prepared reference Φ = 1 to Φ = 1/96, the blocking temperature decreases from 46.1 K to 34.2 K, but higher values reenter upon further diluting the magnetic fluid to Φ = 1/384 (where T B = 42.5 K). We found evidence that cooling below T B within the higher concentration range (Φ > 1/48) leads to the collective freezing of the superspins, whereas individual superspin blocking occurs in the presence of weaker interactions (Φ < 1/96). The unexpected increase of the blocking temperature with the decrease of the inter-particle interactions observed at low nanoparticle concentrations is well described by the Mørup-Tronc model.

  11. AC-susceptibility investigations of superspin blocking and freezing in interacting magnetic nanoparticle ensembles

    NASA Astrophysics Data System (ADS)

    Morris, Joshua Logan

    We have investigated the effect of dipolar interactions on the superspin blocking and freezing of 10 nm average size Fe3O4 magnetic nanoparticle ensembles. Our dynamic susceptibility data reveals a two-regime behavior of the blocking temperature, TB, upon diluting a Fe 3O4/hexane magnetic nanoparticle fluid. As the nanoparticle volume ratio, Phi, is reduced from an as-prepared reference Phi = 1 to Phi = 1/96, the blocking temperature decreases from 46.1 K to 34.2 K, but higher values reenter upon further diluting the magnetic fluid to Phi = 1/384 (where TB = 42.5 K). We show that cooling below TB within the higher concentration range (Phi > 1/48) leads to the collective freezing of the superspins in a spin-glass-like fashion, whereas individual superspin blocking occurs in the presence of weaker dipolar interactions (Phi < 1/96). The unexpected increase of the blocking temperature with the decrease of the interparticle interactions observed at low nanoparticle concentrations is well described by the Morup-Tronc (MT) model.

  12. Ac-susceptibility investigations of superspin blocking and freezing in interacting magnetic nanoparticle ensembles.

    PubMed

    Botez, Cristian E; Morris, Joshua L

    2016-03-18

    We have investigated the effect of dipolar interactions on the superspin blocking and freezing of 9 nm average size Fe3O4 magnetic nanoparticle ensembles. Our dynamic susceptibility data reveals a two-regime behavior of the blocking temperature, T(B), upon diluting a Fe3O4/hexane magnetic fluid. As the nanoparticle volume ratio, Φ, is reduced from an as-prepared reference Φ = 1 to Φ = 1/96, the blocking temperature decreases from 46.1 K to 34.2 K, but higher values reenter upon further diluting the magnetic fluid to Φ = 1/384 (where T(B) = 42.5 K). We found evidence that cooling below T B within the higher concentration range (Φ > 1/48) leads to the collective freezing of the superspins, whereas individual superspin blocking occurs in the presence of weaker interactions (Φ < 1/96). The unexpected increase of the blocking temperature with the decrease of the inter-particle interactions observed at low nanoparticle concentrations is well described by the Mørup-Tronc model. PMID:26876797

  13. Deciphering peculiar protein-protein interacting modules in Deinococcus radiodurans

    PubMed Central

    Mezhoud, Karim; Sghaier, Haïtham; Barkallah, Insaf

    2009-01-01

    Interactomes of proteins under positive selection from ionizing-radiation-resistant bacteria (IRRB) might be a part of the answer to the question as to how IRRB, particularly Deinococcus radiodurans R1 (Deira), resist ionizing radiation. Here, using the Database of Interacting Proteins (DIP) and the Protein Structural Interactome (PSI)-base server for PSI map, we have predicted novel interactions of orthologs of the 58 proteins under positive selection in Deira and other IRRB, but which are absent in IRSB. Among these, 18 domains and their interactomes have been identified in DNA checkpoint and repair; kinases pathways; energy and nucleotide metabolisms were the important biological processes that were found to be involved. This finding provides new clues to the cellular pathways that can to be important for ionizing-radiation resistance in Deira. PMID:19356244

  14. [Chemical libraries dedicated to protein-protein interactions].

    PubMed

    Sperandio, Olivier; Villoutreix, Bruno O; Morelli, Xavier; Roche, Philippe

    2015-03-01

    The identification of complete networks of protein-protein interactions (PPI) within a cell has contributed to major breakthroughs in understanding biological pathways, host-pathogen interactions and cancer development. As a consequence, PPI have emerged as a new class of promising therapeutic targets. However, they are still considered as a challenging class of targets for drug discovery programs. Recent successes have allowed the characterization of structural and physicochemical properties of protein-protein interfaces leading to a better understanding of how they can be disrupted with small molecule compounds. In addition, characterization of the profiles of PPI inhibitors has allowed the development of PPI-focused libraries. In this review, we present the current efforts at developing chemical libraries dedicated to these innovative targets.

  15. Potential disruption of protein-protein interactions by graphene oxide.

    PubMed

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-14

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  16. Potential disruption of protein-protein interactions by graphene oxide

    NASA Astrophysics Data System (ADS)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-01

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  17. Peptiderive server: derive peptide inhibitors from protein-protein interactions.

    PubMed

    Sedan, Yuval; Marcu, Orly; Lyskov, Sergey; Schueler-Furman, Ora

    2016-07-01

    The Rosetta Peptiderive protocol identifies, in a given structure of a protein-protein interaction, the linear polypeptide segment suggested to contribute most to binding energy. Interactions that feature a 'hot segment', a linear peptide with significant binding energy compared to that of the complex, may be amenable for inhibition and the peptide sequence and structure derived from the interaction provide a starting point for rational drug design. Here we present a web server for Peptiderive, which is incorporated within the ROSIE web interface for Rosetta protocols. A new feature of the protocol also evaluates whether derived peptides are good candidates for cyclization. Fast computation times and clear visualization allow users to quickly assess the interaction of interest. The Peptiderive server is available for free use at http://rosie.rosettacommons.org/peptiderive. PMID:27141963

  18. Interaction Energy Based Protein Structure Networks

    PubMed Central

    Vijayabaskar, M.S.; Vishveshwara, Saraswathi

    2010-01-01

    The three-dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino-acid residues of the polypeptide chain. These interactions can be represented collectively in the form of a network. So far, such networks have been investigated by considering the connections based on distances between the amino-acid residues. Here we present a method of constructing the structure network based on interaction energies among the amino-acid residues in the protein. We have investigated the properties of such protein energy-based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability, formation of secondary and super-secondary structural units. Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information, such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein. Finally, the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis. PMID:21112295

  19. Targeting Protein-Protein Interactions for Parasite Control

    PubMed Central

    Taylor, Christina M.; Fischer, Kerstin; Abubucker, Sahar; Wang, Zhengyuan; Martin, John; Jiang, Daojun; Magliano, Marc; Rosso, Marie-Noëlle; Li, Ben-Wen; Fischer, Peter U.; Mitreva, Makedonka

    2011-01-01

    Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific ortholgous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable. PMID

  20. Interaction of very cold neutrons with a polystyrene-polybutadiene-polystyrene block copolymer

    SciTech Connect

    Antonov, A.V.; Gerasimov, V.I.; Isakov, A.I.; Kuznetsov, S.P.; Meshkov, I.V.; Perekrestenko, A.D.; Tarasov, S.G.

    1985-03-10

    The total interaction cross sections have been studied as a function of the wavelength of very cold neutrons (summation/sub t/(lambda)) in a very-cold-neutron spectrometer (A. V. Antonov et al., Kratk. Soobshch. Fiz. 10, 10 (1977)) with a polystyrene-polybutadiene-polystyrene block copolymer at room temperature and at 99 K.

  1. Pentamidine blocks the interaction between mutant S100A5 and RAGE V domain and inhibits the RAGE signaling pathway.

    PubMed

    Cho, Ching Chang; Chou, Ruey Hwang; Yu, Chin

    2016-08-19

    The human S100 protein family contains small, dimeric and acidic proteins that contain two EF-hand motifs and bind calcium. When S100A5 binds calcium, its conformation changes and promotes interaction with the target protein. The extracellular domain of RAGE (Receptor of Advanced Glycation End products) contain three domains: C1, C2 and V. The RAGE V domain is the target protein of S100A5 that promotes cell survival, growth and differentiation by activating several signaling pathways. Pentamidine is an apoptotic and antiparasitic drug that is used to treat or prevent pneumonia. Here, we found that pentamidine interacts with S100A5 using HSQC titration. We elucidated the interactions of S100A5 with RAGE V domain and pentamidine using fluorescence and NMR spectroscopy. We generated two binary models-the S100A5-RAGE V domain and S100A5-Pentamidine complex-and then observed that the pentamidine and RAGE V domain share a similar binding region in mS100A5. We also used the WST-1 assay to investigate the bioactivity of S100A5, RAGE V domain and pentamidine. These results indicated that pentamidine blocks the binding between S100A5 and RAGE V domain. This finding is useful for the development of new anti-proliferation drugs.

  2. Pentamidine blocks the interaction between mutant S100A5 and RAGE V domain and inhibits the RAGE signaling pathway.

    PubMed

    Cho, Ching Chang; Chou, Ruey Hwang; Yu, Chin

    2016-08-19

    The human S100 protein family contains small, dimeric and acidic proteins that contain two EF-hand motifs and bind calcium. When S100A5 binds calcium, its conformation changes and promotes interaction with the target protein. The extracellular domain of RAGE (Receptor of Advanced Glycation End products) contain three domains: C1, C2 and V. The RAGE V domain is the target protein of S100A5 that promotes cell survival, growth and differentiation by activating several signaling pathways. Pentamidine is an apoptotic and antiparasitic drug that is used to treat or prevent pneumonia. Here, we found that pentamidine interacts with S100A5 using HSQC titration. We elucidated the interactions of S100A5 with RAGE V domain and pentamidine using fluorescence and NMR spectroscopy. We generated two binary models-the S100A5-RAGE V domain and S100A5-Pentamidine complex-and then observed that the pentamidine and RAGE V domain share a similar binding region in mS100A5. We also used the WST-1 assay to investigate the bioactivity of S100A5, RAGE V domain and pentamidine. These results indicated that pentamidine blocks the binding between S100A5 and RAGE V domain. This finding is useful for the development of new anti-proliferation drugs. PMID:27297108

  3. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  4. Docking and scoring protein interactions: CAPRI 2009.

    PubMed

    Lensink, Marc F; Wodak, Shoshana J

    2010-11-15

    Protein docking algorithms are assessed by evaluating blind predictions performed during 2007-2009 in Rounds 13-19 of the community-wide experiment on critical assessment of predicted interactions (CAPRI). We evaluated the ability of these algorithms to sample docking poses and to single out specific association modes in 14 targets, representing 11 distinct protein complexes. These complexes play important biological roles in RNA maturation, G-protein signal processing, and enzyme inhibition and function. One target involved protein-RNA interactions not previously considered in CAPRI, several others were hetero-oligomers, or featured multiple interfaces between the same protein pair. For most targets, predictions started from the experimentally determined structures of the free (unbound) components, or from models built from known structures of related or similar proteins. To succeed they therefore needed to account for conformational changes and model inaccuracies. In total, 64 groups and 12 web-servers submitted docking predictions of which 4420 were evaluated. Overall our assessment reveals that 67% of the groups, more than ever before, produced acceptable models or better for at least one target, with many groups submitting multiple high- and medium-accuracy models for two to six targets. Forty-one groups including four web-servers participated in the scoring experiment with 1296 evaluated models. Scoring predictions also show signs of progress evidenced from the large proportion of correct models submitted. But singling out the best models remains a challenge, which also adversely affects the ability to correctly rank docking models. With the increased interest in translating abstract protein interaction networks into realistic models of protein assemblies, the growing CAPRI community is actively developing more efficient and reliable docking and scoring methods for everyone to use.

  5. Finding protein-protein interaction patterns by contact map matching.

    PubMed

    Melo, R C; Ribeiro, C; Murray, C S; Veloso, C J M; da Silveira, C H; Neshich, G; Meira, W; Carceroni, R L; Santoro, M M

    2007-01-01

    We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature. PMID:18058715

  6. Carbohydrate–Aromatic Interactions in Proteins

    PubMed Central

    2015-01-01

    Protein–carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C–H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C–H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C–H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate–aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C–H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein–carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein–carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites. PMID:26561965

  7. PCorral--interactive mining of protein interactions from MEDLINE.

    PubMed

    Li, Chen; Jimeno-Yepes, Antonio; Arregui, Miguel; Kirsch, Harald; Rebholz-Schuhmann, Dietrich

    2013-01-01

    The extraction of information from the scientific literature is a complex task-for researchers doing manual curation and for automatic text processing solutions. The identification of protein-protein interactions (PPIs) requires the extraction of protein named entities and their relations. Semi-automatic interactive support is one approach to combine both solutions for efficient working processes to generate reliable database content. In principle, the extraction of PPIs can be achieved with different methods that can be combined to deliver high precision and/or high recall results in different combinations at the same time. Interactive use can be achieved, if the analytical methods are fast enough to process the retrieved documents. PCorral provides interactive mining of PPIs from the scientific literature allowing curators to skim MEDLINE for PPIs at low overheads. The keyword query to PCorral steers the selection of documents, and the subsequent text analysis generates high recall and high precision results for the curator. The underlying components of PCorral process the documents on-the-fly and are available, as well, as web service from the Whatizit infrastructure. The human interface summarizes the identified PPI results, and the involved entities are linked to relevant resources and databases. Altogether, PCorral serves curator at both the beginning and the end of the curation workflow for information retrieval and information extraction. Database URL: http://www.ebi.ac.uk/Rebholz-srv/pcorral.

  8. A versatile building block: the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins.

    PubMed

    Sun, Yuna; Guo, Yu; Lou, Zhiyong

    2012-12-01

    Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs. PMID:23136065

  9. Interaction of ice binding proteins with ice, water and ions.

    PubMed

    Oude Vrielink, Anneloes S; Aloi, Antonio; Olijve, Luuk L C; Voets, Ilja K

    2016-03-01

    Ice binding proteins (IBPs) are produced by various cold-adapted organisms to protect their body tissues against freeze damage. First discovered in Antarctic fish living in shallow waters, IBPs were later found in insects, microorganisms, and plants. Despite great structural diversity, all IBPs adhere to growing ice crystals, which is essential for their extensive repertoire of biological functions. Some IBPs maintain liquid inclusions within ice or inhibit recrystallization of ice, while other types suppress freezing by blocking further ice growth. In contrast, ice nucleating proteins stimulate ice nucleation just below 0 °C. Despite huge commercial interest and major scientific breakthroughs, the precise working mechanism of IBPs has not yet been unraveled. In this review, the authors outline the state-of-the-art in experimental and theoretical IBP research and discuss future scientific challenges. The interaction of IBPs with ice, water and ions is examined, focusing in particular on ice growth inhibition mechanisms. PMID:26787386

  10. Interaction of ice binding proteins with ice, water and ions.

    PubMed

    Oude Vrielink, Anneloes S; Aloi, Antonio; Olijve, Luuk L C; Voets, Ilja K

    2016-03-19

    Ice binding proteins (IBPs) are produced by various cold-adapted organisms to protect their body tissues against freeze damage. First discovered in Antarctic fish living in shallow waters, IBPs were later found in insects, microorganisms, and plants. Despite great structural diversity, all IBPs adhere to growing ice crystals, which is essential for their extensive repertoire of biological functions. Some IBPs maintain liquid inclusions within ice or inhibit recrystallization of ice, while other types suppress freezing by blocking further ice growth. In contrast, ice nucleating proteins stimulate ice nucleation just below 0 °C. Despite huge commercial interest and major scientific breakthroughs, the precise working mechanism of IBPs has not yet been unraveled. In this review, the authors outline the state-of-the-art in experimental and theoretical IBP research and discuss future scientific challenges. The interaction of IBPs with ice, water and ions is examined, focusing in particular on ice growth inhibition mechanisms.

  11. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  12. Pooled screening for antiproliferative inhibitors of protein-protein interactions.

    PubMed

    Nim, Satra; Jeon, Jouhyun; Corbi-Verge, Carles; Seo, Moon-Hyeong; Ivarsson, Ylva; Moffat, Jason; Tarasova, Nadya; Kim, Philip M

    2016-04-01

    Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide-binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreatic cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action, including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage-dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads, and it provides a rich data set covering phenotypes for the inhibition of thousands of interactions. PMID:26900867

  13. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  14. Low-Temperature Processable Block Copolymers That Preserve the Function of Blended Proteins.

    PubMed

    Iwasaki, Yasuhiko; Takemoto, Kyohei; Tanaka, Shinya; Taniguchi, Ikuo

    2016-07-11

    Low-temperature processable polymers have attracted increasing interest as ecological materials because of their reduced energy consumption during processing and suitability for making composites with heat-sensitive biomolecules at ambient temperature. In the current study, low-temperature processable biodegradable block copolymers were synthesized by ring-opening polymerization of l-lactide (LLA) using polyphosphoester as a macroinitiator. The polymer films could be processed under a hydraulic pressure of 35 MPa. The block copolymer films swelled in water because the polyphosphoester block was partially hydrated. Interestingly, the swelling ratio of the films changed with temperature. The pressure-induced order-to-disorder transition of the block copolymers was characterized by small-angle X-ray scattering; a crystallinity reduction in the block copolymers was observed after application of pressure. The crystallinity of the block copolymers was recovered after removing the applied pressure. The Young's modulus of the block copolymer films increased as the LLA unit content increased. Moreover, the modulus did not change after multiple processing cycles and the recyclability of the block copolymers was also confirmed. Finally, polymer films with embedded proteinase K as a model protein were prepared. The activity of catalase loaded into the polymer films was evaluated after processing at different temperatures. The activity of catalase was preserved when the polymer films were processed at room temperature but was significantly reduced after high-temperature processing. The suitability of low-temperature processable biodegradable polymers for making biofunctional composites without reducing protein activity was clarified. These materials will be useful for biomedical and therapeutic applications. PMID:27280847

  15. Pairwise alignment of protein interaction networks.

    PubMed

    Koyutürk, Mehmet; Kim, Yohan; Topkara, Umut; Subramaniam, Shankar; Szpankowski, Wojciech; Grama, Ananth

    2006-03-01

    With an ever-increasing amount of available data on protein-protein interaction (PPI) networks and research revealing that these networks evolve at a modular level, discovery of conserved patterns in these networks becomes an important problem. Although available data on protein-protein interactions is currently limited, recently developed algorithms have been shown to convey novel biological insights through employment of elegant mathematical models. The main challenge in aligning PPI networks is to define a graph theoretical measure of similarity between graph structures that captures underlying biological phenomena accurately. In this respect, modeling of conservation and divergence of interactions, as well as the interpretation of resulting alignments, are important design parameters. In this paper, we develop a framework for comprehensive alignment of PPI networks, which is inspired by duplication/divergence models that focus on understanding the evolution of protein interactions. We propose a mathematical model that extends the concepts of match, mismatch, and gap in sequence alignment to that of match, mismatch, and duplication in network alignment and evaluates similarity between graph structures through a scoring function that accounts for evolutionary events. By relying on evolutionary models, the proposed framework facilitates interpretation of resulting alignments in terms of not only conservation but also divergence of modularity in PPI networks. Furthermore, as in the case of sequence alignment, our model allows flexibility in adjusting parameters to quantify underlying evolutionary relationships. Based on the proposed model, we formulate PPI network alignment as an optimization problem and present fast algorithms to solve this problem. Detailed experimental results from an implementation of the proposed framework show that our algorithm is able to discover conserved interaction patterns very effectively, in terms of both accuracies and computational

  16. A Tool for Interactive Protein Manipulation

    2005-03-28

    ProteinShop is a graphical environment that facilitates a solution to the protein prediction problem through a combination of unique features and capabilities. These include: 1. Helping researchers automatically generate 3D protein structures from scratcW by using the sequence of amino acids and secondary structure specifications as input. 2. Enabling users to apply their accumulated biochemical knowledge and intuition during the interactive manipulation of structures. 3. FacIlitating interactive comparison and analysis of alternative structures through visualizationmore » of free energy computed during modeling. 4. Accelerating discovery of low-energy configurations by applying local optimizations plug-in to user-selected protein structures. ProteinShop v.2.0 includes the following new features: - Visualizes multiple-domain structures - Automatically creates a user-specified number of beta-sheet configurations - Provides the interface and the libraries for energy visualization and local minimization of protein structures - Reads standard POB files without previous editing« less

  17. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    PubMed

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders. PMID:27136297

  18. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    PubMed

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.

  19. Design of Protein-Peptide Interaction Modules for Assembling Supramolecular Structures in Vivo and in Vitro.

    PubMed

    Speltz, Elizabeth B; Nathan, Aparna; Regan, Lynne

    2015-09-18

    Synthetic biology and protein origami both require protein building blocks that behave in a reliable, predictable fashion. In particular, we require protein interaction modules with known specificity and affinity. Here, we describe three designed TRAP (Tetratricopeptide Repeat Affinity Protein)-peptide interaction pairs that are functional in vivo. We show that each TRAP binds to its cognate peptide and exhibits low cross-reactivity with the peptides bound by the other TRAPs. In addition, we demonstrate that the TRAP-peptide interactions are functional in many cellular contexts. In extensions of these designs, we show that the binding affinity of a TRAP-peptide pair can be systematically varied. The TRAP-peptide pairs we present thus represent a powerful set of new building blocks that are suitable for a variety of applications.

  20. Inhibition of Protein Synthesis by Y Box-Binding Protein 1 Blocks Oncogenic Cell Transformation†

    PubMed Central

    Bader, Andreas G.; Vogt, Peter K.

    2005-01-01

    The multifunctional Y box-binding protein 1 (YB-1) is transcriptionally repressed by the oncogenic phosphoinositide 3-kinase (PI3K) pathway (with P3K as an oncogenic homolog of the catalytic subunit) and, when reexpressed with the retroviral vector RCAS, interferes with P3K- and Akt-induced transformation of chicken embryo fibroblasts. Retrovirally expressed YB-1 binds to the cap of mRNAs and inhibits cap-dependent and cap-independent translation. To determine the requirements for the inhibitory role of YB-1 in P3K-induced transformation, we conducted a mutational analysis, measuring YB-1-induced interference with transformation, subcellular localization, cap binding, mRNA binding, homodimerization, and inhibition of translation. The results show that (i) interference with transformation requires RNA binding and a C-terminal domain that is distinct from the cytoplasmic retention domain, (ii) interference with transformation is tightly correlated with inhibition of translation, and (iii) masking of mRNAs by YB-1 is not sufficient to block transformation or to inhibit translation. We identified a noncanonical nuclear localization signal (NLS) in the C-terminal half of YB-1. A mutant lacking the NLS retains its ability to interfere with transformation, indicating that a nuclear function is not required. These results suggest that YB-1 interferes with P3K-induced transformation by a specific inhibition of translation through its RNA-binding domain and a region in the C-terminal domain. Potential functions of the C-terminal region are discussed. PMID:15743808

  1. Synthesis and characterization of an elastin-mimetic amphiphilic block copolymer protein

    NASA Astrophysics Data System (ADS)

    Lee, Terrence Anita-Talley

    2000-10-01

    The overall goal in material science is to be able to control the molecular architecture of a material and thus its end properties. There is no method that offers greater control than the biological synthesis of proteins. From the DNA sequence to the final synthesized protein, the entire process is finitely controlled. This present work describes methods developed and used to synthesize protein polymers by manipulating this process. From the initial DNA sequence chosen, the end properties that the protein polymer will have are dictated. An amphiphilic diblock copolymer was designed based on environmentally responsive elastin-mimetic peptide sequences [(Val/Ile)-Pro-Gly-Xaa-Gly] (Xaa = Ala or Glu for the hydrophilic block, Val or Phe for the hydrophobic block) and synthesized using a genetic engineering approach. Differential scanning calorimetry measurements in aqueous solution revealed that reversible hydrophobic folding and assembly of the copolymer occurs above the inverse temperature transition, Tt, of the hydrophobic block. This process results in the formation of 50 nm protein-based micellar aggregates, which were characterized by electron microscopy and temperature-dependent dynamic light scattering techniques. The distribution of micellar aggregates can be altered reproducibly through variation of environmental conditions including pH and temperature. The uniform and defined macromolecular architecture of this protein copolymer permits greater control over the physical properties of the micelles, which therefore may facilitate applications in controlled release of small molecules.

  2. Chemical Interactions and Their Role in the Microphase Separation of Block Copolymer Thin Films

    PubMed Central

    Farrell, Richard A.; Fitzgerald, Thomas G.; Borah, Dipu; Holmes, Justin D.; Morris, Michael A.

    2009-01-01

    The thermodynamics of self-assembling systems are discussed in terms of the chemical interactions and the intermolecular forces between species. It is clear that there are both theoretical and practical limitations on the dimensions and the structural regularity of these systems. These considerations are made with reference to the microphase separation that occurs in block copolymer (BCP) systems. BCP systems self-assemble via a thermodynamic driven process where chemical dis-affinity between the blocks driving them part is balanced by a restorative force deriving from the chemical bond between the blocks. These systems are attracting much interest because of their possible role in nanoelectronic fabrication. This form of self-assembly can obtain highly regular nanopatterns in certain circumstances where the orientation and alignment of chemically distinct blocks can be guided through molecular interactions between the polymer and the surrounding interfaces. However, for this to be possible, great care must be taken to properly engineer the interactions between the surfaces and the polymer blocks. The optimum methods of structure directing are chemical pre-patterning (defining regions on the substrate of different chemistry) and graphoepitaxy (topographical alignment) but both centre on generating alignment through favourable chemical interactions. As in all self-assembling systems, the problems of defect formation must be considered and the origin of defects in these systems is explored. It is argued that in these nanostructures equilibrium defects are relatively few and largely originate from kinetic effects arising during film growth. Many defects also arise from the confinement of the systems when they are ‘directed’ by topography. The potential applications of these materials in electronics are discussed. PMID:19865513

  3. Nucleosome assembly proteins and their interacting proteins in neuronal differentiation.

    PubMed

    Attia, Mikaël; Rachez, Christophe; Avner, Philip; Rogner, Ute Christine

    2013-06-01

    Neuronal differentiation from neural stem cells into mature neurons is guided by the concerted action of specific transcription factors that stepwise exercise their role in the context of defined chromatin states. Amongst the classes of proteins that influence chromatin compaction and modification are nucleosome assembly proteins (NAPs). Mammals possess several nucleosome assembly protein 1 like proteins (NAP1L) that show either ubiquitous or neuron-restricted expression. The latter group is presumably involved in the process of neuronal differentiation. Mammalian NAP1Ls can potentially form both homo- and hetero-dimers and octamers, in theory allowing thousands of different combinations to be formed. Detailed studies have been performed on several of the NAP1Ls that point to a range of molecular roles, including transcriptional regulation, nuclear import, and control of cell division. This article aims at summarizing current knowledge of the mammalian NAP1L family and its interactions.

  4. Tools for controlling protein interactions using light.

    PubMed

    Tucker, Chandra L; Vrana, Justin D; Kennedy, Matthew J

    2014-01-01

    Genetically encoded actuators that allow control of protein-protein interactions using light, termed 'optical dimerizers', are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and the coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use in controlling transcription, protein localization, and protein secretion using light. Additionally, we provide instructions and software for constructing a pulse-controlled LED device for use in experiments requiring extended light treatments.

  5. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  6. How Structure Defines Affinity in Protein-Protein Interactions

    PubMed Central

    Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

    2014-01-01

    Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

  7. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

    PubMed

    Ashokkumar, Balasubramaniem; Nabokina, Svetlana M; Ma, Thomas Y; Said, Hamid M

    2009-09-01

    The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

  8. Alignment-free protein interaction network comparison

    PubMed Central

    Ali, Waqar; Rito, Tiago; Reinert, Gesine; Sun, Fengzhu; Deane, Charlotte M.

    2014-01-01

    Motivation: Biological network comparison software largely relies on the concept of alignment where close matches between the nodes of two or more networks are sought. These node matches are based on sequence similarity and/or interaction patterns. However, because of the incomplete and error-prone datasets currently available, such methods have had limited success. Moreover, the results of network alignment are in general not amenable for distance-based evolutionary analysis of sets of networks. In this article, we describe Netdis, a topology-based distance measure between networks, which offers the possibility of network phylogeny reconstruction. Results: We first demonstrate that Netdis is able to correctly separate different random graph model types independent of network size and density. The biological applicability of the method is then shown by its ability to build the correct phylogenetic tree of species based solely on the topology of current protein interaction networks. Our results provide new evidence that the topology of protein interaction networks contains information about evolutionary processes, despite the lack of conservation of individual interactions. As Netdis is applicable to all networks because of its speed and simplicity, we apply it to a large collection of biological and non-biological networks where it clusters diverse networks by type. Availability and implementation: The source code of the program is freely available at http://www.stats.ox.ac.uk/research/proteins/resources. Contact: w.ali@stats.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161230

  9. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  10. Secretion Modification Region-Derived Peptide Disrupts HIV-1 Nef's Interaction with Mortalin and Blocks Virus and Nef Exosome Release

    PubMed Central

    Shelton, Martin N.; Huang, Ming-Bo; Ali, Syed A.; Powell, Michael D.

    2012-01-01

    Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4+ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties. PMID:22013042

  11. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  12. The interactions of peripheral membrane proteins with biological membranes.

    PubMed

    Whited, A M; Johs, A

    2015-11-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  13. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  14. Prediction and integration of regulatory and protein-protein interactions

    SciTech Connect

    Wichadakul, Duangdao; McDermott, Jason E.; Samudrala, Ram

    2009-04-20

    Knowledge of transcriptional regulatory interactions (TRIs) is essential for exploring functional genomics and systems biology in any organism. While several results from genome-wide analysis of transcriptional regulatory networks are available, they are limited to model organisms such as yeast [1] and worm [2]. Beyond these networks, experiments on TRIs study only individual genes and proteins of specific interest. In this chapter, we present a method for the integration of various data sets to predict TRIs for 54 organisms in the Bioverse [3]. We describe how to compile and handle various formats and identifiers of data sets from different sources, and how to predict the TRIs using a homology-based approach, utilizing the compiled data sets. Integrated data sets include experimentally verified TRIs, binding sites of transcription factors, promoter sequences, protein sub-cellular localization, and protein families. Predicted TRIs expand the networks of gene regulation for a large number of organisms. The integration of experimentally verified and predicted TRIs with other known protein-protein interactions (PPIs) gives insight into specific pathways, network motifs, and the topological dynamics of an integrated network with gene expression under different conditions, essential for exploring functional genomics and systems biology.

  15. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation.

    PubMed

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  16. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation

    PubMed Central

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  17. A reliability measure of protein-protein interactions and a reliability measure-based search engine.

    PubMed

    Park, Byungkyu; Han, Kyungsook

    2010-02-01

    Many methods developed for estimating the reliability of protein-protein interactions are based on the topology of protein-protein interaction networks. This paper describes a new reliability measure for protein-protein interactions, which does not rely on the topology of protein interaction networks, but expresses biological information on functional roles, sub-cellular localisations and protein classes as a scoring schema. The new measure is useful for filtering many spurious interactions, as well as for estimating the reliability of protein interaction data. In particular, the reliability measure can be used to search protein-protein interactions with the desired reliability in databases. The reliability-based search engine is available at http://yeast.hpid.org. We believe this is the first search engine for interacting proteins, which is made available to public. The search engine and the reliability measure of protein interactions should provide useful information for determining proteins to focus on.

  18. Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

    PubMed

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

  19. Protein-protein interactions as druggable targets: recent technological advances.

    PubMed

    Higueruelo, Alicia P; Jubb, Harry; Blundell, Tom L

    2013-10-01

    Classical target-based drug discovery, where large chemical libraries are screened using inhibitory assays for a single target, has struggled to find ligands that inhibit protein-protein interactions (PPI). Nevertheless, in the past decade there have been successes that have demonstrated that PPI can be useful drug targets, and the field is now evolving fast. This review focuses on the new approaches and concepts that are being developed to tackle these challenging targets: the use of fragment based methods to explore the chemical space, stapled peptides to regulate intracellular PPI, alternatives to competitive inhibition and the use of antibodies to enable small molecule discovery for these targets.

  20. Automated nanoscale flow cytometry for assessing protein-protein interactions.

    PubMed

    von Kolontaj, Kerstin; Horvath, Gabor L; Latz, Eicke; Büscher, Martin

    2016-09-01

    Despite their importance for signalling events, protein-protein interactions cannot easily be analyzed on a single cell level. We developed a robust automated FRET measurement system implemented on a commercial flow cytometer allowing for rapid profiling of molecular associations in living cells. We used this method to measure the most proximal signaling events on human T lymphocyte activation, which preceded calcium influx, and could automatically detect T cell receptor/CD3 complex clustering defects in immunocompromised patients. © 2016 International Society for Advancement of Cytometry. PMID:27584593

  1. Modular Assembly of Protein Building Blocks to Create Precisely-Defined Megamolecules

    PubMed Central

    Modica, Justin A.; Skarpathiotis, Stratos; Mrksich, Milan

    2013-01-01

    Enzyme promoted assembly offers a simple and straightforward means to construct monodisperse molecular objects too large for classical organic synthesis and too small for top-down techniques. This communication outlines the design and construction of a heterobifunctional protein building block, HaloTag-cutinase, that reacts rapidly and selectively with an appropriately functionalized small molecule linker; and describes the step-wise combination of these building blocks to generate a 300 kDa “megamolecule” that is precisely-defined with respect to domain orientation, connectivity, and composition. PMID:23070998

  2. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene.

    PubMed

    Brzozowska, Agata M; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Cohen Stuart, Martien A

    2010-07-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA(21)-b-PAPEO(14) (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (poly(N-methyl 2-vinyl pyridinium iodide). In solution, these C3Ms partly disintegrate at salt concentrations between 50 and 100 mM NaCl. Adsorption of C3Ms and proteins has been studied with fixed-angle optical reflectometry, at salt concentrations ranging from 1 to 100 mM NaCl. In comparison with the adsorption of PAA(21)-b-PAPEO(14) alone adsorption of C3Ms significantly increases the amount of PAA(21)-b-PAPEO(14) on the surface. This results in a higher surface density of PEO chains. The stability of the C3M coatings and their influence on protein adsorption are determined by the composition and the stability of the C3Ms in solution. A C3M-PAPEO(14)/P2MVPI(43) coating strongly suppresses the adsorption of all proteins on silica and polystyrene. The reduction of protein adsorption is the highest at 100 mM NaCl (>90%). The adsorbed C3M-PAPEO(14)/P2MVPI(43) layer is partly removed from the surface upon exposure to an excess of beta-lactoglobulin solution, due to formation of soluble aggregates consisting of beta-lactoglobulin and P2MVPI(43). In contrast, C3M-PAPEO(14)/P2MVPI(228) which has a fivefold longer cationic block enhances adsorption of the negatively charged proteins on both surfaces at salt concentrations above 1 mM NaCl. A single PAA(21)-b-PAPEO(14) layer causes only a moderate reduction of protein adsorption.

  3. Transferring network topological knowledge for predicting protein-protein interactions.

    PubMed

    Xu, Qian; Xiang, Evan Wei; Yang, Qiang

    2011-10-01

    Protein-protein interactions (PPIs) play an important role in cellular processes within a cell. An important task is to determine the existence of interactions among proteins. Unfortunately, the existing biological experimental techniques are expensive, time-consuming and labor-intensive. The network structures of many such networks are sparse, incomplete and noisy. Thus, state-of-the-art methods for link prediction in these networks often cannot give satisfactory prediction results, especially when some networks are extremely sparse. Noticing that we typically have more than one PPI network available, we naturally wonder whether it is possible to 'transfer' the linkage knowledge from some existing, relatively dense networks to a sparse network, to improve the prediction performance. Noticing that a network structure can be modeled using a matrix model, we introduce the well-known collective matrix factorization technique to 'transfer' usable linkage knowledge from relatively dense interaction network to a sparse target network. Our approach is to establish a correspondence between a source network and a target network via network-wide similarities. We test this method on two real PPI networks, Helicobacter pylori (as a target network) and human (as a source network). Our experimental results show that our method can achieve higher performance as compared with some baseline methods. PMID:21770035

  4. Schizophrenia interactome with 504 novel protein-protein interactions.

    PubMed

    Ganapathiraju, Madhavi K; Thahir, Mohamed; Handen, Adam; Sarkar, Saumendra N; Sweet, Robert A; Nimgaonkar, Vishwajit L; Loscher, Christine E; Bauer, Eileen M; Chaparala, Srilakshmi

    2016-01-01

    Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein-protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities. PMID:27336055

  5. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  6. Notable Aspects of Glycan-Protein Interactions.

    PubMed

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host's immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  7. Notable Aspects of Glycan-Protein Interactions

    PubMed Central

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  8. NMR studies of protein-ligand interactions.

    PubMed

    Maurer, Till

    2005-01-01

    Interaction between biological macromolecules or of macromolecules with low-molecular-weight ligands is a central paradigm in the understanding of function in biological systems. It is also the major goal in pharmaceutical research to find and optimize ligands that modulate the function of biological macromolecules. Both technological advances and new methods in the field of nuclear magnetic resonance (NMR) have led to the development of several tools by which the interaction of proteins or DNA and low molecular weight-ligands can be characterized at an atomic level. Information can be gained quickly and easily with ligand-based techniques. These need only small amounts of nonisotope labeled, and thus readily available target macromolecules. As the focus is on the signals stemming only from the ligand, no further NMR information regarding the target is needed. Techniques based on the observation of isotopically labeled biological macromolecules open the possibility to observe interactions of proteins with low-molecular-weight ligands, DNA or other proteins. With these techniques, the structure of high-molecular-weight complexes can be determined. Here, the resonance signals of the macromolecule must be identified beforehand, which can be time consuming but with the benefit of obtaining more information with respect to the target ligand complex.

  9. Modal interaction in ultrasonic welding block sonotrodes induced by the mistuning of the material properties

    NASA Astrophysics Data System (ADS)

    Brouet, Francois; Twiefel, Jens; Wallaschek, Jörg

    2016-10-01

    In many ultrasonic applications, block sonotrodes are serially coupled with other tuned components to transmit the longitudinal vibration to the workpiece. Uniform vibration amplitude of the output surface is an important criterion for determining quality. It is known that amplitude uniformity is related to other non-tuned modes. In this paper, another type of modal interaction is used to quantify distortion of the first longitudinal mode, when the system exhibits a loss of symmetry (mistuning) and near natural frequencies.

  10. Turning the spotlight on protein-lipid interactions in cells

    PubMed Central

    Peng, Tao; Yuan, Xiaoqiu; Hang, Howard C.

    2014-01-01

    Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. While powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions. PMID:25129056

  11. Control of the PEO chain conformation on nanoparticles by adsorption of PEO-block-poly(L-lysine) copolymers and its significance on colloidal stability and protein repellency.

    PubMed

    Louguet, Stéphanie; Kumar, Anitha C; Guidolin, Nicolas; Sigaud, Gilles; Duguet, Etienne; Lecommandoux, Sébastien; Schatz, Christophe

    2011-11-01

    The physical adsorption of PEO(n)-b-PLL(m) copolymers onto silica nanoparticles and the related properties of poly(ethylene oxide) (PEO)-coated particles were studied as a function of the block copolymer composition. Copolymers adopt an anchor-buoy conformation at the particle surface owing to a preferential affinity of poly(L-lysine) (PLL) blocks with the silica surface over PEO blocks when a large excess of copolymer is used. The interdistance between PEO chains at particle surface is highly dependent on the size of PLL segments; a dense brush of PEO is obtained for short PLL blocks (DP = 10), whereas PEO chains adopt a so-called interacting "mushroom" conformation for large PLL blocks (DP = 270). The size of the PEO blocks does not really influence the copolymer surface density, but it has a strong effect on the PEO layer thickness as expected. Salt and protein stability studies led to similar conclusions about the effectiveness of a PEO layer with a dense brush conformation to prevent colloidal aggregation and protein adsorption. Besides, a minimal PEO length is required to get full stabilization properties; as a matter of fact, both PEO(45)-b-PLL(10) and PEO(113)-b-PLL(10) give rise to a PEO brush conformation but only the latter copolymer efficiently stabilizes the particles in the presence of salt or proteins.

  12. Heparan sulfate and heparin interactions with proteins

    PubMed Central

    Meneghetti, Maria C. Z.; Hughes, Ashley J.; Rudd, Timothy R.; Nader, Helena B.; Powell, Andrew K.; Yates, Edwin A.; Lima, Marcelo A.

    2015-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed. PMID:26289657

  13. Heparan sulfate and heparin interactions with proteins.

    PubMed

    Meneghetti, Maria C Z; Hughes, Ashley J; Rudd, Timothy R; Nader, Helena B; Powell, Andrew K; Yates, Edwin A; Lima, Marcelo A

    2015-09-01

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure-activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure-activity relationships and regulation will be discussed.

  14. Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.

    PubMed

    Xu, Mei; Liu, Chao; Zhou, Mi; Li, Qing; Wang, Renxiao; Kang, Jingwu

    2016-08-16

    A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors. PMID:27425825

  15. PRASA: an integrated web server that analyzes protein interaction types.

    PubMed

    Fan, Chen-Yu; Bai, Yi-Han; Huang, Cheng-Yi; Yao, Tsung-Ju; Chiang, Wen-Hao; Chang, Darby Tien-Hao

    2013-04-10

    This work presents the Protein Association Analyzer (PRASA) (http://zoro.ee.ncku.edu.tw/prasa/) that predicts protein interactions as well as interaction types. Protein interactions are essential to most biological functions. The existence of diverse interaction types, such as physically contacted or functionally related interactions, makes protein interactions complex. Different interaction types are distinct and should not be confused. However, most existing tools focus on a specific interaction type or mix different interaction types. This work collected 7234058 associations with experimentally verified interaction types from five databases and compiled individual probabilistic models for different interaction types. The PRASA result page shows predicted associations and their related references by interaction type. Experimental results demonstrate the performance difference when distinguishing between different interaction types. The PRASA provides a centralized and organized platform for easy browsing, downloading and comparing of interaction types, which helps reveal insights into the complex roles that proteins play in organisms.

  16. Decoupling systems of hydrodynamic type into subsystems with block-triangular interaction

    NASA Astrophysics Data System (ADS)

    Tunitsky, D. V.

    2015-12-01

    This paper is devoted to systems of n inhomogeneous equations of hydrodynamic type with two independent variables. Using a geometric formalism for such systems which goes back to Riemann, one can associate with every system of hydrodynamic type a vector field and a field of linear operators acting on an appropriate tangent bundle. In terms of these fields, we obtain a number of tests for inhomogeneous systems of hydrodynamic type to be decoupled into subsystems with block-triangular interaction. These tests supplement Bogoyavlenskii's well-known results on decoupling of homogeneous systems of hydrodynamic type into non-interacting subsystems.

  17. Comprehensive peptidomimetic libraries targeting protein-protein interactions.

    PubMed

    Whitby, Landon R; Boger, Dale L

    2012-10-16

    Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected

  18. Module organization and variance in protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Lin, Chun-Yu; Lee, Tsai-Ling; Chiu, Yi-Yuan; Lin, Yi-Wei; Lo, Yu-Shu; Lin, Chih-Ta; Yang, Jinn-Moon

    2015-03-01

    A module is a group of closely related proteins that act in concert to perform specific biological functions through protein-protein interactions (PPIs) that occur in time and space. However, the underlying module organization and variance remain unclear. In this study, we collected module templates to infer respective module families, including 58,041 homologous modules in 1,678 species, and PPI families using searches of complete genomic database. We then derived PPI evolution scores and interface evolution scores to describe the module elements, including core and ring components. Functions of core components were highly correlated with those of essential genes. In comparison with ring components, core proteins/PPIs were conserved across multiple species. Subsequently, protein/module variance of PPI networks confirmed that core components form dynamic network hubs and play key roles in various biological functions. Based on the analyses of gene essentiality, module variance, and gene co-expression, we summarize the observations of module organization and variance as follows: 1) a module consists of core and ring components; 2) core components perform major biological functions and collaborate with ring components to execute certain functions in some cases; 3) core components are more conserved and essential during organizational changes in different biological states or conditions.

  19. Channel-interacting PDZ protein, 'CIPP', interacts with proteins involved in cytoskeletal dynamics.

    PubMed

    Alpi, Emanuele; Landi, Elena; Barilari, Manuela; Serresi, Michela; Salvadori, Piero; Bachi, Angela; Dente, Luciana

    2009-04-15

    Neuronal CIPP (channel-interacting PDZ protein) is a multivalent PDZ protein that interacts with specific channels and receptors highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In the present study, we selected a set of potential CIPP interactors that are involved directly or indirectly in mechanisms of cytoskeletal remodelling and membrane protrusion formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in cultured mammalian cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to the IRSp53 C-terminus. Analysis of the puncta nature, using various endocytic markers, revealed that they are not related to cytoplasmic vesicles, but rather represent multi-protein assemblies, where CIPP can tether other potential interactors.

  20. Molecular modeling of protein-glycosaminoglycan interactions.

    PubMed

    Cardin, A D; Weintraub, H J

    1989-01-01

    Forty-nine regions in 21 proteins were identified as potential heparin-binding sites based on the sequence organizations of their basic and nonbasic residues. Twelve known heparin-binding sequences in vitronectin, apolipoproteins E and B-100, and platelet factor 4 were used to formulate two search strings for identifying potential heparin-binding regions in other proteins. Consensus sequences for glycosaminoglycan recognition were determined as [-X-B-B-X-B-X-] and [-X-B-B-B-X-X-B-X-] where B is the probability of a basic residue and X is a hydropathic residue. Predictions were then made as to the heparin-binding domains in endothelial cell growth factor, purpurin, and antithrombin-III. Many of the natural sequences conforming to these consensus motifs show prominent amphipathic periodicities having both alpha-helical and beta-strand conformations as determined by predictive algorithms and circular dichroism studies. The heparin-binding domain of vitronectin was modeled and formed a hydrophilic pocket that wrapped around and folded over a heparin octasaccharide, yielding a complementary structure. We suggest that these consensus sequence elements form potential nucleation sites for the recognition of polyanions in proteins and may provide a useful guide in identifying heparin-binding regions in other proteins. The possible relevance of protein-glycosaminoglycans interactions in atherosclerosis is discussed. PMID:2463827

  1. Weakly Stable Regions and Protein-Protein Interactions in Beta-Barrel Membrane Proteins

    PubMed Central

    Naveed, Hammad; Liang, Jie

    2014-01-01

    We briefly discuss recent progress in computational characterization of the sequence and structural properties of β-barrel membrane properties. We discuss the emerging concept of weakly stable regions in β-barrel membrane proteins, computational methods to identify these regions and mechanisms adopted by β-barrel membrane proteins in nature to stabilize them. We further discuss computational methods to identify protein-protein interactions in β-barrel membrane proteins and recent experimental studies that aim at altering the biophysical properties including oligomerization state and stability of β-barrel membrane proteins based on the emerging organization principles of these proteins from recent computational studies. PMID:23713778

  2. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

    NASA Astrophysics Data System (ADS)

    Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

  3. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

    PubMed

    Huber, Matthias C; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials. PMID:25362355

  4. Prediction of thermodynamic instabilities of protein solutions from simple protein-protein interactions

    NASA Astrophysics Data System (ADS)

    D'Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio

    2013-10-01

    Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein-protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential.

  5. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  6. Impact of blocking and detection chemistries on antibody performance for reverse phase protein arrays.

    PubMed

    Ambroz, Kristi

    2011-01-01

    Careful selection of well-qualified antibodies is critical for accurate data collection from reverse phase protein arrays (RPPA). The most common way to qualify antibodies for RPPA analysis is by Western blotting because the detection mechanism is based on the same immunodetection principles. Western blots of tissue or cell lysates that result in single bands and low cross-reactivity indicate appropriate antibodies for RPPA detection. Western blot conditions used to validate antibodies for RPPA experiments, including blocking and detection reagents, have significant effects on aspects of antibody performance such as cross-reactivity against other proteins in the sample. We have found that there can be a dramatic impact on antibody behavior with changes in blocking reagent and detection method, and offer an alternative method that allows detection reagents and conditions to be held constant in both antibody validation and RPPA experiments. PMID:21901590

  7. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    PubMed Central

    Gannagé, Monique; Schmid, Dorothee; Albrecht, Randy; Dengjel, Jörn; Torossi, Tania; Rämer, Patrick C.; Lee, Monica; Strowig, Till; Arrey, Frida; Conenello, Gina; Pypaert, Marc; Andersen, Jens; García-Sastre, Adolfo; Münz, Christian

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy. PMID:19837376

  8. Interaction of Curcumin with PEO-PPO-PEO block copolymers: a molecular dynamics study.

    PubMed

    Samanta, Susruta; Roccatano, Danilo

    2013-03-21

    Curcumin, a naturally occurring drug molecule, has been extensively investigated for its various potential usages in medicine. Its water insolubility and high metabolism rate require the use of drug delivery systems to make it effective in the human body. Among various types of nanocarriers, block copolymer based ones are the most effective. These polymers are broadly used as drug-delivery systems, but the nature of this process is poorly understood. In this paper, we propose a molecular dynamics simulation study of the interaction of Curcumin with block copolymer based on polyethylene oxide (PEO) and polypropylene oxide (PPO). The study has been conducted considering the smallest PEO and PPO oligomers and multiple chains of the block copolymer Pluronic P85. Our study shows that the more hydrophobic 1,2-dimethoxypropane (DMP) molecules and PPO block preferentially coat the Curcumin molecule. In the case of the Pluronic P85, simulation shows formation of a drug-polymer aggregate within 50 ns. This process leaves exposed the PEO part of the polymers, resulting in better solvation and stability of the drug in water.

  9. Protein assemblies by site-specific avidin-biotin interactions.

    PubMed

    Mori, Yutaro; Minamihata, Kosuke; Abe, Hiroki; Goto, Masahiro; Kamiya, Noriho

    2011-08-21

    Exploiting self-assembly systems with biological building blocks is of significant interest in the fabrication of advanced biomaterials. We assessed the potential use of site-specific ligand labeling of protein building blocks in designing functional protein self-assemblies by combining site-specifically biotinylated bacterial alkaline phosphatase (as a bidentate or tetradentate ligand unit) and streptavidin (as a tetrameric receptor). PMID:21731938

  10. Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

    PubMed

    Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

    2015-10-01

    Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.

  11. Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

    PubMed

    Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

    2015-10-01

    Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins. PMID:26517887

  12. Yeast Three-Hybrid System for the Detection of Protein-Protein Interactions.

    PubMed

    Maruta, Natsumi; Trusov, Yuri; Botella, Jose R

    2016-01-01

    Protein-protein interaction studies provide useful insights into biological processes taking place within the living cell. A number of techniques are available to unravel large structural protein complexes, functional protein modules, and temporary protein associations occurring during signal transduction. The choice of method depends on the nature of the proteins and the interaction being studied. Here we present an optimized and simplified yeast three-hybrid method for analysis of protein interactions involving three components.

  13. Hydrophobic blocks facilitate lipid compatibility and translocon recognition of transmembrane protein sequences.

    PubMed

    Stone, Tracy A; Schiller, Nina; von Heijne, Gunnar; Deber, Charles M

    2015-02-24

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in

  14. Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences

    PubMed Central

    2016-01-01

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity

  15. Cyclin D1 blocks the anti-proliferative function of RUNX3 by interfering with RUNX3-p300 interaction

    SciTech Connect

    Iwatani, Kazunori; Fujimoto, Tetsuhiro; Ito, Takaaki

    2010-09-24

    Research highlights: {yields} Cyclin D1 interacts with RUNX3 and inhibits the interaction and collaboration of RUNX3 with coactivator p300. {yields} Cyclin D1 blocks the ability of RUNX3 to induce the expression of cdk inhibitor p21. {yields} Cyclin D1 releases cancer cells from the inhibition of proliferation induced by RUNX3. -- Abstract: Transcriptional function of cyclin D1, whose deregulation is frequently observed in human cancers, has been suggested to contribute to cancer formation. In the present study, we show that cyclin D1 protein inhibits RUNX3 activity by directly binding to it and interfering with its interaction with p300 interaction in lung cancer cells. Cyclin D1 inhibits p300-dependent RUNX3 acetylation and negatively regulates cyclin-dependent kinase (cdk) inhibitor p21 expression. These transcriptional effects of cyclin D1 do not require cdk4/6 kinase activation. We propose that cyclin D1 provides a transcriptional switch that allows the tumor suppressor activity of RUNX3 to be repressed in cancer cells. Since RUNX3 plays tumor suppressive roles in a wide range of cancers, a non-canonical cyclin D1 function may be critical for neoplastic transformation of the epithelial cells in which RUNX3 regulates proliferation.

  16. Block-adaptive quantum mechanics: an adaptive divide-and-conquer approach to interactive quantum chemistry.

    PubMed

    Bosson, Maël; Grudinin, Sergei; Redon, Stephane

    2013-03-01

    We present a novel Block-Adaptive Quantum Mechanics (BAQM) approach to interactive quantum chemistry. Although quantum chemistry models are known to be computationally demanding, we achieve interactive rates by focusing computational resources on the most active parts of the system. BAQM is based on a divide-and-conquer technique and constrains some nucleus positions and some electronic degrees of freedom on the fly to simplify the simulation. As a result, each time step may be performed significantly faster, which in turn may accelerate attraction to the neighboring local minima. By applying our approach to the nonself-consistent Atom Superposition and Electron Delocalization Molecular Orbital theory, we demonstrate interactive rates and efficient virtual prototyping for systems containing more than a thousand of atoms on a standard desktop computer.

  17. Blocking temperature of interacting magnetic nanoparticles with uniaxial and cubic anisotropies from Monte Carlo simulations

    NASA Astrophysics Data System (ADS)

    Russier, V.

    2016-07-01

    The low temperature behavior of densely packed interacting spherical single domain nanoparticles (MNP) is investigated by Monte Carlo simulations in the framework of an effective one spin model. The particles are distributed through a hard sphere like distribution with periodic boundary conditions and interact through the dipole dipole interaction (DDI) with an anisotropy energy including both cubic and uniaxial symmetry components. The cubic component is shown to play a sizable role on the value of the blocking temperature Tb only when the MNP easy axes are parallel to the cubic easy direction ([111] direction for a negative cubic anisotropy constant). The nature of the collective low temperature state, either ferromagnetic or spin glass like, is found to depend on the ratio of the anisotropy to the dipolar energies characterizing partly the disorder in the system.

  18. Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

    PubMed

    Ouimet, Claire M; Shao, Hao; Rauch, Jennifer N; Dawod, Mohamed; Nordhues, Bryce; Dickey, Chad A; Gestwicki, Jason E; Kennedy, Robert T

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs. PMID:27434096

  19. Developing algorithms for predicting protein-protein interactions of homology modeled proteins.

    SciTech Connect

    Martin, Shawn Bryan; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Roe, Diana C.

    2006-01-01

    The goal of this project was to examine the protein-protein docking problem, especially as it relates to homology-based structures, identify the key bottlenecks in current software tools, and evaluate and prototype new algorithms that may be developed to improve these bottlenecks. This report describes the current challenges in the protein-protein docking problem: correctly predicting the binding site for the protein-protein interaction and correctly placing the sidechains. Two different and complementary approaches are taken that can help with the protein-protein docking problem. The first approach is to predict interaction sites prior to docking, and uses bioinformatics studies of protein-protein interactions to predict theses interaction site. The second approach is to improve validation of predicted complexes after docking, and uses an improved scoring function for evaluating proposed docked poses, incorporating a solvation term. This scoring function demonstrates significant improvement over current state-of-the art functions. Initial studies on both these approaches are promising, and argue for full development of these algorithms.

  20. Protein function prediction using neighbor relativity in protein-protein interaction network.

    PubMed

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network.

  1. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  2. The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells

    PubMed Central

    Caignard, Grégory; Lucas-Hourani, Marianne; Dhondt, Kevin P.; Labernardière, Jean-Louis; Petit, Thierry; Jacob, Yves; Horvat, Branka; Tangy, Frédéric; Vidalain, Pierre-Olivier

    2013-01-01

    The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host. PMID:23342031

  3. Glycosphingolipid–Protein Interaction in Signal Transduction

    PubMed Central

    Russo, Domenico; Parashuraman, Seetharaman; D’Angelo, Giovanni

    2016-01-01

    Glycosphingolipids (GSLs) are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand–receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL–protein interactions and their effects on cell signalling and development. PMID:27754465

  4. Viral and Cellular Proteins Containing FGDF Motifs Bind G3BP to Block Stress Granule Formation

    PubMed Central

    Panas, Marc D.; Schulte, Tim; Thaa, Bastian; Sandalova, Tatiana; Kedersha, Nancy; Achour, Adnane; McInerney, Gerald M.

    2015-01-01

    The Ras-GAP SH3 domain–binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP. PMID:25658430

  5. Viral and cellular proteins containing FGDF motifs bind G3BP to block stress granule formation.

    PubMed

    Panas, Marc D; Schulte, Tim; Thaa, Bastian; Sandalova, Tatiana; Kedersha, Nancy; Achour, Adnane; McInerney, Gerald M

    2015-02-01

    The Ras-GAP SH3 domain-binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.

  6. The Foundations of Protein-Ligand Interaction

    NASA Astrophysics Data System (ADS)

    Klebe, Gerhard

    For the specific design of a drug we must first answer the question: How does a drug achieve its activity? An active ingredient must, in order to develop its action, bind to a particular target molecule in the body. Usually this is a protein, but also nucleic acids in the form of RNA and DNA can be target structures for active agents. The most important condition for binding is at first that the active agent exhibits the correct size and shape in order to optimally fit into a cavity exposed to the surface of the protein, the "bindingpocket". It is further necessary for the surface properties of the ligand and protein to be mutually compatible to form specific interactions. In 1894 Emil Fischer compared the exact fit of a substrate for the catalytic centre of an enzyme with the picture of a "lock-and-key". Paul Ehrlich coined in 1913 "Corpora non agunt nisi fixata", literally "bodies do not work when they are not bound". He wanted to imply that active agents that are meant to kill bacteria or parasites must be "fixed" by them, i.e. linked to their structures. Both concepts form the starting point for any rational concept in the development of active pharmaceutical ingredients. In many respects they still apply today. A drug must, after being administered, reach its target and interact with a biological macromolecule. Specific agents have a large affinity and sufficient selectivity to bind to the macromolecule's active site. This is the only way they can develop the desired biological activity without side-effects.

  7. Expression of Bovine Leukemia Virus Genome is Blocked by a Nonimmunoglobulin Protein in Plasma from Infected Cattle

    NASA Astrophysics Data System (ADS)

    Gupta, P.; Ferrer, J. F.

    1982-01-01

    Plasma of cattle infected with bovine leukemia virus contains a soluble factor that blocks the expression of the viral genome in cultured lymphocytes. The blocking factor is not present in plasma of bovine leukemia virus-free cattle or of cattle infected with common bovine viruses. Blocking of bovine leukemia virus expression by the plasma factor is reversible, and seems to be mediated by a nonimmunoglobulin protein molecule.

  8. Ion-dipole interactions and their functions in proteins.

    PubMed

    Sippel, Katherine H; Quiocho, Florante A

    2015-07-01

    Ion-dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein-protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion-dipole interaction and discuss several prominent examples of their functional roles in nature.

  9. Roles of European blocking and tropical-extratropical interaction in the 2010 Pakistan flooding

    NASA Astrophysics Data System (ADS)

    Hong, Chi-Cherng; Hsu, Huang-Hsiung; Lin, Nai-Hsin; Chiu, Hsun

    2011-07-01

    A sequence of monsoon surges struck Pakistan and Northwestern India during late July-early August 2010. The unusually heavy monsoon rainfall resulted in record-breaking floods, which affected 20 million people with a death toll of near 3000. Simultaneously, a long-lived blocking high appeared over Europe and Russia in middle June and persisted for nearly two months. Extreme flooding occurred when the southward penetration of extratropical potential vorticity in the deep trough east of the European blocking and the tropical monsoon surges arrived concurrently in Pakistan. This study demonstrates that the interaction between the tropical monsoon surges and the extratropical disturbances downstream of the European blocking was the key factor leading to the severe flooding in Pakistan. The 2010 La Niña condition contributed indirectly to the flooding by inducing a low-level easterly anomaly in South and Southeast Asia, which weakened eastward moisture transport and helped enhance moisture transport (convergence) to (in) the Northern Arabian Sea and Pakistan.

  10. Targeting Plant Ethylene Responses by Controlling Essential Protein-Protein Interactions in the Ethylene Pathway.

    PubMed

    Bisson, Melanie M A; Groth, Georg

    2015-08-01

    The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.

  11. Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block.

    PubMed

    Li, Hong; Chen, Xiao Yan; Kong, Qing You; Liu, Jia

    2002-06-01

    The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

  12. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  13. Modelling of the Vajont rockslide displacements by delayed plasticity of interacting sliding blocks

    NASA Astrophysics Data System (ADS)

    Castellanza, riccardo; Hedge, Amarnath; Crosta, Giovanni; di Prisco, Claudio; Frigerio, Gabriele

    2015-04-01

    In order to model complex sliding masses subject to continuous slow movements related to water table fluctuations it is convenient to: i) model the time-dependent mechanical behaviour of the materials by means of a viscous-plastic constitutive law; ii) assume the water table fluctuation as the main input to induce displacement acceleration; iii) consider, the 3D constrains by maintaining a level of simplicity such to allow the implementation into EWS (Early Warning System) for risk management. In this work a 1D pseudo-dynamic visco-plastic model (Secondi et al. 2011), based on Perzyna's delayed plasticity theory is applied. The sliding mass is considered as a rigid block subject to its self weight, inertial forces and seepage forces varying with time. All non-linearities are lumped in a thin layer positioned between the rigid block and the stable bedrock. The mechanical response of this interface is assumed to be visco-plastic. The viscous nucleus is assumed to be of the exponential type, so that irreversible strains develop for both positive and negative values of the yield function; the sliding mass is discretized in blocks to cope with complex rockslide geometries; the friction angle is assumed to reduce with strain rate assuming a sort of strain - rate law (Dietrich-Ruina law). To validate the improvements introduced in this paper the simulation of the displacements of the Vajont rockslide from 1960 to the failure, occurred on October the 9th 1963, is perfomed. It will be shown that, in its modified version, the model satisfactorily fits the Vajont pre-collapse displacements triggered by the fluctuation of the Vajont lake level and the associated groundwater level. The model is able to follow the critical acceleration of the motion with a minimal change in friction properties.The discretization in interacting sliding blocks confirms its suitability to model the complex 3D rockslide behaviour. We are currently implementing a multi-block model capable to include

  14. Predicting protein-protein interactions in unbalanced data using the primary structure of proteins

    PubMed Central

    2010-01-01

    Background Elucidating protein-protein interactions (PPIs) is essential to constructing protein interaction networks and facilitating our understanding of the general principles of biological systems. Previous studies have revealed that interacting protein pairs can be predicted by their primary structure. Most of these approaches have achieved satisfactory performance on datasets comprising equal number of interacting and non-interacting protein pairs. However, this ratio is highly unbalanced in nature, and these techniques have not been comprehensively evaluated with respect to the effect of the large number of non-interacting pairs in realistic datasets. Moreover, since highly unbalanced distributions usually lead to large datasets, more efficient predictors are desired when handling such challenging tasks. Results This study presents a method for PPI prediction based only on sequence information, which contributes in three aspects. First, we propose a probability-based mechanism for transforming protein sequences into feature vectors. Second, the proposed predictor is designed with an efficient classification algorithm, where the efficiency is essential for handling highly unbalanced datasets. Third, the proposed PPI predictor is assessed with several unbalanced datasets with different positive-to-negative ratios (from 1:1 to 1:15). This analysis provides solid evidence that the degree of dataset imbalance is important to PPI predictors. Conclusions Dealing with data imbalance is a key issue in PPI prediction since there are far fewer interacting protein pairs than non-interacting ones. This article provides a comprehensive study on this issue and develops a practical tool that achieves both good prediction performance and efficiency using only protein sequence information. PMID:20361868

  15. Measuring frequency domain granger causality for multiple blocks of interacting time series.

    PubMed

    Faes, Luca; Nollo, Giandomenico

    2013-04-01

    In the past years, several frequency-domain causality measures based on vector autoregressive time series modeling have been suggested to assess directional connectivity in neural systems. The most followed approaches are based on representing the considered set of multiple time series as a realization of two or three vector-valued processes, yielding the so-called Geweke linear feedback measures, or as a realization of multiple scalar-valued processes, yielding popular measures like the directed coherence (DC) and the partial DC (PDC). In the present study, these two approaches are unified and generalized by proposing novel frequency-domain causality measures which extend the existing measures to the analysis of multiple blocks of time series. Specifically, the block DC (bDC) and block PDC (bPDC) extend DC and PDC to vector-valued processes, while their logarithmic counterparts, denoted as multivariate total feedback [Formula: see text] and direct feedback [Formula: see text], represent into a full multivariate framework the Geweke's measures. Theoretical analysis of the proposed measures shows that they: (i) possess desirable properties of causality measures; (ii) are able to reflect either direct causality (bPDC, [Formula: see text] or total (direct + indirect) causality (bDC, [Formula: see text] between time series blocks; (iii) reduce to the DC and PDC measures for scalar-valued processes, and to the Geweke's measures for pairs of processes; (iv) are able to capture internal dependencies between the scalar constituents of the analyzed vector processes. Numerical analysis showed that the proposed measures can be efficiently estimated from short time series, allow to represent in an objective, compact way the information derived from the causal analysis of several pairs of time series, and may detect frequency domain causality more accurately than existing measures. The proposed measures find their natural application in the evaluation of directional

  16. Measuring frequency domain granger causality for multiple blocks of interacting time series.

    PubMed

    Faes, Luca; Nollo, Giandomenico

    2013-04-01

    In the past years, several frequency-domain causality measures based on vector autoregressive time series modeling have been suggested to assess directional connectivity in neural systems. The most followed approaches are based on representing the considered set of multiple time series as a realization of two or three vector-valued processes, yielding the so-called Geweke linear feedback measures, or as a realization of multiple scalar-valued processes, yielding popular measures like the directed coherence (DC) and the partial DC (PDC). In the present study, these two approaches are unified and generalized by proposing novel frequency-domain causality measures which extend the existing measures to the analysis of multiple blocks of time series. Specifically, the block DC (bDC) and block PDC (bPDC) extend DC and PDC to vector-valued processes, while their logarithmic counterparts, denoted as multivariate total feedback [Formula: see text] and direct feedback [Formula: see text], represent into a full multivariate framework the Geweke's measures. Theoretical analysis of the proposed measures shows that they: (i) possess desirable properties of causality measures; (ii) are able to reflect either direct causality (bPDC, [Formula: see text] or total (direct + indirect) causality (bDC, [Formula: see text] between time series blocks; (iii) reduce to the DC and PDC measures for scalar-valued processes, and to the Geweke's measures for pairs of processes; (iv) are able to capture internal dependencies between the scalar constituents of the analyzed vector processes. Numerical analysis showed that the proposed measures can be efficiently estimated from short time series, allow to represent in an objective, compact way the information derived from the causal analysis of several pairs of time series, and may detect frequency domain causality more accurately than existing measures. The proposed measures find their natural application in the evaluation of directional

  17. Perfluorinated Moieties Increase the Interaction of Amphiphilic Block Copolymers with Lipid Monolayers.

    PubMed

    Schwieger, Christian; Blaffert, Jacob; Li, Zheng; Kressler, Jörg; Blume, Alfred

    2016-08-16

    The interaction of amphiphilic and triphilic block copolymers with lipid monolayers has been studied. Amphiphilic triblock copolymer PGMA20-PPO34-PGMA20 (GP) is composed of a hydrophobic poly(propylene oxide) (PPO) middle block that is flanked by two hydrophilic poly(glycerol monomethacrylate) (PGMA) side blocks. The attachment of a perfluoro-n-nonyl residue (F9) to either end of GP yields a triphilic polymer with the sequence F9-PGMA20-PPO34-PGMA20-F9 (F-GP). The F9 chains are fluorophilic, i.e., they have a tendency to demix in hydrophilic as well as in lipophilic environments. We investigated (i) the adsorption of both polymers to differently composed lipid monolayers and (ii) the compression behavior of mixed polymer/lipid monolayers. The lipid monolayers are composed of phospholipids with PC or PE headgroups and acyl chains of different length and saturation. Both polymers interact with lipid monolayers by inserting their hydrophobic moieties (PPO, F9). The interaction is markedly enhanced in the presence of F9 chains, which act as membrane anchors. GP inserts into lipid monolayers up to a surface pressure of 30 mN/m, whereas F-GP inserts into monolayers at up to 45 mN/m, suggesting that F-GP also inserts into lipid bilayer membranes. The adsorption of both polymers to lipid monolayers with short acyl chains is favored. Upon compression, a two-step squeeze-out of F-GP occurs, with PPO blocks being released into the aqueous subphase at 28 mN/m and the F9 chains being squeezed out at 48 mN/m. GP is squeezed out in one step at 28 mN/m because of the lack of F9 anchor groups. The liquid expanded (LE) to liquid condensed (LC) phase transition of DPPC and DMPE is maintained in the presence of the polymers, indicating that the polymers can be accommodated in LE- and LC-phase monolayers. These results show how fluorinated moieties can be included in the rational design of membrane-binding polymers. PMID:27442444

  18. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  19. Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

    SciTech Connect

    Lu, Duo; Bernstein, Douglas A.; Satyshur, Kenneth A.; Keck, James L.

    2010-09-03

    Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

  20. Studying protein-protein interactions via blot overlay/far western blot.

    PubMed

    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  1. Multifaceted capsid proteins: multiple interactions suggest multiple roles for Pepino mosaic virus capsid protein.

    PubMed

    Mathioudakis, Matthaios M; Rodríguez-Moreno, Luis; Sempere, Raquel Navarro; Aranda, Miguel A; Livieratos, Ioannis

    2014-12-01

    Pepino mosaic virus (PepMV) (family Alphaflexiviridae, genus Potexvirus) is a mechanically transmitted tomato pathogen that, over the last decade, has evolved from emerging to endemic worldwide. Here, two heat-shock cognate (Hsc70) isoforms were identified as part of the coat protein (CP)/Hsc70 complex in vivo, following full-length PepMV and CP agroinoculation. PepMV accumulation was severely reduced in Hsp70 virus-induced gene silenced and in quercetin-treated Nicotiana benthamiana plants. Similarly, in vitro-transcribed as well as virion RNA input levels were reduced in quercetin-treated protoplasts, suggesting an essential role for Hsp70 in PepMV replication. As for Potato virus X, the PepMV CP and triple gene-block protein 1 (TGBp1) self-associate and interact with each other in vitro but, unlike in the prototype, both PepMV proteins represent suppressors of transgene-induced RNA silencing with different modes of action; CP is a more efficient suppressor of RNA silencing, sequesters the silencing signal by preventing its spread to neighboring cells and its systemic movement. Here, we provide evidence for additional roles of the PepMV CP and host-encoded Hsp70 in viral infection, the first as a truly multifunctional protein able to specifically bind to a host chaperone and to counterattack an RNA-based defense mechanism, and the latter as an essential factor for PepMV infection. PMID:25162316

  2. Multifaceted capsid proteins: multiple interactions suggest multiple roles for Pepino mosaic virus capsid protein.

    PubMed

    Mathioudakis, Matthaios M; Rodríguez-Moreno, Luis; Sempere, Raquel Navarro; Aranda, Miguel A; Livieratos, Ioannis

    2014-12-01

    Pepino mosaic virus (PepMV) (family Alphaflexiviridae, genus Potexvirus) is a mechanically transmitted tomato pathogen that, over the last decade, has evolved from emerging to endemic worldwide. Here, two heat-shock cognate (Hsc70) isoforms were identified as part of the coat protein (CP)/Hsc70 complex in vivo, following full-length PepMV and CP agroinoculation. PepMV accumulation was severely reduced in Hsp70 virus-induced gene silenced and in quercetin-treated Nicotiana benthamiana plants. Similarly, in vitro-transcribed as well as virion RNA input levels were reduced in quercetin-treated protoplasts, suggesting an essential role for Hsp70 in PepMV replication. As for Potato virus X, the PepMV CP and triple gene-block protein 1 (TGBp1) self-associate and interact with each other in vitro but, unlike in the prototype, both PepMV proteins represent suppressors of transgene-induced RNA silencing with different modes of action; CP is a more efficient suppressor of RNA silencing, sequesters the silencing signal by preventing its spread to neighboring cells and its systemic movement. Here, we provide evidence for additional roles of the PepMV CP and host-encoded Hsp70 in viral infection, the first as a truly multifunctional protein able to specifically bind to a host chaperone and to counterattack an RNA-based defense mechanism, and the latter as an essential factor for PepMV infection.

  3. Electrospun nylon 6/zinc doped hydroxyapatite membrane for protein separation: Mechanism of fouling and blocking model.

    PubMed

    Esfahani, Hamid; Prabhakaran, Molamma P; Salahi, Esmaeil; Tayebifard, Ali; Rahimipour, Mohamad Reza; Keyanpour-Rad, Mansour; Ramakrishna, Seeram

    2016-02-01

    Development of composite nanofibrous membrane via electrospinning a polymer with ceramic nanoparticles (NPs) for application in protein separation systems is explored during this study. Positively charged zinc doped hydroxyapatite (xZH) NPs were prepared in three different compositions via chemical precipitation method. Herein, we created a positively charged surface containing nanoparticles on electrospun Nylon-6 nanofibers (NFs) to improve the separation and selectivity properties for adsorption of negatively charged protein, namely bovine serum albumin (BSA). The decline in permeate flux was analyzed using the framework of classical blocking models and fitting, demonstrated that the transition of fouling mechanisms was dominated during the filtration process. The standard blocking model provided the best fit of the experimental results during the mid-filtration period. The membrane decorated by NPs containing 4at.% zinc cations not only provided maximum BSA separation but also capable of separating higher amounts of BSA molecules (even after 1h filtration) than the pure Nylon membrane. Protein separation was achieved through this membrane with the incorporation of NPs that had high zeta potential (+5.9±0.2mV) and lower particle area (22,155nm(2)). The developed membrane has great potential to act as a high efficiency membrane for capturing BSA. PMID:26652392

  4. Interaction between pregnancy zone protein and plasmin.

    PubMed

    Poulsen, O M; Hau, J

    1988-01-01

    Pregnancy zone protein (PZP, alpha 2-PAG, SP3) was found to bind to plasmin in crossed affino-immunoelectrophoresis using sodium caseinate in the first dimension gel. The plasmin presence in the PZP-plasmin complex was confirmed by addition of antiserum against plasminogen to the gel. In crossed affino-immunoelectrophoresis using plasmin in the first dimension gel a non migrative PZP immunoreactive peak appeared, similar to the peak obtained with casein in the first dimension gel. Incubation of mixtures of PZP and plasmin also demonstrated complex formation between PZP and plasmin. The complex between PZP and plasmin could be precipitated not only by anti-PZP antibodies, but also by anti-plasminogen antibodies, confirming the interaction between the two molecules. The significance of the binding between plasmin and PZP remains to be elucidated, but it is tempting to speculate that PZP, present on the trophoblastic surface, immobilizes plasmin, rendering this molecule able to perform a local fibrinolytic activity.

  5. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    PubMed Central

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  6. Identification of binary interactions between human cytomegalovirus virion proteins.

    PubMed

    Phillips, Stacia L; Bresnahan, Wade A

    2011-01-01

    Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis. PMID:20962080

  7. CPI motif interaction is necessary for capping protein function in cells

    PubMed Central

    Edwards, Marc; McConnell, Patrick; Schafer, Dorothy A.; Cooper, John A.

    2015-01-01

    Capping protein (CP) has critical roles in actin assembly in vivo and in vitro. CP binds with high affinity to the barbed end of actin filaments, blocking the addition and loss of actin subunits. Heretofore, models for actin assembly in cells generally assumed that CP is constitutively active, diffusing freely to find and cap barbed ends. However, CP can be regulated by binding of the ‘capping protein interaction' (CPI) motif, found in a diverse and otherwise unrelated set of proteins that decreases, but does not abolish, the actin-capping activity of CP and promotes uncapping in biochemical experiments. Here, we report that CP localization and the ability of CP to function in cells requires interaction with a CPI-motif-containing protein. Our discovery shows that cells target and/or modulate the capping activity of CP via CPI motif interactions in order for CP to localize and function in cells. PMID:26412145

  8. Categorizing biases in high-confidence high-throughput protein-protein interaction data sets.

    PubMed

    Yu, Xueping; Ivanic, Joseph; Memisević, Vesna; Wallqvist, Anders; Reifman, Jaques

    2011-12-01

    We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not

  9. Ibogaine blocked methamphetamine-induced hyperthermia and induction of heat shock protein in mice.

    PubMed

    Yu, X; Imam, S Z; Newport, G D; Slikker, W; Ali, S F

    1999-03-27

    Body temperature changes and heat shock protein (HSP-72) induction in the caudate nucleus were studied in female C57BL/6N mice pretreated with ibogaine (50 mg/kg) and sacrificed 48 h. after a single dose of methamphetamine (20 mg/kg). Methamphetamine injection resulted in hyperthermia and induced HSP-72 expression, whereas treatment with ibogaine alone produced hypothermia. The ibogaine followed by methamphetamine injection showed no hyperthermia and decreased HSP-72 expression. These data indicate that pretreatment with ibogaine can completely block methamphetamine-induced hyperthermia and HSP-72 expression in the striatum.

  10. Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells.

    PubMed

    Chang, M; Zhang, L; Tam, J P; Sanders-Bush, E

    2000-03-10

    To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals. PMID:10702266

  11. Specificity and non-specificity in RNA–protein interactions

    PubMed Central

    Jankowsky, Eckhard; Harris, Michael E.

    2016-01-01

    Gene expression is regulated by complex networks of interactions between RNAs and proteins. Proteins that interact with RNA have been traditionally viewed as either specific or non-specific; specific proteins interact preferentially with defined RNA sequence or structure motifs, whereas non-specific proteins interact with RNA sites devoid of such characteristics. Recent studies indicate that the binary “specific vs. non-specific” classification is insufficient to describe the full spectrum of RNA–protein interactions. Here, we review new methods that enable quantitative measurements of protein binding to large numbers of RNA variants, and the concepts aimed as describing resulting binding spectra: affinity distributions, comprehensive binding models and free energy landscapes. We discuss how these new methodologies and associated concepts enable work towards inclusive, quantitative models for specific and non-specific RNA–protein interactions. PMID:26285679

  12. Ion–dipole interactions and their functions in proteins

    PubMed Central

    Sippel, Katherine H; Quiocho, Florante A

    2015-01-01

    Ion–dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein–protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion–dipole interaction and discuss several prominent examples of their functional roles in nature. PMID:25866296

  13. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  14. Large-scale identification of yeast integral membrane protein interactions

    PubMed Central

    Miller, John P.; Lo, Russell S.; Ben-Hur, Asa; Desmarais, Cynthia; Stagljar, Igor; Noble, William Stafford; Fields, Stanley

    2005-01-01

    We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions. PMID:16093310

  15. Defining the Protein-Protein Interaction Network of the Human Protein Tyrosine Phosphatase Family.

    PubMed

    Li, Xu; Tran, Kim My; Aziz, Kathryn E; Sorokin, Alexey V; Chen, Junjie; Wang, Wenqi

    2016-09-01

    Protein tyrosine phosphorylation, which plays a vital role in a variety of human cellular processes, is coordinated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Genomic studies provide compelling evidence that PTPs are frequently mutated in various human cancers, suggesting that they have important roles in tumor suppression. However, the cellular functions and regulatory machineries of most PTPs are still largely unknown. To gain a comprehensive understanding of the protein-protein interaction network of the human PTP family, we performed a global proteomic study. Using a Minkowski distance-based unified scoring environment (MUSE) for the data analysis, we identified 940 high confidence candidate-interacting proteins that comprise the interaction landscape of the human PTP family. Through a gene ontology analysis and functional validations, we connected the PTP family with several key signaling pathways or cellular functions whose associations were previously unclear, such as the RAS-RAF-MEK pathway, the Hippo-YAP pathway, and cytokinesis. Our study provides the first glimpse of a protein interaction network for the human PTP family, linking it to a number of crucial signaling events, and generating a useful resource for future studies of PTPs.

  16. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning.

  17. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning. PMID:27400769

  18. Block-diagonal similarity renormalization group and effective nucleon-nucleon interactions

    NASA Astrophysics Data System (ADS)

    Szpigel, S.; Timóteo, V. S.; Ruiz Arriola, E.

    2016-04-01

    We apply the block-diagonal similarity renormalization group to a simple toy-model for the nucleon-nucleon (NN) interaction in the 1 S 0 channel, aiming to analyze the complementarity between the explicit and the implicit renormalization approaches in nuclear physics. By explicit renormalization we mean the methods based on the wilsonian renormalization group in which high-energy modes above a given cutoff scale are integrated out while their effects are replaced by scale dependent effective interactions consistently generated in the process. We call implicit renormalization the usual procedure of cutoff effective theories in which the high-energy modes above the cutoff scale are simply removed and their effects are included through parametrized cutoff dependent counterterms whose strengths are fixed by fitting low-energy data. We compare the effective interactions obtained in both schemes and find a wide range of cutoff scales where they overlap. We further analyze the role played by the one-pion exchange (OPE) considering a δ-shell plus OPE representation for the NN interaction.

  19. Evolution of protein interactions: from interactomes to interfaces.

    PubMed

    Andreani, Jessica; Guerois, Raphael

    2014-07-15

    Protein-protein interactions lie at the heart of most cellular processes. Many experimental and computational studies aim to deepen our understanding of these interactions and improve our capacity to predict them. In this respect, the evolutionary perspective is most interesting, since the preservation of structure and function puts constraints on the evolution of proteins and their interactions. However, uncovering these constraints remains a challenge, and the description and detection of evolutionary signals in protein-protein interactions is currently a very active field of research. Here, we review recent works dissecting the mechanisms of protein-protein interaction evolution and exploring how to use evolutionary information to predict interactions, both at the global level of the interactome and at the detailed level of protein-protein interfaces. We first present to what extent protein-protein interactions are found to be conserved within interactomes and which properties can influence their conservation. We then discuss the evolutionary and co-evolutionary pressures applied on protein-protein interfaces. Finally, we describe how the computational prediction of interfaces can benefit from evolutionary inputs. PMID:24853495

  20. Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

    PubMed Central

    Waheed, Syed Mohsin; Ghosh, Arnab; Chakravarti, Ritu; Biswas, Ashis; Haque, Mohammad Mahfuzul; Panda, Koustubh; Stuehr, Dennis J.

    2010-01-01

    Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time period, and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structure, heme ligation, and function (three NO synthases, two cytochrome P450’s, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme level, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process. PMID:20211245

  1. Assembly of a photosynthetic reaction center with ABA tri-block polymersomes: highlights on protein localization.

    PubMed

    Tangorra, R R; Operamolla, A; Milano, F; Hassan Omar, O; Henrard, J; Comparelli, R; Italiano, F; Agostiano, A; De Leo, V; Marotta, R; Falqui, A; Farinola, G M; Trotta, M

    2015-10-01

    The reconstitution of the integral membrane protein photosynthetic reaction center (RC) in polymersomes, i.e. artificial closed vesicles, was achieved by the micelle-to-vesicle transition technique, a very mild protocol based on size exclusion chromatography often used to drive the incorporation of proteins contemporarily to liposome formation. An optimized protocol was used to successfully reconstitute the protein in a fully active state in polymersomes formed by the tri-block copolymers PMOXA22-PDMS61-PMOXA22. The RC is very sensitive to its solubilizing environment and was used to probe the positioning of the protein in the vesicles. According to charge-recombination experiments and to the enzymatic activity assay, the RC is found to accommodate in the PMOXA22 region of the polymersome, facing the water bulk solution, rather than in the PDMS61 transmembrane-like region. Furthermore, polymersomes were found to preserve protein integrity efficiently as the biomimetic lipid bilayers but show a much longer temporal stability than lipid based vesicles.

  2. RING finger protein PLR-1 blocks Wnt signaling by altering trafficking of Wnt Receptors

    NASA Astrophysics Data System (ADS)

    Robinson, Ryan E.

    Secreted Wnt proteins control a wide range of essential developmental processes, including axon guidance and establishment of anteroposterior neuronal polarity. We identified a transmembrane RING finger protein, PLR-1, that governs the response to Wnts by reducing the cell surface levels of Wnt receptors Frizzled, CAM-1 and LIN-18 in Caenorhabditis elegans. Frizzled, CAM-1 and LIN-18 are normally enriched at the plasma membrane where they are capable of detecting and responding to extracellular Wnts. However, when PLR-1 is expressed Frizzled, CAM-1 and LIN-18 are no longer detected at the cell surface and instead colocalize with PLR-1 in endosomes and Golgi. PLR-1 is related to a broad family of transmembrane proteins that contain a lumenal protease associated domain and a cytosolic RING finger. The RING finger is a hallmark of one type of E3 ubiquitin ligase and monoubiquitination is commonly used to regulate protein trafficking. Protease associated domains are largely thought to mediate interactions between proteins. To identify the domains responsible for PLR-1 regulation of Frizzled from the cell surface we utilized a series of fluorescently tagged fusion proteins and protein truncations containing various domains from PLR-1 and Frizzled. Our data suggests that PLR-1 and Frizzled interact and form a complex via their respective extracellular/lumenal domains, and that ubiqiuitination of Frizzled by PLR-1 targets the Frizzled/PLR-1 complex to the endosome.

  3. Biomimetic block copolymer particles with gated nanopores and ultrahigh protein sorption capacity

    NASA Astrophysics Data System (ADS)

    Yu, Haizhou; Qiu, Xiaoyan; Nunes, Suzana P.; Peinemann, Klaus-Viktor

    2014-06-01

    The design of micro- or nanoparticles that can encapsulate sensitive molecules such as drugs, hormones, proteins or peptides is of increasing importance for applications in biotechnology and medicine. Examples are micelles, liposomes and vesicles. The tiny and, in most cases, hollow spheres are used as vehicles for transport and controlled administration of pharmaceutical drugs or nutrients. Here we report a simple strategy to fabricate microspheres by block copolymer self-assembly. The microsphere particles have monodispersed nanopores that can act as pH-responsive gates. They contain a highly porous internal structure, which is analogous to the Schwarz P structure. The internal porosity of the particles contributes to their high sorption capacity and sustained release behaviour. We successfully separated similarly sized proteins using these particles. The ease of particle fabrication by macrophase separation and self-assembly, and the robustness of the particles makes them ideal for sorption, separation, transport and sustained delivery of pharmaceutical substances.

  4. Biomimetic block copolymer particles with gated nanopores and ultrahigh protein sorption capacity.

    PubMed

    Yu, Haizhou; Qiu, Xiaoyan; Nunes, Suzana P; Peinemann, Klaus-Viktor

    2014-01-01

    The design of micro- or nanoparticles that can encapsulate sensitive molecules such as drugs, hormones, proteins or peptides is of increasing importance for applications in biotechnology and medicine. Examples are micelles, liposomes and vesicles. The tiny and, in most cases, hollow spheres are used as vehicles for transport and controlled administration of pharmaceutical drugs or nutrients. Here we report a simple strategy to fabricate microspheres by block copolymer self-assembly. The microsphere particles have monodispersed nanopores that can act as pH-responsive gates. They contain a highly porous internal structure, which is analogous to the Schwarz P structure. The internal porosity of the particles contributes to their high sorption capacity and sustained release behaviour. We successfully separated similarly sized proteins using these particles. The ease of particle fabrication by macrophase separation and self-assembly, and the robustness of the particles makes them ideal for sorption, separation, transport and sustained delivery of pharmaceutical substances. PMID:24934665

  5. Biomimetic block copolymer particles with gated nanopores and ultrahigh protein sorption capacity.

    PubMed

    Yu, Haizhou; Qiu, Xiaoyan; Nunes, Suzana P; Peinemann, Klaus-Viktor

    2014-01-01

    The design of micro- or nanoparticles that can encapsulate sensitive molecules such as drugs, hormones, proteins or peptides is of increasing importance for applications in biotechnology and medicine. Examples are micelles, liposomes and vesicles. The tiny and, in most cases, hollow spheres are used as vehicles for transport and controlled administration of pharmaceutical drugs or nutrients. Here we report a simple strategy to fabricate microspheres by block copolymer self-assembly. The microsphere particles have monodispersed nanopores that can act as pH-responsive gates. They contain a highly porous internal structure, which is analogous to the Schwarz P structure. The internal porosity of the particles contributes to their high sorption capacity and sustained release behaviour. We successfully separated similarly sized proteins using these particles. The ease of particle fabrication by macrophase separation and self-assembly, and the robustness of the particles makes them ideal for sorption, separation, transport and sustained delivery of pharmaceutical substances.

  6. Nanoporous membrane based on block copolymer thin film for protein drug delivery

    NASA Astrophysics Data System (ADS)

    Yang, Seung Yun; Yang, Jeong-A.; Kim, Eung-Sam; Jeon, Gumhye; Oh, Eun Ju; Choi, Kwan Yong; Hahn, Sei Kwang; Kim, Jin Kon

    2010-03-01

    We studied long term and controlled release of protein drugs by using nanoporous membranes with various pore sizes. Nanoporous membrane consists of the separation layer prepared by polystyrene-block-poly(methylmethacrylate) copolymer thin film and conventional microfiltration membrane as a support. We demonstrate a long-term constant in vitro release of bovine serum albumin (BSA)and human growth hormone ) (hGH) without their denaturation up to 2 months. A nearly constant serum concentration of hGH was maintained up to 3 weeks in SD rats. The long-term constant delivery based on this membrane for protein drugs within the therapeutic range can be highly appreciated for the patients with hormone- deficiency.

  7. MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope.

    PubMed

    Jafferali, Mohammed Hakim; Figueroa, Ricardo A; Hallberg, Einar

    2016-01-01

    The organization and function of the nuclear envelope (NE) involves hundreds of nuclear membrane proteins and myriad protein-protein interactions, most of which are still uncharacterized. Many NE proteins interact stably or dynamically with the nuclear lamina or chromosomes. This can make them difficult to extract under nondenaturing conditions, and greatly limits our ability to explore and identify functional protein interactions at the NE. This knowledge is needed to understand nuclear envelope structure and the mechanisms of human laminopathy diseases. This chapter provides detailed protocols for MCLIP (membrane cross-linking immunoprecipitation) identification of novel protein-protein interactions in mammalian cells.

  8. Selective separation of similarly sized proteins with tunable nanoporous block copolymer membranes.

    PubMed

    Qiu, Xiaoyan; Yu, Haizhou; Karunakaran, Madhavan; Pradeep, Neelakanda; Nunes, Suzana P; Peinemann, Klaus-Viktor

    2013-01-22

    An integral asymmetric membrane was fabricated in a fast and one-step process by combining the self-assembly of an amphiphilic block copolymer (PS-b-P4VP) with nonsolvent-induced phase separation. The structure was found to be composed of a thin layer of densely packed highly ordered cylindrical channels with uniform pore sizes perpendicular to the surface on top of a nonordered sponge-like layer. The as-assembled membrane obtained a water flux of more than 3200 L m(-2) h(-1) bar(-1), which was at least an order of magnitude higher than the water fluxes of commercially available membranes with comparable pore sizes, making this membrane particularly well suited to size-selective and charge-based separation of biomolecules. To test the performance of the membrane, we conducted diffusion experiments at the physiological pH of 7.4 using bovine serum albumin (BSA) and globulin-γ, two proteins with different diameters but too close in size (2-fold difference in molecular mass) to be efficiently separated via conventional dialysis membrane processes. The diffusion rate differed by a factor of 87, the highest value reported to date. We also analyzed charge-based diffusive transport and separation of two proteins of similar molecular weight (BSA and bovine hemoglobin (BHb)) through the membrane as a function of external pH. The membrane achieved a selectivity of about 10 at pH 4.7, the isoelectric point (pI) of BSA. We then positively charged the membrane to improve the separation selectivity. With the modified membrane BSA was completely blocked when the pH was 7.0, the pI of BHb, while BHb was completely blocked at pH 4.7. Our results demonstrate the potential of our asymmetric membrane to efficiently separate biological substances/pharmaceuticals in bioscience, biotechnology, and biomedicine applications.

  9. Selective separation of similarly sized proteins with tunable nanoporous block copolymer membranes.

    PubMed

    Qiu, Xiaoyan; Yu, Haizhou; Karunakaran, Madhavan; Pradeep, Neelakanda; Nunes, Suzana P; Peinemann, Klaus-Viktor

    2013-01-22

    An integral asymmetric membrane was fabricated in a fast and one-step process by combining the self-assembly of an amphiphilic block copolymer (PS-b-P4VP) with nonsolvent-induced phase separation. The structure was found to be composed of a thin layer of densely packed highly ordered cylindrical channels with uniform pore sizes perpendicular to the surface on top of a nonordered sponge-like layer. The as-assembled membrane obtained a water flux of more than 3200 L m(-2) h(-1) bar(-1), which was at least an order of magnitude higher than the water fluxes of commercially available membranes with comparable pore sizes, making this membrane particularly well suited to size-selective and charge-based separation of biomolecules. To test the performance of the membrane, we conducted diffusion experiments at the physiological pH of 7.4 using bovine serum albumin (BSA) and globulin-γ, two proteins with different diameters but too close in size (2-fold difference in molecular mass) to be efficiently separated via conventional dialysis membrane processes. The diffusion rate differed by a factor of 87, the highest value reported to date. We also analyzed charge-based diffusive transport and separation of two proteins of similar molecular weight (BSA and bovine hemoglobin (BHb)) through the membrane as a function of external pH. The membrane achieved a selectivity of about 10 at pH 4.7, the isoelectric point (pI) of BSA. We then positively charged the membrane to improve the separation selectivity. With the modified membrane BSA was completely blocked when the pH was 7.0, the pI of BHb, while BHb was completely blocked at pH 4.7. Our results demonstrate the potential of our asymmetric membrane to efficiently separate biological substances/pharmaceuticals in bioscience, biotechnology, and biomedicine applications. PMID:23252799

  10. Spironolactone blocks Epstein-Barr virus production by inhibiting EBV SM protein function.

    PubMed

    Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

    2016-03-29

    Clinically available drugs active against Epstein-Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses. PMID:26976570

  11. Spironolactone blocks Epstein-Barr virus production by inhibiting EBV SM protein function.

    PubMed

    Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

    2016-03-29

    Clinically available drugs active against Epstein-Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses.

  12. Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

    PubMed Central

    Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

    2016-01-01

    Clinically available drugs active against Epstein–Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses. PMID:26976570

  13. Predicting the protein-protein interactions using primary structures with predicted protein surface

    PubMed Central

    2010-01-01

    Background Many biological functions involve various protein-protein interactions (PPIs). Elucidating such interactions is crucial for understanding general principles of cellular systems. Previous studies have shown the potential of predicting PPIs based on only sequence information. Compared to approaches that require other auxiliary information, these sequence-based approaches can be applied to a broader range of applications. Results This study presents a novel sequence-based method based on the assumption that protein-protein interactions are more related to amino acids at the surface than those at the core. The present method considers surface information and maintains the advantage of relying on only sequence data by including an accessible surface area (ASA) predictor recently proposed by the authors. This study also reports the experiments conducted to evaluate a) the performance of PPI prediction achieved by including the predicted surface and b) the quality of the predicted surface in comparison with the surface obtained from structures. The experimental results show that surface information helps to predict interacting protein pairs. Furthermore, the prediction performance achieved by using the surface estimated with the ASA predictor is close to that using the surface obtained from protein structures. Conclusion This work presents a sequence-based method that takes into account surface information for predicting PPIs. The proposed procedure of surface identification improves the prediction performance with an F-measure of 5.1%. The extracted surfaces are also valuable in other biomedical applications that require similar information. PMID:20122202

  14. Pseudorevertants of a Semliki Forest Virus Fusion-Blocking Mutation Reveal a Critical Interchain Interaction in the Core Trimer▿

    PubMed Central

    Liu, Catherine Y.; Besanceney, Christen; Song, Yifan; Kielian, Margaret

    2010-01-01

    Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein. PMID:20826687

  15. Synoptic/planetary-scale interactions and blocking over the North Atlantic Ocean

    NASA Technical Reports Server (NTRS)

    Smith, Phillip J.; Lupo, Anthony R.; Hunter, Melinda L.; Stettner, David R.

    1993-01-01

    The central theme of this project has been the diagnosis of blocking anticyclogenesis and the corresponding interactions with synoptic-scale circulations. To that end an extensive investigation of the dynamics and energetics of a major blocking anticyclone and two upstream cyclones, all of which occurred over the North Atlantic Ocean and the United States in January 1979, was undertaken. Data for the study were provided by Goddard Laboratory for Atmospheres (GLA) 4 LAT by 5 LON FGGE analyses. The methodology has primarily focused on the diagnosis of circulation forcing mechanisms using the modified forms (referred to as the extended forms) of the height tendency and Zwack-Okossi equations developed by our research group. Calculations use routine second-order finite differencing with boundary layer fraction and sensible heating and latent heat release represented as parameterized quantities. Of particular interest are the latent heat release estimates, which combine convectional parameterized values with estimates derived from satellite IR data. The latter were obtained using an algorithm derived by Dr. Franklin R. Robertson of NASA's Marshall Space Flight Center. Results are contained in project reports, theses and publications identified in previous review summaries and reports, and publications listed at the end of this summary. Significant accomplishments in the past year are presented.

  16. Intermolecular interactions and solvent diffusion in ordered nanostructures formed by self-assembly of block copolymers

    NASA Astrophysics Data System (ADS)

    Gu, Zhiyong

    Hydrogels formed by Poloxamer poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers find various pharmaceutical and biomedical applications. A variety of ordered structures can be exhibited by Poloxamer block copolymers in selective solvents such as water, for example, micellar cubic phase, hexagonal phase, lamellar phase, etc. We are interested in the thermodynamic and transport properties of water in such hydrogels that have an ordered (lyotropic liquid crystalline) structure. We have investigated the time evolution of water loss from Poloxamer gel films under a driving force of known water vapor pressure in the air in contact with the film. The experimental data on the drying process have been fitted to the diffusion equation for water in the film, under a boundary condition that includes the water concentration in the gel at infinite time; the water diffusion coefficient and other parameters have thus been obtained. The water chemical potential and osmotic pressure in the gel have been obtained from osmotic stress measurements. The osmotic pressure (force), together with data on the corresponding lyotropic liquid crystal spacing (distance) that we obtained from Small Angle X-Ray Scattering (SAXS) measurements, have been analyzed to provide information on the prevailing intermolecular (inter-assembly) forces in the gel. The forces in the gel reveal interactions that occur at two levels, that of the PEO coil and that of the PEO segment.

  17. A Laboratory-Intensive Course on the Experimental Study of Protein-Protein Interactions

    ERIC Educational Resources Information Center

    Witherow, D. Scott; Carson, Sue

    2011-01-01

    The study of protein-protein interactions is important to scientists in a wide range of disciplines. We present here the assessment of a lab-intensive course that teaches students techniques used to identify and further study protein-protein interactions. One of the unique elements of the course is that students perform a yeast two-hybrid screen…

  18. Ammonium sulfate co-precipitation of SSB and interacting proteins.

    PubMed

    Marceau, Aimee H

    2012-01-01

    Bacterial single-strand DNA-binding protein (SSB) interacts with many proteins involved in the diverse process of genome maintenance. The interactions are mediated by the essential and conserved amphipathic C-terminus (SSB-Ct). SSB plays a critical role in localizing and stimulating the activity of a wide variety of DNA-processing proteins. The interaction partners have been identified and studied using a variety of methods, one of which, ammonium sulfate co-precipitation, is described here.

  19. Computational Study of Positron-Monovacancy Interaction in d-Block Metals

    NASA Astrophysics Data System (ADS)

    Ishibashi, Shoji

    2015-08-01

    The positron-monovacancy interaction in d-block metals (except for Mn, Tc, and Hg) has been studied by the two-component density-functional-theory formalism [E. Boroński and R. M. Nieminen, Phys. Rev. B 34, 3820 (1986)]. On the unrelaxed structure, the positron lifetime calculated with the presence of a positron is generally longer than that obtained neglecting the positron effect. When the atomic positions are relaxed, the difference is widened, especially for the group V metals. The inward relaxation of the atoms surrounding the monovacancy is suppressed when the positron effect is taken into account. The difference in the positron lifetime can be also related to the bulk modulus and the cohesive energy.

  20. Relationship between blocking temperature and strength of interparticle interaction in magnetic nanoparticle systems

    NASA Astrophysics Data System (ADS)

    Seehra, M. S.; Pisane, K. L.

    2016-06-01

    In magnetic nanoparticle systems, the variation of the blocking temperature TB with the measuring frequency fm is often used to determine the strength of the interparticle interactions (IPI) through a parameter Φ or the Vogel-Fulcher temperature To. Presence of IPI is inferred if To > 0 and Φ = ∆TB/[ TB ∆ log10fm ] < 0.13 where Δ signifies changes in TB and fm. Here it is shown that these two parameters are related by the Eq. Φ = Φo [ 1 - (To/TB(1)) ] where Φo ≈ 0.11-0.15 is a constant of the system depending on the magnitudes of measuring frequency and the attempt frequency fo of the Néel relaxation. Using data on a variety of nanoparticle systems, experimental verification of this relationship is also presented.

  1. Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling

    PubMed Central

    Qiu, Xusheng; Fu, Qiang; Meng, Chunchun; Yu, Shengqing; Zhan, Yuan; Dong, Luna; Song, Cuiping; Sun, Yingjie; Tan, Lei; Hu, Shunlin; Wang, Xiaoquan; Liu, Xiaowen; Peng, Daxin; Liu, Xiufan; Ding, Chan

    2016-01-01

    Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN. PMID:26859759

  2. Interactions of proteins in gels, solutions and on surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Radha Perumal

    2006-12-01

    The study of protein interaction, identification and separation has applications in various fields relating to Biotechnology. In this research these aspects were investigated. The proteins albumin, casein, poly-L-lysine were studied. FITC and TRITC were used to fluorescently tag the proteins. Confocal microscopy was used to image the interaction of proteins. The migration of fluorescently tagged protein-salt aggregates on solid surfaces during electrophoresis was investigated using Confocal microscopy. The secondary structural modifications of proteins in solutions were investigated using FTIR micro spectroscopic imaging. The size of the colloids formed due to protein-protein interactions as a function of the protein concentrations were studied using DLS and their charges were found using zeta potential measurements. Based on DL.S and zeta potential measurements, a model is proposed for interactions of oppositely charged proteins. The nature of interaction was found using UV - Visual spectroscopy. It was found that oppositely charged proteins formed ionic bonds. It was also found that FITC molecule influenced the surface charge of albumin more than TRITC molecule. The effects of the influence of cell geometries upon Electro Osmotic Flow (EOF) were studied using neutrally charged fluorescent Polystyrene beads. Results showed that tagging proteins with fluorescent molecules influenced their mobility and interactions with other proteins. However no secondary structural modifications of the proteins were observed when oppositely charged proteins interacted. It was also observed that electrostatic interactions made oppositely charged proteins form large aggregates. The EOF was found to be dependent upon the ionic strength of the buffer, conductivity of the solid surfaces, distance from the surface and position of the electrodes in the electrophoretic cell.

  3. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample.

  4. Examining post-translational modification-mediated protein-protein interactions using a chemical proteomics approach.

    PubMed

    Li, Xiang; Foley, Emily A; Kawashima, Shigehiro A; Molloy, Kelly R; Li, Yinyin; Chait, Brian T; Kapoor, Tarun M

    2013-03-01

    Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein-protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein-protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein-protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein-protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me₃)-dependent protein-protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein-protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone "mark" appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation "mark". Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein-protein interactions mediated by PTMs. PMID:23281010

  5. Encoding protein-ligand interaction patterns in fingerprints and graphs.

    PubMed

    Desaphy, Jérémy; Raimbaud, Eric; Ducrot, Pierre; Rognan, Didier

    2013-03-25

    We herewith present a novel and universal method to convert protein-ligand coordinates into a simple fingerprint of 210 integers registering the corresponding molecular interaction pattern. Each interaction (hydrophobic, aromatic, hydrogen bond, ionic bond, metal complexation) is detected on the fly and physically described by a pseudoatom centered either on the interacting ligand atom, the interacting protein atom, or the geometric center of both interacting atoms. Counting all possible triplets of interaction pseudoatoms within six distance ranges, and pruning the full integer vector to keep the most frequent triplets enables the definition of a simple (210 integers) and coordinate frame-invariant interaction pattern descriptor (TIFP) that can be applied to compare any pair of protein-ligand complexes. TIFP fingerprints have been calculated for ca. 10,000 druggable protein-ligand complexes therefore enabling a wide comparison of relationships between interaction pattern similarity and ligand or binding site pairwise similarity. We notably show that interaction pattern similarity strongly depends on binding site similarity. In addition to the TIFP fingerprint which registers intermolecular interactions between a ligand and its target protein, we developed two tools (Ishape, Grim) to align protein-ligand complexes from their interaction patterns. Ishape is based on the overlap of interaction pseudoatoms using a smooth Gaussian function, whereas Grim utilizes a standard clique detection algorithm to match interaction pattern graphs. Both tools are complementary and enable protein-ligand complex alignments capitalizing on both global and local pattern similarities. The new fingerprint and companion alignment tools have been successfully used in three scenarios: (i) interaction-biased alignment of protein-ligand complexes, (ii) postprocessing docking poses according to known interaction patterns for a particular target, and (iii) virtual screening for bioisosteric

  6. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    PubMed

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  7. Protein-Protein Interactions Inferred from Domain-Domain Interactions in Genogroup II Genotype 4 Norovirus Sequences

    PubMed Central

    Huang, Chuan-Ching

    2013-01-01

    Severe gastroenteritis and foodborne illness caused by Noroviruses (NoVs) during the winter are a worldwide phenomenon. Vulnerable populations including young children and elderly and immunocompromised people often require hospitalization and may die. However, no efficient vaccine for NoVs exists because of their variable genome sequences. This study investigates the infection processes in protein-protein interactions between hosts and NoVs. Protein-protein interactions were collected from related Pfam NoV domains. The related Pfam domains were accumulated incrementally from the protein domain interaction database. To examine the influence of domain intimacy, the 7 NoV domains were grouped by depth. The number of domain-domain interactions increased exponentially as the depth increased. Many protein-protein interactions were relevant; therefore, cloud techniques were used to analyze data because of their computational capacity. The infection relationship between hosts and NoVs should be used in clinical applications and drug design. PMID:23738320

  8. Folding superfunnel to describe cooperative folding of interacting proteins.

    PubMed

    Smeller, László

    2016-07-01

    This paper proposes a generalization of the well-known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein-protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein-protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009-1016. © 2016 Wiley Periodicals, Inc.

  9. What Evidence Is There for the Homology of Protein-Protein Interactions?

    PubMed Central

    Lewis, Anna C. F.; Jones, Nick S.; Porter, Mason A.; Deane, Charlotte M.

    2012-01-01

    The notion that sequence homology implies functional similarity underlies much of computational biology. In the case of protein-protein interactions, an interaction can be inferred between two proteins on the basis that sequence-similar proteins have been observed to interact. The use of transferred interactions is common, but the legitimacy of such inferred interactions is not clear. Here we investigate transferred interactions and whether data incompleteness explains the lack of evidence found for them. Using definitions of homology associated with functional annotation transfer, we estimate that conservation rates of interactions are low even after taking interactome incompleteness into account. For example, at a blastp -value threshold of , we estimate the conservation rate to be about between S. cerevisiae and H. sapiens. Our method also produces estimates of interactome sizes (which are similar to those previously proposed). Using our estimates of interaction conservation we estimate the rate at which protein-protein interactions are lost across species. To our knowledge, this is the first such study based on large-scale data. Previous work has suggested that interactions transferred within species are more reliable than interactions transferred across species. By controlling for factors that are specific to within-species interaction prediction, we propose that the transfer of interactions within species might be less reliable than transfers between species. Protein-protein interactions appear to be very rarely conserved unless very high sequence similarity is observed. Consequently, inferred interactions should be used with care. PMID:23028270

  10. An Inhibitory Antibody Blocks Interactions between Components of the Malarial Invasion Machinery

    PubMed Central

    Collins, Christine R.; Withers-Martinez, Chrislaine; Hackett, Fiona; Blackman, Michael J.

    2009-01-01

    Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1. PMID:19165323

  11. Design of High-Affinity Stapled Peptides To Target the Repressor Activator Protein 1 (RAP1)/Telomeric Repeat-Binding Factor 2 (TRF2) Protein-Protein Interaction in the Shelterin Complex.

    PubMed

    Ran, Xu; Liu, Liu; Yang, Chao-Yie; Lu, Jianfeng; Chen, Yong; Lei, Ming; Wang, Shaomeng

    2016-01-14

    Shelterin, a six-protein complex, plays a fundamental role in protecting both the length and the stability of telomeres. Repressor activator protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are two subunits in shelterin that interact with each other. Small-molecule inhibitors that block the RAP1/TRF2 protein-protein interaction can disrupt the structure of shelterin and may be employed as pharmacological tools to investigate the biology of shelterin. On the basis of the cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class triazole-stapled peptides that block the protein-protein interaction between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1 protein with a Ki value of 7 nM and is >100 times more potent than the corresponding wild-type TRF2 peptide. On the basis of our high-affinity peptides, we have developed and optimized a competitive, fluorescence polarization (FP) assay for accurate and rapid determination of the binding affinities of our designed compounds and this assay may also assist in the discovery of non-peptide, small-molecule inhibitors capable of blocking the RAP1/TRF2 protein-protein interaction.

  12. Direct detection of CH/pi interactions in proteins.

    PubMed

    Plevin, Michael J; Bryce, David L; Boisbouvier, Jérôme

    2010-06-01

    XH/pi interactions make important contributions to biomolecular structure and function. These weakly polar interactions, involving pi-system acceptor groups, are usually identified from the three-dimensional structures of proteins. Here, nuclear magnetic resonance spectroscopy has been used to directly detect methyl/pi (Me/pi) interactions in proteins at atomic resolution. Density functional theory calculations predict the existence of weak scalar (J) couplings between nuclei involved in Me/pi interactions. Using an optimized isotope-labelling strategy, these J couplings have been detected in proteins using nuclear magnetic resonance spectroscopy. The resulting spectra provide direct experimental evidence of Me/pi interactions in proteins and allow a simple and unambiguous assignment of donor and acceptor groups. The use of nuclear magnetic resonance spectroscopy is an elegant way to identify and experimentally characterize Me/pi interactions in proteins without the need for arbitrary geometric descriptions or pre-existing three-dimensional structures. PMID:20489715

  13. VirusMINT: a viral protein interaction database

    PubMed Central

    Chatr-aryamontri, Andrew; Ceol, Arnaud; Peluso, Daniele; Nardozza, Aurelio; Panni, Simona; Sacco, Francesca; Tinti, Michele; Smolyar, Alex; Castagnoli, Luisa; Vidal, Marc; Cusick, Michael E.; Cesareni, Gianni

    2009-01-01

    Understanding the consequences on host physiology induced by viral infection requires complete understanding of the perturbations caused by virus proteins on the cellular protein interaction network. The VirusMINT database (http://mint.bio.uniroma2.it/virusmint/) aims at collecting all protein interactions between viral and human proteins reported in the literature. VirusMINT currently stores over 5000 interactions involving more than 490 unique viral proteins from more than 110 different viral strains. The whole data set can be easily queried through the search pages and the results can be displayed with a graphical viewer. The curation effort has focused on manuscripts reporting interactions between human proteins and proteins encoded by some of the most medically relevant viruses: papilloma viruses, human immunodeficiency virus 1, Epstein–Barr virus, hepatitis B virus, hepatitis C virus, herpes viruses and Simian virus 40. PMID:18974184

  14. Globular and disordered—the non-identical twins in protein-protein interactions

    PubMed Central

    Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B.

    2015-01-01

    In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol−1. PMID:26217672

  15. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  16. 14-3-3 proteins in plant-pathogen interactions.

    PubMed

    Lozano-Durán, Rosa; Robatzek, Silke

    2015-05-01

    14-3-3 proteins define a eukaryotic-specific protein family with a general role in signal transduction. Primarily, 14-3-3 proteins act as phosphosensors, binding phosphorylated client proteins and modulating their functions. Since phosphorylation regulates a plethora of different physiological responses in plants, 14-3-3 proteins play roles in multiple signaling pathways, including those controlling metabolism, hormone signaling, cell division, and responses to abiotic and biotic stimuli. Increasing evidence supports a prominent role of 14-3-3 proteins in regulating plant immunity against pathogens at various levels. In this review, potential links between 14-3-3 function and the regulation of plant-pathogen interactions are discussed, with a special focus on the regulation of 14-3-3 proteins in response to pathogen perception, interactions between 14-3-3 proteins and defense-related proteins, and 14-3-3 proteins as targets of pathogen effectors.

  17. A second-generation protein-protein interaction network of Helicobacter pylori.

    PubMed

    Häuser, Roman; Ceol, Arnaud; Rajagopala, Seesandra V; Mosca, Roberto; Siszler, Gabriella; Wermke, Nadja; Sikorski, Patricia; Schwarz, Frank; Schick, Matthias; Wuchty, Stefan; Aloy, Patrick; Uetz, Peter

    2014-05-01

    Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

  18. Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein.

    PubMed

    Kang, Su-Min; Shin, Min-Jung; Kim, Jung-Hee; Oh, Jong-Won

    2005-05-01

    Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.

  19. Blocking the Interaction between Apolipoprotein E and Aβ Reduces Intraneuronal Accumulation of Aβ and Inhibits Synaptic Degeneration

    PubMed Central

    Kuszczyk, Magdalena A.; Sanchez, Sandrine; Pankiewicz, Joanna; Kim, Jungsu; Duszczyk, Malgorzata; Guridi, Maitea; Asuni, Ayodeji A.; Sullivan, Patrick M.; Holtzman, David M.; Sadowski, Martin J.

    2014-01-01

    Accumulation of β-amyloid (Aβ) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Aβ and has been implicated in the receptor-mediated Aβ uptake by neurons. To characterize ApoE involvement in the intraneuronal Aβ accumulation and to investigate whether blocking the ApoE/Aβ interaction could reduce intraneuronal Aβ buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Aβ peptides were added into the media without or with cotreatment with Aβ12-28P, which is a nontoxic peptide antagonist of ApoE/Aβ binding. Compared with neurons cultured alone, intraneuronal Aβ content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Aβ, increased level of intraneuronal Aβ oligomers, and marked down-regulation of several synaptic proteins. Aβ12-28P treatment significantly reduced intraneuronal Aβ accumulation, including Aβ oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Aβ accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Aβ accumulation and provide evidence that ApoE/Aβ binding antagonists can effectively prevent this process. PMID:23499462

  20. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    PubMed

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-10-22

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria.

  1. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

    PubMed Central

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-01-01

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

  2. Combination of neutralizing monoclonal antibodies against Hepatitis C virus E2 protein effectively blocks virus infection.

    PubMed

    Bose, Mihika; Mullick, Ranajoy; Das, Soma; Das, Saumitra; Karande, Anjali A

    2016-09-15

    Hepatitis C virus (HCV) represents a major global health threat. The envelope glycoproteins, E1-E2 of HCV play an important role in infection by binding to hepatocyte surface receptors leading to viral entry. Several regions on the E1-E2 are conserved for maintaining structural stability, despite the high mutation rate of HCV. Identification of antigenic determinants in these domains would aid in the development of anti-virals. The present study was aimed to delineate neutralizing epitopes by generating monoclonal antibodies (mAbs) to envelope proteins that can block virus binding and entry. Using HCV-like particles (HCV-LPs) corresponding to genotype 3a (prevalent in India), we obtained three mAbs specific for the E2 protein that significantly inhibited virus binding to hepatoma cells. Using overlapping protein fragments and peptides of the E2 protein, the epitopes corresponding to the mAbs were delineated. MAbs H6D3 and A10F2 recognise sequential linear epitopes, whereas, mAb E3D8 recognises a discontinuous epitope. The epitope of mAb E3D8 overlaps with the CD81 receptor-binding site and that of mAb A10F2 with the hypervariable region 2 of the E2 protein. The epitopes corresponding to these mAbs are distinct and unique. A combination of these antibodies significantly inhibited HCV binding and entry in both HCV pseudoparticle (in vitro) and HCV cell culture (ex vivo) system compared to the mAbs alone (P<0.0001). In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs in the management of HCV infection. PMID:27574733

  3. The simulation approach to lipid-protein interactions.

    PubMed

    Paramo, Teresa; Garzón, Diana; Holdbrook, Daniel A; Khalid, Syma; Bond, Peter J

    2013-01-01

    The interactions between lipids and proteins are crucial for a range of biological processes, from the folding and stability of membrane proteins to signaling and metabolism facilitated by lipid-binding proteins. However, high-resolution structural details concerning functional lipid/protein interactions are scarce due to barriers in both experimental isolation of native lipid-bound complexes and subsequent biophysical characterization. The molecular dynamics (MD) simulation approach provides a means to complement available structural data, yielding dynamic, structural, and thermodynamic data for a protein embedded within a physiologically realistic, modelled lipid environment. In this chapter, we provide a guide to current methods for setting up and running simulations of membrane proteins and soluble, lipid-binding proteins, using standard atomistically detailed representations, as well as simplified, coarse-grained models. In addition, we outline recent studies that illustrate the power of the simulation approach in the context of biologically relevant lipid/protein interactions. PMID:23404287

  4. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    PubMed

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

  5. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    PubMed Central

    Olijve, Luuk L. C.; Meister, Konrad; DeVries, Arthur L.; Duman, John G.; Guo, Shuaiqi; Bakker, Huib J.; Voets, Ilja K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application. PMID:26936953

  6. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    PubMed

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application. PMID:26936953

  7. From networks of protein interactions to networks of functional dependencies

    PubMed Central

    2012-01-01

    Background As protein-protein interactions connect proteins that participate in either the same or different functions, networks of interacting and functionally annotated proteins can be converted into process graphs of inter-dependent function nodes (each node corresponding to interacting proteins with the same functional annotation). However, as proteins have multiple annotations, the process graph is non-redundant, if only proteins participating directly in a given function are included in the related function node. Results Reasoning that topological features (e.g., clusters of highly inter-connected proteins) might help approaching structured and non-redundant understanding of molecular function, an algorithm was developed that prioritizes inclusion of proteins into the function nodes that best overlap protein clusters. Specifically, the algorithm identifies function nodes (and their mutual relations), based on the topological analysis of a protein interaction network, which can be related to various biological domains, such as cellular components (e.g., peroxisome and cellular bud) or biological processes (e.g., cell budding) of the model organism S. cerevisiae. Conclusions The method we have described allows converting a protein interaction network into a non-redundant process graph of inter-dependent function nodes. The examples we have described show that the resulting graph allows researchers to formulate testable hypotheses about dependencies among functions and the underlying mechanisms. PMID:22607727

  8. Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins.

    PubMed

    Jaberi, Aala; Hooker, Erika; Guillemette, Julie; Papillon, Joan; Kristof, Arnold S; Cybulsky, Andrey V

    2015-10-01

    Expression and activity of the Ste20-like kinase, SLK, are increased during kidney development and recovery from ischemia-reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, α-actinin-4, as potential SLK-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and α-actinin-4. The association of SLK with Tpr and α-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and α-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by α-actinin-4. Thus, SLK interacts with Tpr and α-actinin-4 in cells, and these protein-protein interactions may control the subcellular localization and the biological activity of SLK.

  9. Replisome fate upon encountering a leading strand block and clearance from DNA by recombination proteins.

    PubMed

    McInerney, Peter; O'Donnell, Mike

    2007-08-31

    Replication forks that collapse upon encountering a leading strand lesion are reactivated by a recombinative repair process called replication restart. Using rolling circle DNA substrates to model replication forks, we examine the fate of the helicase and both DNA polymerases when the leading strand polymerase is blocked. We find that the helicase continues over 0.5 kb but less than 3 kb and that the lagging strand DNA polymerase remains active despite its connection to a stalled leading strand enzyme. Furthermore, the blocked leading strand polymerase remains stably bound to the replication fork, implying that it must be dismantled from DNA in order for replication restart to initiate. Genetic studies have identified at least four gene products required for replication restart, RecF, RecO, RecR, and RecA. We find here that these proteins displace a stalled polymerase at a DNA template lesion. Implications of these results for replication fork collapse and recovery are discussed. PMID:17609212

  10. The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration.

    PubMed

    Gdynia, Georg; Sauer, Sven W; Kopitz, Jürgen; Fuchs, Dominik; Duglova, Katarina; Ruppert, Thorsten; Miller, Matthias; Pahl, Jens; Cerwenka, Adelheid; Enders, Markus; Mairbäurl, Heimo; Kamiński, Marcin M; Penzel, Roland; Zhang, Christine; Fuller, Jonathan C; Wade, Rebecca C; Benner, Axel; Chang-Claude, Jenny; Brenner, Hermann; Hoffmeister, Michael; Zentgraf, Hanswalter; Schirmacher, Peter; Roth, Wilfried

    2016-01-01

    The high-mobility group box 1 (HMGB1) protein has a central role in immunological antitumour defense. Here we show that natural killer cell-derived HMGB1 directly eliminates cancer cells by triggering metabolic cell death. HMGB1 allosterically inhibits the tetrameric pyruvate kinase isoform M2, thus blocking glucose-driven aerobic respiration. This results in a rapid metabolic shift forcing cells to rely solely on glycolysis for the maintenance of energy production. Cancer cells can acquire resistance to HMGB1 by increasing glycolysis using the dimeric form of PKM2, and employing glutaminolysis. Consistently, we observe an increase in the expression of a key enzyme of glutaminolysis, malic enzyme 1, in advanced colon cancer. Moreover, pharmaceutical inhibition of glutaminolysis sensitizes tumour cells to HMGB1 providing a basis for a therapeutic strategy for treating cancer. PMID:26948869

  11. The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration

    PubMed Central

    Gdynia, Georg; Sauer, Sven W.; Kopitz, Jürgen; Fuchs, Dominik; Duglova, Katarina; Ruppert, Thorsten; Miller, Matthias; Pahl, Jens; Cerwenka, Adelheid; Enders, Markus; Mairbäurl, Heimo; Kamiński, Marcin M.; Penzel, Roland; Zhang, Christine; Fuller, Jonathan C.; Wade, Rebecca C.; Benner, Axel; Chang-Claude, Jenny; Brenner, Hermann; Hoffmeister, Michael; Zentgraf, Hanswalter; Schirmacher, Peter; Roth, Wilfried

    2016-01-01

    The high-mobility group box 1 (HMGB1) protein has a central role in immunological antitumour defense. Here we show that natural killer cell-derived HMGB1 directly eliminates cancer cells by triggering metabolic cell death. HMGB1 allosterically inhibits the tetrameric pyruvate kinase isoform M2, thus blocking glucose-driven aerobic respiration. This results in a rapid metabolic shift forcing cells to rely solely on glycolysis for the maintenance of energy production. Cancer cells can acquire resistance to HMGB1 by increasing glycolysis using the dimeric form of PKM2, and employing glutaminolysis. Consistently, we observe an increase in the expression of a key enzyme of glutaminolysis, malic enzyme 1, in advanced colon cancer. Moreover, pharmaceutical inhibition of glutaminolysis sensitizes tumour cells to HMGB1 providing a basis for a therapeutic strategy for treating cancer. PMID:26948869

  12. The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration.

    PubMed

    Gdynia, Georg; Sauer, Sven W; Kopitz, Jürgen; Fuchs, Dominik; Duglova, Katarina; Ruppert, Thorsten; Miller, Matthias; Pahl, Jens; Cerwenka, Adelheid; Enders, Markus; Mairbäurl, Heimo; Kamiński, Marcin M; Penzel, Roland; Zhang, Christine; Fuller, Jonathan C; Wade, Rebecca C; Benner, Axel; Chang-Claude, Jenny; Brenner, Hermann; Hoffmeister, Michael; Zentgraf, Hanswalter; Schirmacher, Peter; Roth, Wilfried

    2016-03-07

    The high-mobility group box 1 (HMGB1) protein has a central role in immunological antitumour defense. Here we show that natural killer cell-derived HMGB1 directly eliminates cancer cells by triggering metabolic cell death. HMGB1 allosterically inhibits the tetrameric pyruvate kinase isoform M2, thus blocking glucose-driven aerobic respiration. This results in a rapid metabolic shift forcing cells to rely solely on glycolysis for the maintenance of energy production. Cancer cells can acquire resistance to HMGB1 by increasing glycolysis using the dimeric form of PKM2, and employing glutaminolysis. Consistently, we observe an increase in the expression of a key enzyme of glutaminolysis, malic enzyme 1, in advanced colon cancer. Moreover, pharmaceutical inhibition of glutaminolysis sensitizes tumour cells to HMGB1 providing a basis for a therapeutic strategy for treating cancer.

  13. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells

    PubMed Central

    Suzuki, Atsushi; Raya, Ángel; Kawakami, Yasuhiko; Morita, Masanobu; Matsui, Takaaki; Nakashima, Kinichi; Gage, Fred H.; Rodríguez-Esteban, Concepción; Izpisúa Belmonte, Juan Carlos

    2006-01-01

    ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cell-specific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process. PMID:16801560

  14. Design of Protein-Protein Interactions with a Novel Ensemble-Based Scoring Algorithm

    NASA Astrophysics Data System (ADS)

    Roberts, Kyle E.; Cushing, Patrick R.; Boisguerin, Prisca; Madden, Dean R.; Donald, Bruce R.

    Protein-protein interactions (PPIs) are vital for cell signaling, protein trafficking and localization, gene expression, and many other biological functions. Rational modification of PPI targets provides a mechanism to understand their function and importance. However, PPI systems often have many more degrees of freedom and flexibility than the small-molecule binding sites typically targeted by protein design algorithms. To handle these challenging design systems, we have built upon the computational protein design algorithm K * [8,19] to develop a new design algorithm to study protein-protein and protein-peptide interactions. We validated our algorithm through the design and experimental testing of novel peptide inhibitors.

  15. A physical interaction network of dengue virus and human proteins.

    PubMed

    Khadka, Sudip; Vangeloff, Abbey D; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J; Perera, Rushika; LaCount, Douglas J

    2011-12-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

  16. Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

    PubMed

    Lee, Hong-Won; Ryu, Ji Young; Yoo, Janghyun; Choi, Byungsan; Kim, Kipom; Yoon, Tae-Young

    2013-10-01

    Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

  17. Detection and quantification of protein-protein interactions by far-western blotting.

    PubMed

    Jadwin, Joshua A; Mayer, Bruce J; Machida, Kazuya

    2015-01-01

    Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

  18. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms"). PMID:26871637

  19. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").

  20. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network

    PubMed Central

    Hu, Yang; Zhang, Ying; Ren, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence.

  1. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network.

    PubMed

    Hu, Yang; Zhang, Ying; Ren, Jun; Wang, Yadong; Wang, Zhenzhen; Zhang, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence. PMID:27648447

  2. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network

    PubMed Central

    Hu, Yang; Zhang, Ying; Ren, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence. PMID:27648447

  3. Structural study of surfactant-dependent interaction with protein

    SciTech Connect

    Mehan, Sumit; Aswal, Vinod K.; Kohlbrecher, Joachim

    2015-06-24

    Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

  4. Structural study of surfactant-dependent interaction with protein

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, Vinod K.; Kohlbrecher, Joachim

    2015-06-01

    Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

  5. Targeting the K-Ras/PDEδ protein-protein interaction: the solution for Ras-driven cancers or just another therapeutic mirage?

    PubMed

    Frett, Brendan; Wang, Yuanxiang; Li, Hong-Yu

    2013-10-01

    The holy grail, finally? After years of unsuccessful attempts at drugging the Ras oncogene, a recent paper by Zimmerman et al. has revealed the possibility of inhibiting Ras signaling on a clinically relevant level by blocking the K-Ras/PDEδ protein-protein interaction. The results, reported in Nature, are highlighted herein with future implications and directions to evaluate the full clinical potential of this research. PMID:23939923

  6. Dual stimuli-responsive coating designed through layer-by-layer assembly of PAA-b-PNIPAM block copolymers for the control of protein adsorption.

    PubMed

    Osypova, A; Magnin, D; Sibret, P; Aqil, A; Jérôme, C; Dupont-Gillain, C; Pradier, C-M; Demoustier-Champagne, S; Landoulsi, J

    2015-11-01

    In this paper, we describe the successful construction, characteristics and interaction with proteins of stimuli-responsive thin nanostructured films prepared by layer-by-layer (LbL) sequential assembly of PNIPAM-containing polyelectrolytes and PAH. PAA-b-PNIPAM block copolymers were synthesized in order to benefit from (i) the ionizable properties of PAA, to be involved in the LbL assembly, and (ii) the sensitivity of PNIPAM to temperature stimulus. The impact of parameters related to the structure and size of the macromolecules (their molecular weight and the relative degree of polymerization of PAA and PNIPAM), and the interaction with proteins under physico-chemical stimuli, such as pH and temperature, are carefully investigated. The incorporation of PAA-b-PNIPAM into multilayered films is shown to be successful whatever the block copolymer used, resulting in slightly thicker films than the corresponding (PAA/PAH)n film. Importantly, the protein adsorption studies demonstrate that it is possible to alter the adsorption behavior of proteins on (PAA-b-PNIPAM/PAH)n surfaces by varying the temperature and/or the pH of the medium, which seems to be intimately related to two key factors: (i) the ability of PNIPAM units to undergo conformational changes and (ii) the structural changes of the film made of weak polyelectrolytes. The simplicity of construction of these PNIPAM block copolymer-based LbL coatings on a large range of substrates, combined with their highly tunable features, make them ideal candidates to be employed for various biomedical applications requiring the control of protein adsorption.

  7. Dual stimuli-responsive coating designed through layer-by-layer assembly of PAA-b-PNIPAM block copolymers for the control of protein adsorption.

    PubMed

    Osypova, A; Magnin, D; Sibret, P; Aqil, A; Jérôme, C; Dupont-Gillain, C; Pradier, C-M; Demoustier-Champagne, S; Landoulsi, J

    2015-11-01

    In this paper, we describe the successful construction, characteristics and interaction with proteins of stimuli-responsive thin nanostructured films prepared by layer-by-layer (LbL) sequential assembly of PNIPAM-containing polyelectrolytes and PAH. PAA-b-PNIPAM block copolymers were synthesized in order to benefit from (i) the ionizable properties of PAA, to be involved in the LbL assembly, and (ii) the sensitivity of PNIPAM to temperature stimulus. The impact of parameters related to the structure and size of the macromolecules (their molecular weight and the relative degree of polymerization of PAA and PNIPAM), and the interaction with proteins under physico-chemical stimuli, such as pH and temperature, are carefully investigated. The incorporation of PAA-b-PNIPAM into multilayered films is shown to be successful whatever the block copolymer used, resulting in slightly thicker films than the corresponding (PAA/PAH)n film. Importantly, the protein adsorption studies demonstrate that it is possible to alter the adsorption behavior of proteins on (PAA-b-PNIPAM/PAH)n surfaces by varying the temperature and/or the pH of the medium, which seems to be intimately related to two key factors: (i) the ability of PNIPAM units to undergo conformational changes and (ii) the structural changes of the film made of weak polyelectrolytes. The simplicity of construction of these PNIPAM block copolymer-based LbL coatings on a large range of substrates, combined with their highly tunable features, make them ideal candidates to be employed for various biomedical applications requiring the control of protein adsorption. PMID:26338028

  8. Inhibition of the p53/hDM2 protein-protein interaction by cyclometallated iridium(III) compounds

    PubMed Central

    Liu, Li-Juan; He, Bingyong; Miles, Jennifer A.; Wang, Wanhe; Mao, Zhifeng; Che, Weng Ian; Lu, Jin-Jian; Chen, Xiu-Ping; Wilson, Andrew J.; Ma, Dik-Lung; Leung, Chung-Hang

    2016-01-01

    Inactivation of the p53 transcription factor by mutation or other mechanisms is a frequent event in tumorigenesis. One of the major endogenous negative regulators of p53 in humans is hDM2, a ubiquitin E3 ligase that binds to p53 causing proteasomal p53 degradation. In this work, a library of organometallic iridium(III) compounds were synthesized and evaluated for their ability to disrupt the p53/hDM2 protein-protein interaction. The novel cyclometallated iridium(III) compound 1 [Ir(eppy)2(dcphen)](PF6) (where eppy = 2-(4-ethylphenyl)pyridine and dcphen = 4, 7-dichloro-1, 10-phenanthroline) blocked the interaction of p53/hDM2 in human amelanotic melanoma cells. Finally, 1 exhibited anti-proliferative activity and induced apoptosis in cancer cell lines consistent with inhibition of the p53/hDM2 interaction. Compound 1 represents the first reported organometallic p53/hDM2 protein-protein interaction inhibitor. PMID:26883110

  9. Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Gao, Yuan; Yu, Li-Rong; Alla, Ramani; Shmookler Reis, Robert

    2015-01-01

    Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly

  10. Computational biology for target discovery and characterization: a feasibility study in protein-protein interaction detection

    SciTech Connect

    Zhou, C; Zemla, A

    2009-02-25

    In this work we developed new code for detecting putative multi-domain protein-protein interactions for a small network of bacterial pathogen proteins, and determined how structure-driven domain-fusion (DF) methods should be scaled up for whole-proteome analysis. Protein-protein interactions are of great interest in structural biology and are important for understanding the biology of pathogens. The ability to predict protein-protein interactions provides a means for development of anti-microbials that may interfer with key processes in pathogenicity. The function of a protein-protein complex can be elucidated through knowledge of its structure. The overall goal of this project was to determine the feasibility of extending current LLNL capabilities to produce a high-throughput systems bio-informatics capability for identification and characterization of putative interacting protein partners within known or suspected small protein networks. We extended an existing LLNL methodology for identification of putative protein-protein interacting partners (Chakicherla et al (in review)) by writing a new code to identify multi-domain-fusion linkages (3 or more per complex). We applied these codes to the proteins in the Yersinia pestis quorum sensing network, known as the lsr operon, which comprises a virulence mechanism in this pathogen. We determined that efficient application of our computational algorithms in high-throughput for detection of putative protein-protein complexes genome wide would require pre-computation of PDB domains and construction of a domain-domain association database.

  11. Making the LINC: SUN and KASH protein interactions

    PubMed Central

    Kim, Dae In; Birendra, KC; Roux, Kyle J.

    2015-01-01

    Cell nuclei are physically integrated with the cytoskeleton through the LINC complex (for LInker of Nucleoskeleton and Cytoskeleton), a structure that spans the nuclear envelope to link the nucleoskeleton and cytoskeleton. Outer nuclear membrane KASH domain proteins and inner nuclear membrane SUN domain proteins interact to form the core of the LINC complex. In this review we provide a comprehensive analysis of the reported protein-protein interactions for KASH and SUN domain proteins. This critical structure, directly connecting the genome with the rest of the cell, contributes to a myriad of cellular functions and, when perturbed, is associated with human disease. PMID:25720065

  12. Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins.

    PubMed

    Wang, Li; Xu, Yuan; Zhou, Sumei; Qian, Haifeng; Zhang, Hui; Qi, Xiguang; Fan, Meihua

    2016-03-01

    In this study, we investigated the interaction of Vaccinium bracteatum Thunb. leaf (VBTL) pigment and rice proteins. In the presence of rice protein, VBTL pigment antioxidant activity and free polyphenol content decreased by 67.19% and 68.11%, respectively, and L(∗) of the protein-pigment complex decreased significantly over time. L(∗) values of albumin, globulin and glutelin during 60-min pigment exposure decreased by 55.00, 57.14, and 54.30%, respectively, indicating that these proteins had bound to the pigment. A significant difference in protein surface hydrophobicity was observed between rice proteins and pigment-protein complexes, indicating that hydrophobic interaction is a major binding mechanism between VBTL pigment and rice proteins. A significant difference in secondary structures between proteins and protein-pigment complexes was also uncovered, indicating that hydrogen bonding may be another mode of interaction between VBTL pigment and rice proteins. Our results indicate that VBTL pigment can stain rice proteins with hydrophobic and hydrogen interactions. PMID:26471554

  13. PPLook: an automated data mining tool for protein-protein interaction

    PubMed Central

    2010-01-01

    Background Extracting and visualizing of protein-protein interaction (PPI) from text literatures are a meaningful topic in protein science. It assists the identification of interactions among proteins. There is a lack of tools to extract PPI, visualize and classify the results. Results We developed a PPI search system, termed PPLook, which automatically extracts and visualizes protein-protein interaction (PPI) from text. Given a query protein name, PPLook can search a dataset for other proteins interacting with it by using a keywords dictionary pattern-matching algorithm, and display the topological parameters, such as the number of nodes, edges, and connected components. The visualization component of PPLook enables us to view the interaction relationship among the proteins in a three-dimensional space based on the OpenGL graphics interface technology. PPLook can also provide the functions of selecting protein semantic class, counting the number of semantic class proteins which interact with query protein, counting the literature number of articles appearing the interaction relationship about the query protein. Moreover, PPLook provides heterogeneous search and a user-friendly graphical interface. Conclusions PPLook is an effective tool for biologists and biosystem developers who need to access PPI information from the literature. PPLook is freely available for non-commercial users at http://meta.usc.edu/softs/PPLook. PMID:20550717

  14. An ontology-based search engine for protein-protein interactions

    PubMed Central

    2010-01-01

    Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

  15. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

    2002-10-15

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  16. Searching the MINT database for protein interaction information.

    PubMed

    Cesareni, Gianni; Chatr-aryamontri, Andrew; Licata, Luana; Ceol, Arnaud

    2008-06-01

    The Molecular Interactions Database (MINT) is a relational database designed to store information about protein interactions. Expert curators extract the relevant information from the scientific literature and deposit it in a computer readable form. Currently (April 2008), MINT contains information on almost 29,000 proteins and more than 100,000 interactions from more than 30 model organisms. This unit provides protocols for searching MINT over the Internet, using the MINT Viewer.

  17. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  18. Cyclic Limulus anti-lipopolysaccharide (LPS) factor-derived peptide CLP-19 antagonizes LPS function by blocking binding to LPS binding protein.

    PubMed

    Liu, Yao; Ni, Bing; Ren, Jian-dong; Chen, Jian-hong; Tian, Zhi-qiang; Tang, Min; Li, Di; Xia, Peiyuan

    2011-01-01

    Inflammation and septic shock due to endotoxins from Gram-negative bacteria infection continue to pose significant challenges to human healthcare. It is, therefore, necessary to develop therapeutic strategies targeting endotoxins, such as lipopolysaccharide (LPS), to prevent their potentially systemic effects. Pathogenesis due to Gram-negative bacteria involves LPS binding to the host LPS-binding protein (LBP), causing detrimental downstream signaling cascades. Our previous study showed that CLP-19, a synthetic peptide derived from the Limulus anti-LPS factor (LALF), could effectively neutralize LPS toxicity; however, the detailed mechanisms underlying this anti-LPS effect remained unexplained. Thus, we carried out investigations to determine how the CLP-19 neutralizes LPS toxicity. CLP-19 was found to block LPS binding to LBP in a dose-dependent manner, as evidenced by competitive enzyme-linked immunosorbent assay (ELISA). In peripheral blood mononuclear cells, CLP-19 blocked LPS-induced phosphorylation of mitogen activated protein kinase (MAPK) signaling proteins p38, extracellular signal-regulating kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2. Furthermore, CLP-19 potency in LPS antagonism in vitro and in vivo was directly associated with its ability to block the LPS-LBP interaction. Taken together, the results suggested that CLP-19's inhibitory effect on LPS-LBP binding and on the subsequent MAPK pathway signaling may be responsible for its anti-LPS mechanism. This peptide appears to represent a potential therapeutic agent for clinical treatment of sepsis. PMID:22040879

  19. Filamin A Protein Interacts with Human Immunodeficiency Virus Type 1 Gag Protein and Contributes to Productive Particle Assembly*

    PubMed Central

    Cooper, JoAnn; Liu, Ling; Woodruff, Elvin A.; Taylor, Harry E.; Goodwin, J. Shawn; D'Aquila, Richard T.; Spearman, Paul; Hildreth, James E. K.; Dong, Xinhong

    2011-01-01

    HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection. PMID:21705339

  20. Protein--nanoparticle interaction: identification of the ubiquitin--gold nanoparticle interaction site.

    PubMed

    Calzolai, Luigi; Franchini, Fabio; Gilliland, Douglas; Rossi, François

    2010-08-11

    We demonstrate that it is possible to identify the protein--nanoparticle interaction site at amino acid scale in solution. Using NMR, chemical shift perturbation analysis, and dynamic light scattering we have identified a specific domain of human ubiquitin that interacts with gold nanoparticles. This method allows a detailed structural analysis of proteins absorbed onto surfaces of nanoparticles in physiological conditions and it will provide much needed experimental data for better modeling and prediction of protein--nanoparticle interactions. PMID:20698623

  1. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  2. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    PubMed

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species. PMID:25787689

  3. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    PubMed

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species.

  4. Information-driven structural modelling of protein-protein interactions.

    PubMed

    Rodrigues, João P G L M; Karaca, Ezgi; Bonvin, Alexandre M J J

    2015-01-01

    Protein-protein docking aims at predicting the three-dimensional structure of a protein complex starting from the free forms of the individual partners. As assessed in the CAPRI community-wide experiment, the most successful docking algorithms combine pure laws of physics with information derived from various experimental or bioinformatics sources. Of these so-called "information-driven" approaches, HADDOCK stands out as one of the most successful representatives. In this chapter, we briefly summarize which experimental information can be used to drive the docking prediction in HADDOCK, and then focus on the docking protocol itself. We discuss and illustrate with a tutorial example a "classical" protein-protein docking prediction, as well as more recent developments for modelling multi-body systems and large conformational changes. PMID:25330973

  5. Lead-protein interactions as a basis for lead toxicity.

    PubMed

    Goering, P L

    1993-01-01

    The interaction of lead (Pb) with proteins may represent a fundamental mechanism by which Pb exerts toxicity. In this overview, various factors which influence the interaction of Pb with proteins will be discussed. Pb interacts with enzyme functional groups, and high-affinity metal-binding proteins, such as Pb-binding proteins and metallothioneins, can mediate this Pb-enzyme interaction. Many other factors influence Pb-protein interactions including ligand competition and binding affinities; protein folding and the nature of the metal-binding site; rates of protein synthesis and degradation; and intracellular localization of the ligand and metal. The remainder of this overview will focus on specific examples of important proteins known to be influenced by Pb or which hypothetically may be influenced by Pb. Gaps in knowledge and important research needs are emphasized. Many of the factors discussed play a role in the relative sensitivity of various enzymes in heme biosynthesis to Pb. Disruption of this critical pathway by Pb may result in neuropathologies and accumulation of neurotoxic heme precursors. High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase. Knowledge of regional localization in brain and the postnatal ontogeny of the high-affinity metal-binding proteins may be pivotal in understanding Pb neurotoxicity. Other specific examples related to or potentially related to Pb toxicity which are discussed include nucleic acid binding proteins, calmodulin, protein kinase C, and carbonic anhydrase. These proteins will serve as models to understand some basic principles and differences in Pb-protein interactions.

  6. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins.

    PubMed

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  7. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  8. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

    PubMed

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping

  9. Arctigenin blocks the unfolded protein response and shows therapeutic antitumor activity.

    PubMed

    Kim, Ju-Young; Hwang, Ji-Hwan; Cha, Mi-Ran; Yoon, Mi-Young; Son, Eun-Soon; Tomida, Akihiro; Ko, Bosung; Song, Si-Whan; Shin-ya, Kazuo; Hwang, Yong-il; Park, Hae-Ryong

    2010-07-01

    Cancer cells in poorly vascularized solid tumors are constantly or intermittently exposed to stressful microenvironments, including glucose deprivation, hypoxia, and other forms of nutrient starvation. These tumor-specific conditions, especially glucose deprivation, activate a signaling pathway called the unfolded protein response (UPR), which enhances cell survival by induction of the stress proteins. We have established a screening method to discover anticancer agents that could preferentially inhibit tumor cell viability under glucose-deprived conditions. Here we identify arctigenin (ARC-G) as an active compound that shows selective cytotoxicity and inhibits the UPR during glucose deprivation. Indeed, ARC-G blocked expression of UPR target genes such as phosphorylated-PERK, ATF4, CHOP, and GRP78, which was accompanied by enhanced phosphorylation of eIF2 alpha during glucose deprivation. The UPR inhibition led to apoptosis involving a mitochondrial pathway by activation of caspase-9 and -3. Furthermore, ARC-G suppressed tumor growth of colon cancer HT-29 xenografts. Our results demonstrate that ARC-G can be served as a novel type of antitumor agent targeting the UPR in glucose-deprived solid tumors.

  10. Magnetically Directed Two-Dimensional Crystallization of OmpF Membrane Proteins in Block Copolymers.

    PubMed

    Klara, Steven S; Saboe, Patrick O; Sines, Ian T; Babaei, Mahnoush; Chiu, Po-Lin; DeZorzi, Rita; Dayal, Kaushik; Walz, Thomas; Kumar, Manish; Mauter, Meagan S

    2016-01-13

    Two-dimensional (2D) alignment and crystallization of membrane proteins (MPs) is increasingly important in characterizing their three-dimensional (3D) structure, in designing pharmacological agents, and in leveraging MPs for biomimetic devices. Large, highly ordered MP 2D crystals in block copolymer (BCP) matrices are challenging to fabricate, but a facile and scalable technique for aligning and crystallizing MPs in thin-film geometries would rapidly translate into applications. This work introduces a novel method to grow larger and potentially better ordered 2D crystals by performing the crystallization process in the presence of a strong magnetic field. We demonstrate the efficacy of this approach using a β-barrel MP, outer membrane protein F (OmpF), in short-chain polybutadiene-poly(ethylene oxide) (PB-PEO) membranes. Crystals grown in a magnetic field were up to 5 times larger than conventionally grown crystals, and a signal-to-noise (SNR) analysis of diffraction peaks in Fourier transforms of specimens imaged by negative-stain electron microscopy (EM) and cryo-EM showed twice as many high-SNR diffraction peaks, indicating that the magnetic field also improves crystal order. PMID:26677866

  11. Surface array proteins of Campylobacter fetus block lectin-mediated binding to type A lipopolysaccharide.

    PubMed Central

    Fogg, G C; Yang, L Y; Wang, E; Blaser, M J

    1990-01-01

    Campylobacter fetus strains with type A lipopolysaccharide (LPS) and a surface array protein layer (S+) have been found to be pathogenic in humans and animals. Spontaneous laboratory mutants that lack surface array proteins (S-) are sensitive to the bactericidal activity of normal human serum. The ability of lectins to determine the presence of the S-layer and differentiate LPS type was assessed. We screened 14 lectins and found 3 (wheat germ agglutinin, Bandeiraea simplicifolia II, and Helix pomatia agglutinin) that agglutinated S- C. fetus strains with type A LPS but not S- strains with type B or type C LPS or S+ strains. However, the S+ type A strains were agglutinated after sequential water extraction, heat, or pronase treatment, all of which remove the S-layer, whereas there was no effect on the control strains. Specific carbohydrates for each lectin and purified LPS from a type A C. fetus strain specifically inhibited agglutination of an S- type A strain. In a direct enzyme-linked lectin assay, binding to the S- type A LPS strain was significantly greater than binding to the S+ strain (P = 0.01) or to a Campylobacter jejuni strain (P = 0.008). Consequently, these results indicate that the three lectins bind to the O side chains of C. fetus type A LPS but that the presence of the S-layer on intact cells blocks binding. Images PMID:2387622

  12. Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

    SciTech Connect

    Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

    2008-03-14

    Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3{beta} (GSK-3{beta}), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3{beta}, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3{beta}, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

  13. Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting Dectin-1/syk kinase signaling

    PubMed Central

    Kyrmizi, Irene; Gresnigt, Mark S.; Akoumianaki, Tonia; Samonis, George; Sidiropoulos, Prodromos; Boumpas, Dimitrios; Netea, Mihai G; van de Veerdonk, Frank L; Kontoyiannis, Dimitrios P; Chamilos, Georgios

    2013-01-01

    Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease; CGD) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link Toll-like receptor activation with phagosome maturation via the engagement of autophagy proteins. Herein, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus-phagosomes required syk kinase-dependent production of reactive oxygen species (ROS) and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In-vivo and ex-vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of syk kinase phosphorylation and downstream production of ROS. Our studies link Dectin-1/syk kinase signaling with maturation of A. fumigatus phagosomes and uncover a mechanism for development of invasive fungal disease. PMID:23817424

  14. Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors.

    PubMed

    Sable, Rushikesh; Jois, Seetharama

    2015-01-01

    Blocking protein-protein interactions (PPI) using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

  15. The role of electrostatics in protein-protein interactions of a monoclonal antibody.

    PubMed

    Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

    2014-07-01

    Understanding how protein-protein interactions depend on the choice of buffer, salt, ionic strength, and pH is needed to have better control over protein solution behavior. Here, we have characterized the pH and ionic strength dependence of protein-protein interactions in terms of an interaction parameter kD obtained from dynamic light scattering and the osmotic second virial coefficient B22 measured by static light scattering. A simplified protein-protein interaction model based on a Baxter adhesive potential and an electric double layer force is used to separate out the contributions of longer-ranged electrostatic interactions from short-ranged attractive forces. The ionic strength dependence of protein-protein interactions for solutions at pH 6.5 and below can be accurately captured using a Deryaguin-Landau-Verwey-Overbeek (DLVO) potential to describe the double layer forces. In solutions at pH 9, attractive electrostatics occur over the ionic strength range of 5-275 mM. At intermediate pH values (7.25 to 8.5), there is a crossover effect characterized by a nonmonotonic ionic strength dependence of protein-protein interactions, which can be rationalized by the competing effects of long-ranged repulsive double layer forces at low ionic strength and a shorter ranged electrostatic attraction, which dominates above a critical ionic strength. The change of interactions from repulsive to attractive indicates a concomitant change in the angular dependence of protein-protein interaction from isotropic to anisotropic. In the second part of the paper, we show how the Baxter adhesive potential can be used to predict values of kD from fitting to B22 measurements, thus providing a molecular basis for the linear correlation between the two protein-protein interaction parameters.

  16. Screening for protein-DNA interactions by automatable DNA-protein interaction ELISA.

    PubMed

    Brand, Luise H; Henneges, Carsten; Schüssler, Axel; Kolukisaoglu, H Üner; Koch, Grit; Wallmeroth, Niklas; Hecker, Andreas; Thurow, Kerstin; Zell, Andreas; Harter, Klaus; Wanke, Dierk

    2013-01-01

    DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding

  17. Screening for Protein-DNA Interactions by Automatable DNA-Protein Interaction ELISA

    PubMed Central

    Schüssler, Axel; Kolukisaoglu, H. Üner; Koch, Grit; Wallmeroth, Niklas; Hecker, Andreas; Thurow, Kerstin; Zell, Andreas; Harter, Klaus; Wanke, Dierk

    2013-01-01

    DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding

  18. Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

    PubMed Central

    Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

    2016-01-01

    To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

  19. Molecular transport through channels and pores: Effects of in-channel interactions and blocking

    PubMed Central

    Bauer, Wolfgang R.; Nadler, Walter

    2006-01-01

    Facilitated translocation of molecules through channels and pores is of fundamental importance for transmembrane transport in biological systems. Several such systems have specific binding sites inside the channel, but a clear understanding of how the interaction between channel and molecules affects the flow is still missing. We present a generic analytical treatment of the problem that relates molecular flow to the first passage time across and the number of particles inside the channel. Both quantities depend in different ways on the channel properties. For the idealized case of noninteracting molecules, we find an increased flow whenever there is a binding site in the channel, despite an increased first passage time. In the more realistic case that molecules may block the channel, we find an increase of flow only up to a certain threshold value of the binding strength and a dependence on the sign of the concentration gradient, i.e., asymmetric transport. The optimal binding strength in that case is analyzed. In all cases the reason for transport facilitation is an increased occupation probability of a particle inside the channel that overcomes any increase in the first passage time because of binding. PMID:16861303

  20. Molecular transport through channels and pores: effects of in-channel interactions and blocking.

    PubMed

    Bauer, Wolfgang R; Nadler, Walter

    2006-08-01

    Facilitated translocation of molecules through channels and pores is of fundamental importance for transmembrane transport in biological systems. Several such systems have specific binding sites inside the channel, but a clear understanding of how the interaction between channel and molecules affects the flow is still missing. We present a generic analytical treatment of the problem that relates molecular flow to the first passage time across and the number of particles inside the channel. Both quantities depend in different ways on the channel properties. For the idealized case of noninteracting molecules, we find an increased flow whenever there is a binding site in the channel, despite an increased first passage time. In the more realistic case that molecules may block the channel, we find an increase of flow only up to a certain threshold value of the binding strength and a dependence on the sign of the concentration gradient, i.e., asymmetric transport. The optimal binding strength in that case is analyzed. In all cases the reason for transport facilitation is an increased occupation probability of a particle inside the channel that overcomes any increase in the first passage time because of binding.

  1. Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function1

    PubMed Central

    Jaiswal, Aruna S; Armas, Melissa L; Izumi, Tadahide; Strauss, Phyllis R; Narayan, Satya

    2012-01-01

    In previous studies, we found that adenomatous polyposis coli (APC) blocks the base excision repair (BER) pathway by interacting with 5′-flap endonuclease 1 (Fen1). In this study, we identify the molecular features that contribute to the formation and/or stabilization of the APC/Fen1 complex that determines the extent of BER inhibition, and the subsequent accumulation of DNA damage creates mutagenic lesions leading to transformation susceptibility. We show here that APC binds to the nuclear localization sequence of Fen1 (Lys365Lys366Lys367), which prevents entry of Fen1 into the nucleus and participation in Pol-β-directed long-patch BER. We also show that levels of the APC/Fen1 complex are higher in breast tumors than in the surrounding normal tissues. These studies demonstrate a novel role for APC in the suppression of Fen1 activity in the BER pathway and a new biomarker profile to be explored to identify individuals who may be susceptible to the development of mammary and other tumors. PMID:22787431

  2. Molecular interactions and solubilization of structurally related meso-porphyrin photosensitizers by amphiphilic block copolymers (Pluronics).

    PubMed

    Sobczyński, Jan; Smistad, Gro; Hegge, Anne Bee; Kristensen, Solveig

    2015-01-01

    The influence of four Pluronics block copolymers (i.e. F68, P123, F127, and L44) on the aggregation and solubilization of five structurally related meso-tetraphenyl porphyrin photosensitizers (PS) as model compounds for use in Photodynamic Therapy of cancer (PDT) was evaluated. Interactions between the PSs and Pluronics were studied at micromolar concentration by means of UV-Vis absorption spectrometry and by kinematic viscosity (υ) and osmolarity measurements at millimolar concentrations. Pluronic micelles were characterized by size and zeta potential (ζ) measurements. The morphology of selected PS-Pluronic assemblies was studied by atomic force microscopy (AFM). While hydrophobic 5,10,15,20-Tetrakis(4-hydroxyphenyl) porphine (THPP) seemed to be solubilized in the Pluronic micellar cores, amphiphilic di(monoethanolammonium) meso-tetraphenyl porphine disulphonate (TPPS2a) was likely bound to the micellar palisade layer. Hydrophilic PSs like 5,10,15,20-Tetrakis (4-trimethylaniliniumphenyl) porphine (TAPP) seemed to form complexes with Pluronic unimers and to be distributed among the micellar coronas. TPPS2a aggregated into a network which could be broken at Pluronic concentration [Formula: see text] cmc, but would reconstitute in the presence of tonicity adjusting agents, e.g. sodium chloride (NaCl) or glucose. PMID:25027806

  3. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  4. Evolution of biomolecular networks: lessons from metabolic and protein interactions.

    PubMed

    Yamada, Takuji; Bork, Peer

    2009-11-01

    Despite only becoming popular at the beginning of this decade, biomolecular networks are now frameworks that facilitate many discoveries in molecular biology. The nodes of these networks are usually proteins (specifically enzymes in metabolic networks), whereas the links (or edges) are their interactions with other molecules. These networks are made up of protein-protein interactions or enzyme-enzyme interactions through shared metabolites in the case of metabolic networks. Evolutionary analysis has revealed that changes in the nodes and links in protein-protein interaction and metabolic networks are subject to different selection pressures owing to distinct topological features. However, many evolutionary constraints can be uncovered only if temporal and spatial aspects are included in the network analysis.

  5. Cannabinoid receptor 2 expression modulates Gβ(1)γ(2) protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1.

    PubMed

    Nagler, Marina; Palkowitsch, Lysann; Rading, Sebastian; Moepps, Barbara; Karsak, Meliha

    2016-01-01

    The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ. The Tctex-1-Gβγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gβγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.

  6. Protein-engineered block-copolymers as stem cell delivery vehicles

    NASA Astrophysics Data System (ADS)

    Heilshorn, Sarah

    2015-03-01

    Stem cell transplantation is a promising therapy for a myriad of debilitating diseases and injuries; however, current delivery protocols are inadequate. Transplantation by direct injection, which is clinically preferred for its minimal invasiveness, commonly results in less than 5% cell viability, greatly inhibiting clinical outcomes. We demonstrate that mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we show that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. Building on these fundamental studies, we have designed a reproducible, bio-resorbable, customizable hydrogel using protein-engineering technology. In our Mixing-Induced Two-Component Hydrogel (MITCH), network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the network crosslinking density, sol-gel phase behavior, and gel mechanics. This is in contrast to many other physical hydrogels, where predictable tuning of bulk mechanics from the molecular level remains elusive due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. Furthermore, the hydrogel network can be easily modified to deliver a variety of bioactive payloads including growth factors, peptide drugs, and hydroxyapatite nanoparticles. Through a series of in vitro and in vivo studies, we demonstrate that these materials may significantly improve transplanted stem cell retention and function.

  7. Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

    PubMed Central

    Gauliard, Emilie; Ouellette, Scot P.; Rueden, Kelsey J.; Ladant, Daniel

    2015-01-01

    Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins). Incs represent 7–10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH) method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF, and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia. PMID:25717440

  8. Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

    PubMed Central

    Andorf, Carson M.; Honavar, Vasant; Sen, Taner Z.

    2013-01-01

    Background Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners). Methodology Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. Conclusions Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. Availability We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu. PMID:23431393

  9. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    PubMed

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth. PMID:24773089

  10. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    PubMed

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  11. Stabilized helical peptides: a strategy to target protein-protein interactions.

    PubMed

    Klein, Mark A

    2014-08-14

    Protein-protein interactions are critical for cell proliferation, differentiation, and function. Peptides hold great promise for clinical applications focused on targeting protein-protein interactions. Advantages of peptides include a large chemical space and potential diversity of sequences and structures. However, peptides do present well-known challenges for drug development. Progress has been made in the development of stabilizing alpha helices for potential therapeutic applications. Advantages and disadvantages of different methods of helical peptide stabilization are discussed.

  12. Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

    PubMed Central

    DeBlasio, Stacy L.; Chavez, Juan D.; Alexander, Mariko M.; Ramsey, John; Eng, Jimmy K.; Mahoney, Jaclyn; Gray, Stewart M.; Bruce, James E.

    2015-01-01

    ABSTRACT Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection—hallmarks of host-pathogen interactions—were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCE The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used

  13. Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling.

    PubMed

    Xing, Qiong; Huang, Peng; Yang, Ju; Sun, Jian-Qiang; Gong, Zhou; Dong, Xu; Guo, Da-Chuan; Chen, Shao-Min; Yang, Yu-Hong; Wang, Yan; Yang, Ming-Hui; Yi, Ming; Ding, Yi-Ming; Liu, Mai-Li; Zhang, Wei-Ping; Tang, Chun

    2014-10-20

    Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a K(D)≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III)-based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration.

  14. Metabotropic Glutamate Receptors and Interacting Proteins in Epileptogenesis.

    PubMed

    Qian, Feng; Tang, Feng-Ru

    2016-01-01

    Neurotransmitter and receptor systems are involved in different neurological and neuropsychological disorders such as Parkinson's disease, depression, Alzheimer's disease and epilepsy. Recent advances in studies of signal transduction pathways or interacting proteins of neurotransmitter receptor systems suggest that different receptor systems may share the common signal transduction pathways or interacting proteins which may be better therapeutic targets for development of drugs to effectively control brain diseases. In this paper, we reviewed metabotropic glutamate receptors (mGluRs) and their related signal transduction pathways or interacting proteins in status epilepticus and temporal lobe epilepsy, and proposed some novel therapeutical drug targets for controlling epilepsy and epileptogenesis. PMID:27030135

  15. Discovering novel protein-protein interactions by measuring the protein semantic similarity from the biomedical literature.

    PubMed

    Chiang, Jung-Hsien; Ju, Jiun-Huang

    2014-12-01

    Protein-protein interactions (PPIs) are involved in the majority of biological processes. Identification of PPIs is therefore one of the key aims of biological research. Although there are many databases of PPIs, many other unidentified PPIs could be buried in the biomedical literature. Therefore, automated identification of PPIs from biomedical literature repositories could be used to discover otherwise hidden interactions. Search engines, such as Google, have been successfully applied to measure the relatedness among words. Inspired by such approaches, we propose a novel method to identify PPIs through semantic similarity measures among protein mentions. We define six semantic similarity measures as features based on the page counts retrieved from the MEDLINE database. A machine learning classifier, Random Forest, is trained using the above features. The proposed approach achieve an averaged micro-F of 71.28% and an averaged macro-F of 64.03% over five PPI corpora, an improvement over the results of using only the conventional co-occurrence feature (averaged micro-F of 68.79% and an averaged macro-F of 60.49%). A relation-word reinforcement further improves the averaged micro-F to 71.3% and averaged macro-F to 65.12%. Comparing the results of the current work with other studies on the AIMed corpus (ranging from 77.58% to 85.1% in micro-F, 62.18% to 76.27% in macro-F), we show that the proposed approach achieves micro-F of 81.88% and macro-F of 64.01% without the use of sophisticated feature extraction. Finally, we manually examine the newly discovered PPI pairs based on a literature review, and the results suggest that our approach could extract novel protein-protein interactions.

  16. Conductance and block of hair-cell mechanotransducer channels in transmembrane channel-like protein mutants.

    PubMed

    Beurg, Maryline; Kim, Kyunghee X; Fettiplace, Robert

    2014-07-01

    Transmembrane channel-like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This "reversed stimulus-polarity" current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel's large unitary conductance, high Ca(2+) permeability, and pharmacological profile, and that this vestibule

  17. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  18. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    SciTech Connect

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-08-12

    Highlights: {yields} Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. {yields} BAI2 interaction with GIP was revealed by yeast two-hybrid assay. {yields} Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. {yields} BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, {beta}-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  19. Protein-protein interactions between proteins of Citrus tristeza virus isolates.

    PubMed

    Nchongboh, Chofong Gilbert; Wu, Guan-Wei; Hong, Ni; Wang, Guo-Ping

    2014-12-01

    Citrus tristeza virus (CTV) is one of the most devastating pathogens of citrus. Its genome is organized into 12 open reading frames (ORFs), of which ten ORFs located at the 3'-terminus of the genome have multiple biological functions. The ten genes at the 3'-terminus of the genome of a severe isolate (CTV-S4) and three ORFs (CP, CPm and p20) of three other isolates (N4, S45 and HB1) were cloned into pGBKT7 and pGADT7 yeast shuttle vectors. Yeast two-hybridization (Y2H) assays results revealed a strong self-interaction for CP and p20, and a unique interaction between the CPm of CTV-S4 (severe) and CP of CTV-N4 (mild) isolates. Bimolecular fluorescence complementation also confirmed these interactions. Analysis of the deletion mutants delineated the domains of CP and p20 self-interaction. Furthermore, the domains responsible for CP and p20 self-interactions were mapped at the CP amino acids sites 41-84 and p20 amino acids sites 1-21 by Y2H. This study provided new information on CTV protein interactions which will help for further understanding the biological functions.

  20. Improved method for evaluating the dead volume and protein-protein interactions by self-interaction chromatography.

    PubMed

    Binabaji, Elaheh; Rao, Suma; Zydney, Andrew L

    2013-10-01

    Self-interaction chromatography (SIC) is a well-established method for studying protein-protein interactions. The second virial coefficient in SIC is evaluated directly from the measured retention coefficient for the protein using a column packed with resin on which the same protein has been immobilized on the pore surface. One of the challenges in determining the retention coefficient is the evaluation of the dead volume, which is the retention volume that would be measured for a noninteracting solute with the same effective size as the protein of interest. Previous studies of SIC have used a "dead column" packed with the same resin but without the immobilized protein to evaluate the dead volume, but this creates several experimental and theoretical challenges. We have developed a new approach using a dextran standard with effective size equal to that of the protein (as determined by size exclusion chromatography). The second virial coefficient was evaluated for a monoclonal antibody over a range of buffer conditions using this new approach. The data were in good agreement with independent measurements obtained by membrane osmometry under conditions dominated by repulsive interactions. The simplicity and accuracy of this method should facilitate the use of self-interaction chromatography for quantifying protein-protein interactions.

  1. A protein-protein interaction map of the Trypanosoma brucei paraflagellar rod.

    PubMed

    Lacomble, Sylvain; Portman, Neil; Gull, Keith

    2009-01-01

    We have conducted a protein interaction study of components within a specific sub-compartment of a eukaryotic flagellum. The trypanosome flagellum contains a para-crystalline extra-axonemal structure termed the paraflagellar rod (PFR) with around forty identified components. We have used a Gateway cloning approach coupled with yeast two-hybrid, RNAi and 2D DiGE to define a protein-protein interaction network taking place in this structure. We define two clusters of interactions; the first being characterised by two proteins with a shared domain which is not sufficient for maintaining the interaction. The other cohort is populated by eight proteins, a number of which possess a PFR domain and sub-populations of this network exhibit dependency relationships. Finally, we provide clues as to the structural organisation of the PFR at the molecular level. This multi-strand approach shows that protein interactome data can be generated for insoluble protein complexes. PMID:19888464

  2. Painting proteins blue: β-(1-azulenyl)-L-alanine as a probe for studying protein-protein interactions.

    PubMed

    Moroz, Yurii S; Binder, Wolfgang; Nygren, Patrik; Caputo, Gregory A; Korendovych, Ivan V

    2013-01-18

    We demonstrated that β-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

  3. Characterization of serum resistance of Neisseria gonorrhoeae that disseminate. Roles of blocking antibody and gonococcal outer membrane proteins.

    PubMed

    Rice, P A; Kasper, D L

    1982-07-01

    Neisseria gonorrhoeae isolated from patients with disseminated infection (DGI) often resist complement (C')-dependent killing by normal human serum (NHS) and less commonly by convalescent DGI serum. 7 of 10 NHS specimens completely inhibited killing of serum-resistant (ser(r)) gonococci by convalescent or immune DGI serum. Immunoglobulin G (IgG) purified from NHS was shown to be the blocking agent. In addition, IgM (plus C') purified from NHS was shown to be fivefold more effective (wt/wt) in killing serum-sensitive (ser(s)) gonococci than equivalent amounts of IgM tested in the presence of IgG (whole serum). Although inhibition of NHS killing of ser(s) gonococci required a 640% excess of IgG, only a 40% excess was required to block immune serum killing of ser(r) gonococci. F(ab')(2) prepared from IgG also blocked killing of ser(r) gonococci by immune serum indicating antigenic specificity of blocking IgG.IgG immunoconcentrated against outer membrane protein (OMP) derived from ser(r) gonococci showed 40-fold increased blocking activity over normal IgG (wt/wt) and lacked antibody activity directed against gonococcal lipopolysaccharide by ELISA. Using direct immunoabsorption of IgG with purified gonococcal OMP; ser(r)-OMP was found sixfold more effective than ser(s)-OMP in neutralizing the blocking of immune serum killing of ser(r) gonococci, and 10-fold more effective in systems that used excess blocking IgG, NHS, and ser(s) gonococci. Blocking IgG preabsorbed with whole ser(r) gonococci lost 75% of its ability to block immune serum killing compared with no loss in this system using a similar absorption with ser(s) gonococci. IgG purified from NHS contained fivefold higher titers of antibody against ser(r)-OMP than ser(s)-OMP by ELISA.

  4. Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.

    PubMed Central

    Heimburg, T; Marsh, D

    1995-01-01

    The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally

  5. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

    PubMed

    Eyckerman, Sven; Lemmens, Irma; Catteeuw, Dominiek; Verhee, Annick; Vandekerckhove, Joel; Lievens, Sam; Tavernier, Jan

    2005-06-01

    Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53. PMID:15908921

  6. Choosing negative examples for the prediction of protein-protein interactions

    PubMed Central

    Ben-Hur, Asa; Noble, William Stafford

    2006-01-01

    The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions. PMID:16723005

  7. A Yeast Two-Hybrid approach for probing protein-protein interactions at the centrosome

    PubMed Central

    Galletta, Brian J.; Rusan, Nasser M.

    2016-01-01

    As a large, non-membrane bound organelle, the centrosome must rely heavily on protein-protein interactions to assemble itself in the cytoplasm and perform its functions as a microtubule-organizing center. Therefore, to understand how this organelle is built and functions, one must understand the protein-protein interactions made by each centrosome protein. Unfortunately, the highly interconnected nature of the centrosome, combined with its predicted unstructured, coil-rich proteins, has made the use of many standard approaches to studying protein-protein interactions very challenging. The yeast-two hybrid (Y2H) system is well suited for studying the centrosome and is an important complement to other biochemical approaches. In this chapter we describe how to carry out a directed Y2H screen to identify the direct interactions between a given centrosome protein and a library of others. Specifically, we detail using a bioinformatics based approach (structure prediction programs) to subdivide proteins and screen for interactions using an array-based Y2H approach. We also describe how to use the interaction information garnered from this screen to generate mutations to disrupt specific interactions using mutagenic-PCR and a “reverse” Y2H screen. Finally, we discuss how information from such a screen can be integrated into existing models of centrosome assembly and how it can initiate and guide extensive in vitro and in vivo experimentation to test these models. PMID:26175443

  8. Protein function prediction using guilty by association from interaction networks.

    PubMed

    Piovesan, Damiano; Giollo, Manuel; Ferrari, Carlo; Tosatto, Silvio C E

    2015-12-01

    Protein function prediction from sequence using the Gene Ontology (GO) classification is useful in many biological problems. It has recently attracted increasing interest, thanks in part to the Critical Assessment of Function Annotation (CAFA) challenge. In this paper, we introduce Guilty by Association on STRING (GAS), a tool to predict protein function exploiting protein-protein interaction networks without sequence similarity. The assumption is that whenever a protein interacts with other proteins, it is part of the same biological process and located in the same cellular compartment. GAS retrieves interaction partners of a query protein from the STRING database and measures enrichment of the associated functional annotations to generate a sorted list of putative functions. A performance evaluation based on CAFA metrics and a fair comparison with optimized BLAST similarity searches is provided. The consensus of GAS and BLAST is shown to improve overall performance. The PPI approach is shown to outperform similarity searches for biological process and cellular compartment GO predictions. Moreover, an analysis of the best practices to exploit protein-protein interaction networks is also provided.

  9. Characterization of the interactions between protein and carbon black.

    PubMed

    Chen, Tzu-Tao; Chuang, Kai-Jen; Chiang, Ling-Ling; Chen, Chun-Chao; Yeh, Chi-Tai; Wang, Liang-Shun; Gregory, Clive; Jones, Tim; BéruBé, Kelly; Lee, Chun-Nin; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

    2014-01-15

    A considerable amount of studies have been conducted to investigate the interactions of biological fluids with nanoparticle surfaces, which exhibit a high affinity for proteins and particles. However, the mechanisms underlying these interactions have not been elucidated, particularly as they relate to human health. Using bovine serum albumin (BSA) and mice bronchoalveolar lavage fluid (BALF) as models for protein-particle conjugates, we characterized the physicochemical modifications of carbon blacks (CB) with 23nm or 65nm in diameter after protein treatment. Adsorbed BALF-containing proteins were quantified and identified by pathways, biological analyses and protein classification. Significant modifications of the physicochemistry of CB were induced by the addition of BSA. Enzyme modulators and hydrolase predominately interacted with CB, with protein-to-CB interactions that were associated with the coagulation pathways. Additionally, our results revealed that an acute-phase response could be activated by these proteins. With regard to human health, the present study revealed that the CB can react with proteins (∼55kDa and 70kDa) after inhalation and may modify the functional structures of lung proteins, leading to the activation of acute-inflammatory responses in the lungs. PMID:24291665

  10. Scalable prediction of compound-protein interactions using minwise hashing.

    PubMed

    Tabei, Yasuo; Yamanishi, Yoshihiro

    2013-01-01

    The identification of compound-protein interactions plays key roles in the drug development toward discovery of new drug leads and new therapeutic protein targets. There is therefore a strong incentive to develop new efficient methods for predicting compound-protein interactions on a genome-wide scale. In this paper we develop a novel chemogenomic method to make a scalable prediction of compound-protein interactions from heterogeneous biological data using minwise hashing. The proposed method mainly consists of two steps: 1) construction of new compact fingerprints for compound-protein pairs by an improved minwise hashing algorithm, and 2) application of a sparsity-induced classifier to the compact fingerprints. We test the proposed method on its ability to make a large-scale prediction of compound-protein interactions from compound substructure fingerprints and protein domain fingerprints, and show superior performance of the proposed method compared with the previous chemogenomic methods in terms of prediction accuracy, computational efficiency, and interpretability of the predictive model. All the previously developed methods are not computationally feasible for the full dataset consisting of about 200 millions of compound-protein pairs. The proposed method is expected to be useful for virtual screening of a huge number of compounds against many protein targets.

  11. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

    SciTech Connect

    Chowdhury, Saiful M.; Shi, Liang; Yoon, Hyunjin; Ansong, Charles; Rommereim, Leah M.; Norbeck, Angela D.; Auberry, Kenneth J.; Moore, R. J.; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.

    2009-02-10

    We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

  12. PPISEARCHENGINE: gene ontology-based search for protein-protein interactions.

    PubMed

    Park, Byungkyu; Cui, Guangyu; Lee, Hyunjin; Huang, De-Shuang; Han, Kyungsook

    2013-01-01

    This paper presents a new search engine called PPISearchEngine which finds protein-protein interactions (PPIs) using the gene ontology (GO) and the biological relations of proteins. For efficient retrieval of PPIs, each GO term is assigned a prime number and the relation between the terms is represented by the product of prime numbers. This representation is hidden from users but facilitates the search for the interactions of a query protein by unique prime factorisation of the number that represents the query protein. For a query protein, PPISearchEngine considers not only the GO term associated with the query protein but also the GO terms at the lower level than the GO term in the GO hierarchy, and finds all the interactions of the query protein which satisfy the search condition. In contrast, the standard keyword-matching or ID-matching search method cannot find the interactions of a protein unless the interactions involve a protein with explicit annotations. To the best of our knowledge, this search engine is the first method that can process queries like 'for protein p with GO [Formula: see text], find p's interaction partners with GO [Formula: see text]'. PPISearchEngine is freely available to academics at http://search.hpid.org/.

  13. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGES

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  14. The herpes simplex virus 1 US3 protein kinase blocks caspase-dependent double cleavage and activation of the proapoptotic protein BAD.

    PubMed

    Benetti, Luca; Munger, Joshua; Roizman, Bernard

    2003-06-01

    An earlier report showed that the U(S)3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the U(S)3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the U(S)3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the U(S)3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the U(S)3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the U(S)3 protein kinase. (iv) The U(S)3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M(r) of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of U(S)3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.

  15. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

    PubMed

    Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano

    2013-02-01

    Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

  16. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  17. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N P; Riedmayr, Lisa M; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  18. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  19. Biophysics of protein-DNA interactions and chromosome organization

    PubMed Central

    Marko, John F.

    2014-01-01

    The function of DNA in cells depends on its interactions with protein molecules, which recognize and act on base sequence patterns along the double helix. These notes aim to introduce basic polymer physics of DNA molecules, biophysics of protein-DNA interactions and their study in single-DNA experiments, and some aspects of large-scale chromosome structure. Mechanisms for control of chromosome topology will also be discussed. PMID:25419039

  20. Biophysics of protein-DNA interactions and chromosome organization

    NASA Astrophysics Data System (ADS)

    Marko, John F.

    2015-01-01

    The function of DNA in cells depends on its interactions with protein molecules, which recognize and act on base sequence patterns along the double helix. These notes aim to introduce basic polymer physics of DNA molecules, biophysics of protein-DNA interactions and their study in single-DNA experiments, and some aspects of large-scale chromosome structure. Mechanisms for control of chromosome topology will also be discussed.

  1. Proteins differentially interact with grapefruit furanocoumarins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapefruit juice (GFJ) interferes with the cytochrome P450 3A4 activity responsible for metabolizing certain medications. This interference is referred to as the "grapefruit-drug interaction". Grapefruit furanocoumarins (FCs), such as 6', 7'-dihydroxybergamottin (DHB) and bergamottin (BM), have been...

  2. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  3. Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins.

    PubMed

    Lumeng, Carey N; Deyoung, Stephanie M; Saltiel, Alan R

    2007-01-01

    Obesity leads to a proinflammatory state with immune responses that include infiltration of adipose tissue with macrophages. These macrophages are believed to alter insulin sensitivity in adipocytes, but the mechanisms that underlie this effect have not been characterized. We have explored the interaction between macrophages and adipocytes in the context of both indirect and direct coculture. Macrophage-secreted factors blocked insulin action in adipocytes via downregulation of GLUT4 and IRS-1, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane. GLUT1 was upregulated with a concomitant increase in basal glucose uptake. These changes recapitulate those seen in adipose tissue from insulin-resistant humans and animal models. TNF-alpha-neutralizing antibodies partially reversed the insulin resistance produced by macrophage-conditioned media. Peritoneal macrophages and macrophage-enriched stromal vascular cells from adipose tissue also attenuated responsiveness to insulin in a manner correlating with inflammatory cytokine secretion. Adipose tissue macrophages from obese mice have an F4/80(+)CD11b(+)CD68(+)CD14(-) phenotype and form long cellular extensions in culture. Peritoneal macrophages take on similar characteristics in direct coculture with adipocytes and induce proinflammatory cytokines, suggesting that macrophage activation state is influenced by contact with adipocytes. Thus both indirect/secreted and direct/cell contact-mediated factors derived from macrophages influence insulin sensitivity in adipocytes.

  4. Blocking the interaction between S100A9 and RAGE V domain using CHAPS molecule: A novel route to drug development against cell proliferation.

    PubMed

    Chang, Chin-Chi; Khan, Imran; Tsai, Kun-Lin; Li, Hongchun; Yang, Lee-Wei; Chou, Ruey-Hwang; Yu, Chin

    2016-11-01

    Human S100A9 (Calgranulin B) is a Ca(2+)-binding protein, from the S100 family, that often presents as a homodimer in myeloid cells. It becomes an important mediator during inflammation once calcium binds to its EF-hand motifs. Human RAGE protein (receptor for advanced glycation end products) is one of the target-proteins. RAGE binds to a hydrophobic surface on S100A9. Interactions between these proteins trigger signal transduction cascades, promoting cell growth, proliferation, and tumorigenesis. Here, we present the solution structure of mutant S100A9 (C3S) homodimer, determined by multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain via NMR spectroscopy. CHAPS is a zwitterionic and non-denaturing molecule widely used for protein solubilizing and stabilization. We found out that CHAPS and RAGE V domain would interact with mS100A9 by using (1)H-(15)N HSQC NMR titrations. Therefore, using the HADDOCK program, we superimpose two binary complex models mS100A9-RAGE V domain and mS100A9-CHAPS and demonstrate that CHAPS molecules could play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support the conclusion that CHAPS inhibits the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.

  5. Quantification of protein interaction kinetics in a micro droplet

    SciTech Connect

    Yin, L. L.; Wang, S. P. E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J. E-mail: njtao@asu.edu; Zhang, S. T.

    2015-11-15

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  6. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling.

  7. Quantification of protein interaction kinetics in a micro droplet

    NASA Astrophysics Data System (ADS)

    Yin, L. L.; Wang, S. P.; Shan, X. N.; Zhang, S. T.; Tao, N. J.

    2015-11-01

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  8. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling. PMID:25873628

  9. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  10. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  11. Identification of Redox and Glucose-Dependent Txnip Protein Interactions

    PubMed Central

    Neuharth, Skyla; Kim, Dae In; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip. PMID:27437069

  12. Diffusing Colloidal Probes of Protein and Synthetic Macromolecule Interactions

    PubMed Central

    Everett, W. Neil; Wu, Hung-Jen; Anekal, Samartha G.; Sue, Hung-Jue; Bevan, Michael A.

    2007-01-01

    A new approach is described for measuring kT and nanometer scale protein-protein and protein-synthetic macromolecule interactions. The utility of this method is demonstrated by measuring interactions of bovine serum albumin (BSA) and copolymers with exposed polyethyleneoxide (PEO) moieties adsorbed to hydrophobically modified colloids and surfaces. Total internal reflection and video microscopy are used to track three-dimensional trajectories of many single diffusing colloids that are analyzed to yield interaction potentials, mean-square displacements, and colloid-surface association lifetimes. A criterion is developed to identify colloids as being levitated, associated, or deposited based on energetic, spatial, statistical, and temporal information. Whereas levitation and deposition occur for strongly repulsive or attractive potentials, association is exponentially sensitive to weak interactions influenced by adsorbed layer architectures and surface heterogeneity. Systematic experiments reveal how BSA orientation and PEO molecular weight produce adsorbed layers that either conceal or expose substrate heterogeneities to generate a continuum of colloid-surface association lifetimes. These measurements provide simultaneous access to a broad range of information that consistently indicates purely repulsive BSA and PEO interactions and a role for surface heterogeneity in colloid-surface association. The demonstrated capability to measure nonspecific protein interactions provides a basis for future measurements of specific protein interactions. PMID:17098785

  13. Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

    PubMed Central

    2012-01-01

    Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed. PMID:22789293

  14. Use and application of hydrophobic interaction chromatography for protein purification.

    PubMed

    McCue, Justin T

    2014-01-01

    The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.

  15. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a practical…

  16. Rare disease relations through common genes and protein interactions.

    PubMed

    Fernandez-Novo, Sara; Pazos, Florencio; Chagoyen, Monica

    2016-06-01

    ODCs (Orphan Disease Connections), available at http://csbg.cnb.csic.es/odcs, is a novel resource to explore potential molecular relations between rare diseases. These molecular relations have been established through the integration of disease susceptibility genes and human protein-protein interactions. The database currently contains 54,941 relations between 3032 diseases.

  17. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormon...

  18. Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein-Protein Interaction Data.

    PubMed

    Pokharel, Yuba Raj; Saarela, Jani; Szwajda, Agnieszka; Rupp, Christian; Rokka, Anne; Lal Kumar Karna, Shibendra; Teittinen, Kaisa; Corthals, Garry; Kallioniemi, Olli; Wennerberg, Krister; Aittokallio, Tero; Westermarck, Jukka

    2015-12-01

    High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges in translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a relevance ranking platform (RRP), which consist of modular functional and bioinformatic filters to provide relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data, highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validated the importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition. The RRP framework is modular and can be modified to answer versatile research problems depending on the nature of the biological question under study. Based on comparison of RRP to other existing filtering tools, the presented data indicate that RRP offers added value especially for the analysis of interacting proteins for which there is no sufficient prior knowledge available. Finally, we encourage the use of RRP in combination with either SAINT or CRAPome computational tools for selecting the candidate interactors that fulfill the both important requirements, functional relevance, and high confidence interaction detection.

  19. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  20. Examination of Interactions of Oppositely Charged Proteins in Gels

    SciTech Connect

    Ramasamy,P.; El-Maghrabi, M.; Halada, G.; Miller, L.; Rafailovich, M.

    2007-01-01

    Understanding the interactions of proteins with one another serves as an important step for developing faster protein separation methods. To examine protein-protein interactions of oppositely charged proteins, fluorescently labeled albumin and poly-L-lysine were subjected to electrophoresis in agarose gels, in which the cationic albumin and the anionic poly-L-lysine were allowed to migrate toward each other and interact. Fluorescence microscopy was used to image fluorescently tagged proteins in the gel. The secondary structure of the proteins in solution was studied using conventional FTIR spectroscopy. Results showed that sharp interfaces were formed where FITC tagged albumin met poly-L-lysine and that the interfaces did not migrate after they had been formed. The position of the interface in the gel was found to be linearly dependent upon the relative concentration of the proteins. The formation of the interface also depended upon the fluorescent tag attached to the protein. The size of the aggregates at the interface, the fluorescence intensity modifications, and the mobility of the interface for different pore sizes of the gel were investigated. It was observed that the interface was made up of aggregates of about 1 {mu}m in size. Using dynamic light scattering, it was observed that the size of the aggregates that formed due to interactions of oppositely charged proteins depended upon the fluorescent tags attached to the proteins. The addition of small amounts of poly-L-lysine to solutions containing FITC albumin decreased the zeta potential drastically. For this, we propose a model suggesting that adding small amounts of poly-L-lysine to solutions containing FITC -albumin favors the formation of macromolecular complexes having FITC albumin molecules on its surface. Although oppositely charged FITC tagged poly-L-lysine and FITC tagged albumin influence each other's migration velocities by forming aggregates, there were no observable secondary structural

  1. Computational Prediction of Protein–Protein Interaction Networks: Algo-rithms and Resources

    PubMed Central

    Zahiri, Javad; Bozorgmehr, Joseph Hannon; Masoudi-Nejad, Ali

    2013-01-01

    Protein interactions play an important role in the discovery of protein functions and pathways in biological processes. This is especially true in case of the diseases caused by the loss of specific protein-protein interactions in the organism. The accuracy of experimental results in finding protein-protein interactions, however, is rather dubious and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. Computational methods have attracted tremendous attention among biologists because of the ability to predict protein-protein interactions and validate the obtained experimental results. In this study, we have reviewed several computational methods for protein-protein interaction prediction as well as describing major databases, which store both predicted and detected protein-protein interactions, and the tools used for analyzing protein interaction networks and improving protein-protein interaction reliability. PMID:24396273

  2. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  3. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  4. Mechanisms of Peroxynitrite Interactions with Heme Proteins

    PubMed Central

    Su, Jia; Groves, John T.

    2010-01-01

    Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. In this Forum we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve nitric oxide and peroxynitrite. We have recently described direct evidence that ferrylMb and NO2 are both produced during the reaction of PN and metmyolgobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [FeIV=O .NO2]. This caged pair reacts mainly via internal return with a rate constant kr to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, kobs of heme-mediated PN decomposition. Freely-diffusing NO2, which is liberated concomitantly from the radical pair (ke), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a β-sheet structure, at the expense of turns and helices at low pH. It is proposed that a short β-sheet segment, comprising residues 37-39 and 58-61, extends itself into the large 37-61 loop when the latter is destabilized by protonation of H26, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in

  5. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  6. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  7. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  8. Neuronal cell-surface protein neurexin 1 interaction with multi-PDZ domain protein MUPP1.

    PubMed

    Jang, Won Hee; Choi, Sun Hee; Jeong, Joo Young; Park, Jung-Hwa; Kim, Sang-Jin; Seog, Dae-Hyun

    2014-01-01

    Location of membrane proteins is often stabilized by PDZ domain-containing scaffolding proteins. Using the yeast two-hybrid screening, we found that neurexin 1 interacted with multi-PDZ domain protein 1 (MUPP1) through PDZ domain. Neurexin 2 and 3 also interacted with MUPP1. MUPP1 and neurexin 1 were co-localized in cultured cells. These results suggest a novel mechanism for localizing neurexin 1 to synaptic sites.

  9. Computational large-scale mapping of protein-protein interactions using structural complexes.

    PubMed

    Shoemaker, Benjamin; Wuchty, Stefan; Panchenko, Anna R

    2013-01-01

    Although the identification of protein interactions by high-throughput methods progresses at a fast pace, "interactome" datasets still suffer from high rates of false positives and low coverage. To map the interactome of any organism, this unit presents a computational framework to predict protein-protein or gene-gene interactions utilizing experimentally determined evidence of structural complexes, atomic details of binding interfaces and evolutionary conservation.

  10. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  11. The Rift Valley Fever virus protein NSm and putative cellular protein interactions

    PubMed Central

    2012-01-01

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection. PMID:22838834

  12. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  13. [Study of decision tree in the application of predicting protein-protein interactions].

    PubMed

    Guo, Xiaolong; Jiang, Yan; Qui, Lu

    2013-10-01

    Proteins are the final executive actor of cell viability and function. Protein-protein interactions determine the complexity of the organism. Research on the protein interactions can help us understand the function of the protein at the molecular level, learn the cell growth, development, differentiation, apoptosis and understand biological regulation mechanisms and other activities. They are essential for understanding the pathologies of diseases and helpful in the prevention and treatment of diseases, as well as in the development of new drugs. In this paper, we employ the single decision-tree classification model to predict protein-protein interactions in the yeast. The original data came from the existing literature. Using software Clementine, this paper analyzes how these attributes affect the accuracy of the model by adjusting the predicted attributes. The result shows that a single decision tree is a good classification model and it has higher accuracy compared to those in the previous researches.

  14. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    PubMed

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  15. ProteinShop: A tool for interactive protein manipulation and steering

    SciTech Connect

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  16. Proteins interactions implicated in AMPA receptor trafficking: a clear destination and an improving route map||

    PubMed Central

    Henley, Jeremy M.

    2012-01-01

    The mechanisms that regulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), synthesis, transport, targeting and surface expression are of fundamental importance to understand the molecular basis of fast excitatory neurotransmission and synaptic plasticity in the mammalian CNS. An area of intense current interest is how AMPARs are directed to the correct locations in the neuron as and when required. This is a multi-layered problem, which involves complex spatiotemporal coordination of multiple protein interactions. Considerable progress has been achieved in identifying a number of proteins that bind directly to AMPAR subunits and the functional consequences of blocking some of these interactions have been determined. This review highlights recent developments in the field. PMID:12631461

  17. Sequence-based prediction of protein-protein interaction sites with L1-logreg classifier.

    PubMed

    Dhole, Kaustubh; Singh, Gurdeep; Pai, Priyadarshini P; Mondal, Sukanta

    2014-05-01

    Protein-protein interactions are of central importance for virtually every process in a living cell. Information about the interaction sites in proteins improves our understanding of disease mechanisms and can provide the basis for new therapeutic approaches. Since a multitude of unique residue-residue contacts facilitate the interactions, protein-protein interaction sites prediction has become one of the most important and challenging problems of computational biology. Although much progress in this field has been reported, this problem is yet to be satisfactorily solved. Here, a novel method (LORIS: L1-regularized LOgistic Regression based protein-protein Interaction Sites predictor) is proposed, that identifies interaction residues, using sequence features and is implemented via the L1-logreg classifier. Results show that LORIS is not only quite effective, but also, performs better than existing state-of-the art methods. LORIS, available as standalone package, can be useful for facilitating drug-design and targeted mutation related studies, which require a deeper knowledge of protein interactions sites. PMID:24486250

  18. AKAP350 Interaction with cdc42 Interacting Protein 4 at the Golgi Apparatus

    PubMed Central

    Larocca, M. Cecilia; Shanks, Ryan A.; Tian, Lan; Nelson, David L.; Stewart, Donn M.; Goldenring, James R.

    2004-01-01

    The A kinase anchoring protein 350 (AKAP350) is a multiply spliced type II protein kinase A anchoring protein that localizes to the centrosomes in most cells and to the Golgi apparatus in epithelial cells. In the present study, we sought to identify AKAP350 interacting proteins that could yield insights into AKAP350 function at the Golgi