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Brian K Kay

Publications and source records attributed to Brian K Kay.

17 recordsLinked to original sources
PubMed2006

Mapping protease substrates by using a biotinylated phage substrate library.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Bacteriophage M13↗
PubMed2005

Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries.

Materials that combine inorganic components and biological molecules provide a new paradigm for synthesizing nanoscale and larger structures with tailored physical properties. These synthesis techniques utilize the molecular recognition properties of many biological molecules to nucleate and control growth of the nanoscale structure. Phage-displayed peptide libraries are a powerful tool to identify peptides that selectively recognize and bind to a variety of inorganic surfaces that are utilized in electronic and photonic devices. These libraries have been used extensively to study the peptide-mediated nucleation and growth of some metallic and semiconducting materials, and the application to designed nanostructures has been demonstrated.

Bacteriophage M13↗
PubMed2005

Efficient construction of a large collection of phage-displayed combinatorial peptide libraries.

Selections from phage-displayed combinatorial peptide libraries are an effective strategy for identifying peptide ligands to target proteins. Existing protocols for constructing phage-displayed libraries utilize either ligation into double-stranded phage DNA or Kunkel mutagenesis with single-stranded phagemid DNA. Although the Kunkel approach rapidly provides library sizes of up to 10(11), as many as 20% of the phagemids may be non-recombinant. With several modifications to current Kunkel protocols, we have generated peptide libraries with sizes of up to 10(11) clones and recombination frequencies approaching 100%. The production of phage libraries, as opposed to phagemid libraries, simplifies selection experiments by eliminating the need for helper phage. Our approach relies upon the presence of an amber stop codon in the coding region of gene III of bacteriophage M13. Oligonucleotides containing randomized stretches of DNA are annealed to the phage genome such that the randomized region forms a heteroduplex with the stop codon. The oligonucleotide is then enzymatically extended to generate covalently-closed, circular DNA, which is electroporated into a non-suppressor strain of Escherichia coli. If the amber stop codon is present in the DNA molecule, protein III is not synthesized and the phage cannot propagate itself. This method is customizable for the display of either random or focused peptide libraries. To date, we have constructed 22 different libraries ranging from 8-20 amino acids in length, utilizing complete or reduced codon sets.

Amino Acid Sequence↗
PubMed2004

Fas-associated protein with death domain (FADD)-independent recruitment of c-FLIPL to death receptor 5.

Here we show a novel mechanism by which FLICE-like inhibitory protein (c-FLIP) regulates apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and one of its receptors, DR5. c-FLIP is a critical regulator of the TNF family of cytokine receptor signaling. c-FLIP has been postulated to prevent formation of the competent death-inducing signaling complex (DISC) in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. In order to identify regulators of TRAIL function, we used the intracellular death domain (DD) of DR5 as a target to screen a phage-displayed combinatorial peptide library. The DD of DR5 selected from the library a peptide that showed sequence similarity to a stretch of amino acids in the C terminus of c-FLIP(L). The phage-displayed peptide selectively interacted with the DD of DR5 in in vitro binding assays. Similarly, full-length c-FLIP (c-FLIP(L)) and the C-terminal p12 domain of c-FLIP interacted with DR5 both in in vitro pull-down assays and in mammalian cells. This interaction was independent of TRAIL. To the contrary, TRAIL treatment released c-FLIP(L) from DR5, permitting the recruitment of FADD to the active DR5 signaling complex. By employing FADD-deficient Jurkat cells, we demonstrate that DR5 and c-FLIP(L) interact in a FADD-independent manner. Moreover, we show that a cellular membrane permeable version of the peptide corresponding to the DR5 binding domain of c-FLIP induces apoptosis in mammalian cells. Taken together, these findings indicate that c-FLIP interacts with the DD of DR5, thus preventing death (L)signaling by DR5 prior to the formation of an active DISC. Because TRAIL and DR5 are ubiquitously expressed, the interaction of c-FLIP(L) and DR5 indicates a mechanism by which tumor selective apoptosis can be achieved through protecting normal cells from undergoing death receptor-induced apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers.

Antibody arrays hold considerable potential in a variety of applications including proteomics research, drug discovery, and diagnostics. Many of the schemes used to fabricate the arrays fail to immobilize the antibodies at a uniform density or in a single orientation; consequently, the immobilized antibodies recognize their antigens with variable efficiency. This paper describes a strategy to immobilize antibodies in a single orientation, with a controlled density, using the covalent interaction between cutinase and its suicide substrate. Protein fusions between cutinase and five antibodies of three different types (scFv, V(HH), and FN3) were prepared and immobilized upon self-assembled monolayers (SAMs) presenting a phosphonate capture ligand. The immobilized antibodies exhibit high affinity and selectivity for their target antigens, as monitored by surface plasmon resonance and fluorescence scanning. Furthermore, by changing the density of capture ligand on the SAM the density of the immobilized antibody could be controlled. The monolayers, which also present a tri(ethylene glycol) group, are inert to nonspecific adsorption of proteins and allow the detection of a specific antigen in a complex mixture. The demonstration of cutinase-directed antibody immobilization with insert SAMs provides a straightforward and robust method for preparing antibody chips.

Antibodies↗
PubMed2004

Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain.

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.

Animals↗
PubMed2004

In vivo biotinylated proteins as targets for phage-display selection experiments.

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus (pMCSG16 and 15) via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase. To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, also contain a ligase-independent cloning site (LIC), which permits up to 100% cloning efficiency. The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries. From selections using the N-terminally biotinylated version of one target protein, a peptide ligand (Kd= 9 microM) was recovered that bound in a format-dependent manner. To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries.

Bacterial Proteins↗
PubMed2004

Identification of a peptide binding motif for secreted frizzled-related protein-1.

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.

Alanine↗
PubMed2004

Accelerated screening of phage-display output with alkaline phosphatase fusions.

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.

Alkaline Phosphatase↗
PubMed2003

Characterization of a gamma-adaptin ear-binding motif in enthoprotin.

Enthoprotin, a newly identified component of clathrin-coated vesicles, interacts with the trans-Golgi network (TGN) clathrin adapters AP-1 and GGA2. Here we perform a multi-faceted analysis of the site in enthoprotin that is responsible for the binding to the gamma-adaptin ear (gamma-ear) domain of AP-1. Alanine scan mutagenesis and nuclear magnetic resonance (NMR) studies reveal the full extent of the site as well as critical residues for this interaction. NMR studies of the gamma-ear in complex with a synthetic peptide from enthoprotin provide structural details of the binding site for TGN accessory proteins within the gamma-ear.

Adaptor Protein Complex gamma Subunits↗
PubMed2003

A role for epsin N-terminal homology/AP180 N-terminal homology (ENTH/ANTH) domains in tubulin binding.

The epsin N-terminal homology (ENTH) domain is a protein module of approximately 150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple alpha-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of approximately 1 microm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling.

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.

3T3 Cells↗
PubMed2002

A fluorescence polarization assay for the identification of inhibitors of the p53-DM2 protein-protein interaction.

Improper function of the tumor suppressor protein p53 is a contributing factor in many human cancers. In normal cells, p53 acts to arrest the cell cycle in response to DNA damage or nucleotide depletion. One mechanism of regulating the amount of p53 in the cell is through the action of the Double Minute 2 protein, DM2 (also known as MDM2), which ubiquitinates p53 and targets it for proteosomal degradation. In a number of human cancers, the DM2 gene is amplified or overexpressed, leading to inadequate levels of p53 for cell cycle arrest or apoptosis. With the goal of restoring p53 function in cancers that overexpress DM2, we are developing inhibitors of the p53-DM2 protein-protein interaction that structurally mimic the N-terminal segment of p53 that binds to DM2. To assist this effort, we have devised a fluorescence polarization assay that quantifies the interaction between the N-terminal regions of both proteins in 384-well microtiter plates. Using this assay, we have demonstrated that a peptide with a nonhydrolyzable beta-amino acid substitution binds DM2 with an affinity comparable to a p53 peptide that is composed of only alpha-amino acids.

Amino Acid Sequence↗
PubMed2002

One from column A and two from column B: the benefits of phage display in molecular-recognition studies.

Recent uses of phage-displayed combinatorial peptide and cDNA libraries have proven invaluable in mapping protein-protein interactions, protein-drug interactions, and the generation of 'molecular therapeutics'. This article reviews some of the findings of the past year and points out some of the pros and cons of phage display as compared with those of yeast two-hybrid screening.

Animals↗