The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Michael B. Yaffe - One of the best experts on this subject based on the ideXlab platform.

  • substrate specificity analysis of protein kinase complex dbf2 mob1 by Peptide Library and proteome array screening
    BMC Biochemistry, 2005
    Co-Authors: Angie S Mah, Michael B. Yaffe, Andrew E H Elia, Geeta Devgan, Jason Ptacek, Mike Schutkowski, Michael Snyder, Raymond J Deshaies
    Abstract:

    The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. Using a combination of Peptide Library selection, phosphorylation of opitmal Peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in Peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for Peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. We have determined by Peptide Library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site

  • the use of in vitro Peptide Library screens in the analysis of phosphoserine threonine binding domain structure and function
    Annual Review of Biophysics and Biomolecular Structure, 2004
    Co-Authors: Michael B. Yaffe, Stephen J Smerdon
    Abstract:

    ▪ Abstract Phosphoserine/threonine-binding domains integrate intracellular signal transduction events by forming multiprotein complexes with substrates of protein serine/threonine kinases. These phosphorylation-dependent molecular recognition events are responsible for coordinating the precise temporal and spatial response of cells to a wide range of stimuli, particularly those involved in cell cycle control and the response to DNA damage. The known families of phosphoserine/threonine-binding modules include 14-3-3 proteins, WW domains, FHA domains, WD40 repeats, and the Polo-box domains of Polo-like kinases. Peptide-Library experiments reveal the optimal sequence motifs recognized by these domains, and facilitate high-resolution structural studies elucidating the mechanisms of phospho-dependent binding and the molecular basis for domain function within intricate signaling networks. Information emerging from these studies is critical for the design of novel experimental and therapeutic tools aimed at alte...

  • the use of in vitro Peptide Library screens in the analysis of phosphoserine threonine binding domain structure and function
    Annual Review of Biophysics and Biomolecular Structure, 2004
    Co-Authors: Michael B. Yaffe, Stephen J Smerdon
    Abstract:

    Phosphoserine/threonine-binding domains integrate intracellular signal transduction events by forming multiprotein complexes with substrates of protein serine/threonine kinases. These phosphorylation-dependent molecular recognition events are responsible for coordinating the precise temporal and spatial response of cells to a wide range of stimuli, particularly those involved in cell cycle control and the response to DNA damage. The known families of phosphoserine/threonine-binding modules include 14-3-3 proteins, WW domains, FHA domains, WD40 repeats, and the Polo-box domains of Polo-like kinases. Peptide-Library experiments reveal the optimal sequence motifs recognized by these domains, and facilitate high-resolution structural studies elucidating the mechanisms of phospho-dependent binding and the molecular basis for domain function within intricate signaling networks. Information emerging from these studies is critical for the design of novel experimental and therapeutic tools aimed at altering signal transduction cascades in normal and diseased cells.

  • Peptide and protein Library screening defines optimal substrate motifs for akt pkb
    Journal of Biological Chemistry, 2000
    Co-Authors: Michael B. Yaffe, Toshiyuki Obata, German Leparc, Elizabeth T Piro, Hiroshi Maegawa, Atsunori Kashiwagi, Ryuichi Kikkawa, Lewis C. Cantley
    Abstract:

    AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented Peptide Library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal Peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for highV max/K m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a λGEX phage HeLa cell cDNA expression Library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by Peptide Library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.

  • a Peptide Library approach identifies a specific inhibitor for the zap 70 protein tyrosine kinase
    Molecular Cell, 2000
    Co-Authors: Kiyotaka Nishikawa, Michael B. Yaffe, Sansana Sawasdikosol, David A Fruman, Jack Lai, Zhou Songyang, Steven J Burakoff, Lewis C. Cantley
    Abstract:

    Summary cally blocked signaling downstream of ZAP-70. Our studies extend the range of experimental approaches We utilized a novel Peptide Library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase in- for probing ZAP-70 function in vivo and introduce a technique for developing protein kinase inhibitors that volved in T cell activation. By screening more than 6 billion Peptides oriented by a common Tyr residue could be useful for a wide range of protein kinases. for their ability to bind to ZAP-70, we determined a consensus optimal Peptide. A Phe-for-Tyr substituted Results and Discussion version of the Peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates In order to identify high-affinity inhibitors of the ZAP-70 (KI of 2 mM). The related protein Tyr kinases, Lck and protein Tyr kinase, we screened a Tyr-oriented Peptide Syk, were not significantly inhibited by the Peptide. Library by affinity purification rather than by catalytic When introduced into intact T cells, the Peptide conversion. The Peptide Library used contained the seblocked signaling downstream of ZAP-70, including quence Met-Ala-X-X-X-X-Tyr-X-X-X-X-Ala-Lys-Lys-Lys ZAP-70-dependent gene induction, without affecting where X indicates all amino acids except Trp, Cys, or upstream Tyr phosphorylation. Thus, screening Tyr- Tyr. The predicted degeneracy of this Library is 178 < oriented Peptide libraries can identify selective pep- 6.9 billion. Screening was performed in the presence of tide inhibitors of protein Tyr kinases. 100 mM ATP but in the absence of Mg 21 to prevent

Emmanouil D. Karagiannis - One of the best experts on this subject based on the ideXlab platform.

  • Rational Design of a Biomimetic Cell Penetrating Peptide Library
    ACS Nano, 2013
    Co-Authors: Emmanouil D. Karagiannis, Jeisa M. Pelet, Siddharth Jhunjhunwala, Aleksandra M Urbanska, Gaurav Sahay, Robert Langer, Dustin Anderson
    Abstract:

    Cell penetrating Peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating Peptide based formulations with potential to deliver small interfering RNAs intracellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different Peptides was evaluated both in vitro and in vivo and correlated with the Peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the Peptides were presented to bone marrow-derived dendritic cells they induce low immunoactivation relative to control cell penetrating Peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86 and major histocompatibility complex class II. In vivo, Peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human Peptides derived from surfactant protein B; a lung surfactant protein, orexin; a neuroPeptide hormone and lactoferricin; a globular glycoprotein that exist in many physiological fluids, facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.

  • Rational design of a biomimetic cell penetrating Peptide Library
    ACS Nano, 2013
    Co-Authors: Emmanouil D. Karagiannis, Jeisa M. Pelet, Siddharth Jhunjhunwala, Aleksandra M Urbanska, Gaurav Sahay, Robert Langer, D. G. Anderson
    Abstract:

    Cell penetrating Peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating Peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different Peptides was evaluated both in vitro and in vivo and correlated with the Peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the Peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating Peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. In vivo, Peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human Peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuroPeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.

Lewis C. Cantley - One of the best experts on this subject based on the ideXlab platform.

  • a rapid method for determining protein kinase phosphorylation specificity
    Nature Methods, 2004
    Co-Authors: Jessica E Hutti, Lewis C. Cantley, Emily T Jarrell, James Chang, Derek W Abbott, Peter Storz, Alex Toker, Benjamin E Turk
    Abstract:

    Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred Peptide phosphorylation motif for a given kinase permits the production of efficient Peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial Peptide Library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.

  • Peptide and protein Library screening defines optimal substrate motifs for akt pkb
    Journal of Biological Chemistry, 2000
    Co-Authors: Michael B. Yaffe, Toshiyuki Obata, German Leparc, Elizabeth T Piro, Hiroshi Maegawa, Atsunori Kashiwagi, Ryuichi Kikkawa, Lewis C. Cantley
    Abstract:

    AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented Peptide Library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal Peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for highV max/K m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a λGEX phage HeLa cell cDNA expression Library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by Peptide Library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.

  • a Peptide Library approach identifies a specific inhibitor for the zap 70 protein tyrosine kinase
    Molecular Cell, 2000
    Co-Authors: Kiyotaka Nishikawa, Michael B. Yaffe, Sansana Sawasdikosol, David A Fruman, Jack Lai, Zhou Songyang, Steven J Burakoff, Lewis C. Cantley
    Abstract:

    Summary cally blocked signaling downstream of ZAP-70. Our studies extend the range of experimental approaches We utilized a novel Peptide Library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase in- for probing ZAP-70 function in vivo and introduce a technique for developing protein kinase inhibitors that volved in T cell activation. By screening more than 6 billion Peptides oriented by a common Tyr residue could be useful for a wide range of protein kinases. for their ability to bind to ZAP-70, we determined a consensus optimal Peptide. A Phe-for-Tyr substituted Results and Discussion version of the Peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates In order to identify high-affinity inhibitors of the ZAP-70 (KI of 2 mM). The related protein Tyr kinases, Lck and protein Tyr kinase, we screened a Tyr-oriented Peptide Syk, were not significantly inhibited by the Peptide. Library by affinity purification rather than by catalytic When introduced into intact T cells, the Peptide conversion. The Peptide Library used contained the seblocked signaling downstream of ZAP-70, including quence Met-Ala-X-X-X-X-Tyr-X-X-X-X-Ala-Lys-Lys-Lys ZAP-70-dependent gene induction, without affecting where X indicates all amino acids except Trp, Cys, or upstream Tyr phosphorylation. Thus, screening Tyr- Tyr. The predicted degeneracy of this Library is 178 < oriented Peptide libraries can identify selective pep- 6.9 billion. Screening was performed in the presence of tide inhibitors of protein Tyr kinases. 100 mM ATP but in the absence of Mg 21 to prevent

  • 12 mapping specificity determinants for protein protein association using protein fusions and random Peptide libraries
    Methods in Enzymology, 2000
    Co-Authors: Michael B. Yaffe, Lewis C. Cantley
    Abstract:

    Publisher Summary Many signaling proteins and small modular domains such as Src homology 2 (SH2), SH3, 14-3-3, WW, protein tyrosine binding (PTB), and PSD-90/disc-large/zol (PDZ) domains interact with protein ligands by recognizing short linear stretches of amino acids. Determining the precise amino-acid sequence that constitutes the binding motif for these modular signaling elements can be a technical challenge, and a variety of techniques are available for this purpose including phage display, one-Peptide/one-pin type techniques, and alanine-scanning mutagenesis of the target proteins. Oriented Peptide Library screening is a technique that differs from these techniques by the rapidity with which sequence motifs can be determined for a protein or domain of interest. Once an immobilized form of the protein or domain is available, usually as a glutathione S -transferase (GST) or maltose-binding protein (MBP) fusion, soluble Peptide Library screening can be performed in an afternoon and a motif determined within 1–2 days. An additional advantage of the soluble Peptide Library technique is that the results provide a quantitative measure of the binding affinity for any amino acid within the deduced binding motif.

  • use of an oriented Peptide Library to determine the optimal substrates of protein kinases
    Current Biology, 1994
    Co-Authors: Zhou Songyang, Steven Blechner, Nicole Hoagland, Merl F Hoekstra, Helen Piwnicaworms, Lewis C. Cantley
    Abstract:

    Abstract Background: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals. A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly. Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking. Results We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented Library of more than 2.5 billion Peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates. The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B–Cdc2 and cyclin A–CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity. We also discuss a model incorporating the optimal cyclin B–Cdc2 substrate into the known crystal structure of this kinase. Conclusion Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.

Kiyotaka Hitomi - One of the best experts on this subject based on the ideXlab platform.

  • Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed Peptide Library
    Archives of Biochemistry and Biophysics, 2008
    Co-Authors: Yoshiaki Sugimura, Masatoshi Maki, Keiichi Yokoyama, Kiyotaka Hitomi
    Abstract:

    Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred Peptide sequences using a phage-displayed random Peptide Library. Most of the Peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the Peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.

  • Screening for the preferred substrate sequence of transglutaminase using a phage-displayed Peptide Library: Identification of Peptide substrates for TGase 2 and factor XIIIa
    Journal of Biological Chemistry, 2006
    Co-Authors: Yoshiaki Sugimura, Masayo Hosono, Fumitaka Wada, Masatoshi Maki, Tohru Yoshimura, Kiyotaka Hitomi
    Abstract:

    Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of Peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random Peptide Library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing Peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII ( Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxP phi D( P), QxP phi, and Qxx phi DP; Factor XIIIa, Qxx phi xWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant Peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Dehua Pei - One of the best experts on this subject based on the ideXlab platform.

  • synthesis and screening of a cyclic Peptide Library discovery of small molecule ligands against human prolactin receptor
    Bioorganic & Medicinal Chemistry, 2009
    Co-Authors: Tao Liu, Sang Hoon Joo, Jeffrey L Voorhees, Charles L Brooks, Dehua Pei
    Abstract:

    Prolactin receptor is involved in normal lactation and reproduction; however, excessive prolactin levels can cause various reproductive disorders such as prolactinomas. Small-molecule antagonists against the human prolactin receptor (hPRLr) thus have potential clinical applications and may serve as useful molecular probes in biomedical research. In this work, we synthesized a large, supportbound cyclic Peptide Library (theoretical diversity of 1.2 x 10 7 ) on 90-μm TentaGel beads and screened it against the extracellular domain of hPRLr. To facilitate hit identification, each TentaGel bead was spatially segregated into outer and inner layers, with a cyclic Peptide displayed on the bead surface while the bead interior contained the corresponding linear Peptide. The identity of a positive bead was revealed by sequencing the linear encoding Peptide within the bead by partial Edman degradation/mass spectrometry. Screening of the Library resulted in 20 hits, two of which were selected for further analysis and shown to bind to hPRLr with dissociation constants of 2–3 μM.

  • substrate profiling of protein tyrosine phosphatase ptp1b by screening a combinatorial Peptide Library
    Journal of the American Chemical Society, 2007
    Co-Authors: Mathieu Garaud, Dehua Pei
    Abstract:

    Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. To date, the in vivo substrates and physiological functions of PTPs remain poorly defined. In this work, we have developed a novel combinatorial Library method to systematically determine the substrate specificity of PTPs. A one-bead-one-compound Peptide Library containing five randomized residues, Fmoc-XXXXXpYAA (where X is norleucine or 17 proteinogenic amino acids excluding Tyr, Cys, and Met), was chemically synthesized on 90-μm TentaGel resin by the split-and-pool method. Limited treatment of the Library with a PTP removed the phosphoryl group from beads that carry the most preferred substrates. The exposed tyrosine side chain was selectively oxidized into an orthoquinone by the treatment with tyrosinase in the presence of atmospheric oxygen. The orthoquinone was then selectively derivatized with biotin-hydrazide, followed by on-bead colorim...

  • high throughput sequence determination of cyclic Peptide Library members by partial edman degradation mass spectrometry
    Journal of the American Chemical Society, 2006
    Co-Authors: Sang Hoon Joo, Qing Xiao, Yun Ling, Bhaskar Gopishetty, Dehua Pei
    Abstract:

    Cyclic Peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic Peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit Peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic Peptide Library members has been developed. TentaGel microbeads (90 μm) were spatially segregated into outer and inner layers; cyclic Peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear Peptide as the encoding sequence. After screening of the cyclic Peptide Library against a macromolecular target, the identity of hit Peptides was determined by sequencing the linear encoding Peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octaPeptide Library (theore...